Advances in Plant Breeding

Volume - 3
Chief Editor
Dr. Pidigam Saidaiah
Department of Genetics and Plant Breeding, College of Horticulture,
Sri Konda Laxman Telangana State Horticultural University Rajendranagar,
Hyderabad, Telangana, India
AkiNik Publications
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Publication Year: 2019
Pages: 166
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Contents
Chapters Page No.
1. Different Vector Systems and Their Utilization in Gene Transfer of

Eukaryotes 01-23
(B. Prasanna Kumar, R. Gireesh Kumar and Dr. A. Vijaya Gopal)
2. Basic Concepts of Heterosis 25-37

(Manav, Jaswant Singh Khokhar and Bharat Taindu Jain)
3. Introduction of Plant Breeding 39-47

(Madhuri Grace Minz and Anjum Ahmad)
4. Conventional vs. Marker Assisted Backcross Breeding 49-75

(Dr. Sree Latha Edpuganti)
5. Study of Induced Polygenic Variability in M3 Population of

Aromatic Rice 77-93
(Sanjeev Singh, Rishi Kumar Sharma, Satish Kumar Chakravarti and Prakash Singh)
6. Plant Breeding: A Tool for Potential Exploitation of Genetic Gain

in Crop Plants 95-105
(Manish Kumar, Ravi P Singh, Onkar Nath Singh, Vineeta Singh, Prakash Singh,
Dahiphale A.V., Debarchana Jena, Diptibala Rout, J.L. Katara, S. Samantaray and
Ramlakhan Verma)
7. Flowering Biology of Horticultural Crops 107-133

(Dr. Sable P.A., Sushma Sable (Sonpure) and R.R. Lipane)
8. Unfruitfulness 135-166
(Dr. Sable P.A., Sushma Sable (Sonpure) and R.R. Lipane)
Chapter - 1
Different Vector Systems and Their Utilization in
Gene Transfer of Eukaryotes
Authors
B. Prasanna Kumar
Ph.D. Research Scholar, Department of Agricultural
Microbiology, Acharya N.G Ranga Agricultural University,
Advanced Post Graduate Centre, Lam, Guntur, Andhra
Pradesh, India
R. Gireesh Kumar
Pradesh, India
Dr. A. Vijaya Gopal
Pradesh, India
Page | 1
Page | 2
Chapter - 1
Different Vector Systems and Their Utilization in Gene
Transfer of Eukaryotes
B. Prasanna Kumar, R. Gireesh Kumar and Dr. A. Vijaya Gopal
Abstract
A vector is a DNA molecule used as a vehicle to carry foreign genetic
material into another cell, where it can be replicated and/or expressed. A
vector containing foreign DNA is termed recombinant DNA. The four major
types of vectors are plasmids, viral vectors, cosmids, and artificial
chromosomes. Of these, the most commonly used vectors are plasmids.
Common to all engineered vectors are an origin of replication, a multi
cloning site, and a selectable marker.
Generally, there are two main approaches: the one utilizing biological,
viral vectors and the other utilizing either chemical or physical methods to
introduce gene of interest into target cells. The first one, the viral gene
transfer is a viral-mediated process referred to as an infection. The second,
non-viral gene transfer involves treatment of cell by chemical or physical
means and the process itself is named transfection. Gene delivery by
infection is more complicated than transfection. Infection requires more
steps and more times than does transfection and biosafety issues may also
arise, depending on the virus used. A much safer alternative to infection-
transfection, is also faster and requires only a few reagents including plasmid
DNA containing the gene of interest under the control of a strong cell-
specific promoter. However, inefficient gene delivery and poor sustained
gene expression are its major drawbacks (Mitrovic, T. 2003).
Approaches for delivering DNA into plant cells and gene transformation
can be divided into two major categories: direct and indirect DNA deliveries.
Direct method does not employ bacterial cells as mediators, as an alternative,
it uses a chemical alteration or physical force such as electric discharge or
pressure to deliver the vector DNA into a host cell. Whereas, in indirect
approach, genes of interest are introduced into the target cell through
bacteria, for instance, Agrobacterium tumefaciens or Agrobacterium
rhizogenes. For introducing genes into plants, microprojectile bombardment
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and Agrobacterium-mediated gene transfer is the most widely exploited
methods because of their ability to transform intact, re-generable tissues and
organs (Rashid and Lateef, 2016).
In view of the above, the vector system for gene transfer is very
important. Vectors in which can take up large channels of DNA and
movement across the genomes in the future is important.
Keywords: Transfection, transformation, infection, biosafety, gene delivery,
Agrobacterium, rhizogenes
Introduction
What is a Vector?
The cloning vehicles are called vectors. The vector is a vehicle (or)
carrier which is used for cloning foreign DNA in bacteria. Any extra-
chromosomal small genome.
E.g. Plasmid, Phage and Virus may be used as vector.
Vectors Components
1) The right border sequence of T-DNA which is absolutely required for T-
DNA integration into plant cell DNA, 2. A multiple cloning site
(polylinker DNA) that promotes the insertion of cloned gene into the
region between T-DNA borders, 3. An origin of DNA replication that
allow the plasmids to multiply in E. coli, 4. A selectable marker gene for
appropriate selection of the transformed cells.
(E.g. Neomycin phosphotransferase)
Properties of a Good Vector

1) It should be able to replicate autonomously.
2) It should be easy to isolate and purify.
3) It should be easily introduced into the host cells.
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4) The vector should have suitable marker genes that allow easy
detection or/and selection of the transformed host cell, E.g. Genes
for Ampicillin and Tetracycline resistance.
5) The cells transform with recombination DNA should be identifiable
(or) selectable from those transformed by the unaltered vector.
6) A vector should contain unique target sites for as many restriction
enzymes as possible into which the DNA insert can be integrated.
Major Types of Cloning Vectors
 Plasmid: Circular extra chromosomal DNA that autonomously
replicates inside the bacterial cell. Plasmids generally have a high
copy number, such as pUC19 which has a copy number of 500-700
copies per cell.
 Phage: Linear DNA molecules derived of bacteriophage lambda.
Can be replaced with foreign DNA without disrupting its life cycle.
 Cosmids: Another circular extra chromosomal DNA molecule that
combines features of plasmids and phage.
 Bacterial Artificial Chromosomes: Based on bacterial mini-F
plasmids.
 Yeast Artificial Chromosomes: Artificial chromosome that
contains telomeres (disposable buffers at the ends of chromosomes
which are cut off during cell division), origin of replication, a yeast
centromere (part of a chromosome that links sister chromatids or a
dyad), and a selectable marker for identification in yeast cells.
 Human Artificial Chromosome: This type of vector is potentially
useful for gene delivery into human cells, and a tool for expression
studies and determining human chromosome function. It can carry
very large DNA fragment.
Plasmid
Plasmids are double-stranded, generally circular DNA sequences
capable of automatically replicating in a host cell. Plasmid vectors minimally
consist of the transgene insert and an origin of replication, which allows for
semi-independent replication of the plasmid in the host. Plasmids may be
conjugative/transmissible or non-conjugative. Conjugative plasmids mediate
DNA transfer through conjugation and therefore spread rapidly among the
bacterial cells of a population. Non-conjugative plasmids do not mediate
DNA through conjugation.The size of plasmids ranges from a few kb to near
100 kb.
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pBR322 plasmid, one of the first plasmids to be used widely as
a cloning vector. The genes encoded (amp and tet for Ampicillin and
Tetracycline resistance respectively), its origin of replication (ori), and
various restriction sites (indicated in blue).
It contains a polylinker which can recognize several different restriction

enzymes, an ampicillin-resistance gene (ampr) for selective amplification,
and a replication origin (ORI) for proliferation in the host cell.
Phage Vector
Phages are very simple in structure, consisting merely of aDNA (or
occasionally RNA) molecule carrying a number of genes, surrounded by a
Page | 6
protective coat or capsidmade up of protein molecules. It can accept very
large pieces of foreign DNA. Genetic engineers have constructed numerous
derivatives of phage vectors that contain only one or two sites for a variety
of restriction enzymes. Phage that have astuffer fragment are called
substitution vectors because they are designed to have a piece removed and
substituted with something else. E.g.: Lambda phage, M13 phage, T4, T7
phage, P1 phage etc.
Genes (III, VI, VII, VIII and IX encode structural proteins

Genes (II, V and X) for phage DNA replication
Genes (I, IV and XI) encode products for assembly and secretion
Lambda Phage
λ phages are viruses that can infect bacteria. The major advantage of the
λ phage vector is its high transformation efficiency, about 1000 times more
efficient than the plasmid vector.
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DNA cloning using λ phages as vectors. The DNA to be cloned is first
inserted into the λ DNA, replacing a nonessential region. Then, by an in vitro
assembly system, the λ virion carrying the recombinant DNA can be formed.
The λ genome is 49 kb in length which can carry up to 25 kb foreign DNA.
Cosmids
The cosmid vector is a combination of the plasmid vector and the COS
site which allows the target DNA to be inserted into the λ head. The cos site
is the only requirement for DNA to be packaged into a phage particle. Since
phage particles can accept between 38 and 53 kb of DNA and since most
cosmids are about 5 kb, between 33 and 48 kb of DNA can cloned in these
vectors.
It has the Following Advantages
High transformation efficiency. The cosmid vector can carry up to 45 kb
whereas plasmid and λ phage vectors are limited to 25kb.
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Cloning by using cosmid vectors. (a) In addition to ampr, ORI, and
polylinker as in the plasmid vector, the cosmid vector also contains a COS
site. (b) After cosmid vectors are cleaved with restriction enzyme, they are
ligated with DNA fragments. The subsequent assembly and transformation
steps are the same as cloning with λ phages.
Bacterial Artificial Chromosomes (BAC)

Bacterial Artificial Chromosome vector used to clone a target DNA
piece and planted in bacterial content. The vector acts as a parent host, which
can acts like a guide to carry out the gene for cloning. Bacterial artificial
chromosomes can be as large as insects. In order to amplify a gene, DNA
sequence must be extracted from a desired source using restriction enzymes
to cleave the vector and the target DNA and attach into a host bact.
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Yeast Artificial Chromosomes
Yeast Artificial Chromosomes (YACs) are synthetic double stranded
linear constructs containing the elements necessary for replication as
independent chromosomes in yeast. Minimal size for a YAC is between 50
kb and 100 kb, while maximum sizes are 1 Mb to 3 Mb.A common tool for
constructing YACs is a shuttle plasmid such as pYAC4 which replicates in
E. coli, has a multiple cloning site, and a pair of telomeres which can be
cleaved to form a linear fragment.
Page | 10
An Autonomous Replication Sequence (ARS): ARS1, chromosome III
ARS, ARSH4, A Centromere: CEN4, CEN6: CEN4 is found in most yeast
centromere-containing vectors, such as pYAC4. These vectors typically use
ARS1 sequences. Centromeres consist of three centromere determining
elements: CDE I, CDE II, and CDE III. A telomeric sequence at each end
typically the chromosome also contains a selection marker such as TRP1,
Lys2 or Ura3.
Human Artificial Chromosome (HAC)
A Human Artificial Chromosome (HAC) is a mini-chromosome that is
constructed artificially in human cells. Using its own self-replicating and
segregating systems, a HAC can behave as a stable chromosome that is
independent from the chromosomes of host cells.The essential elements for
chromosome maintenance and transmission are the following three regions:
The “replication origin,” from which the duplication of DNA begins, the
“centromere,” which functions in proper chromosome segregation during
cell division. The “telomere,” which protects the ends of linear
chromosomes.
Page | 11
Transformation of Plant Cells
The process of transfer, integration and expression of transgene in the
host cells is known as genetic transformation. The foreign gene (termed the
"transgene") is incorporated into the host plant genome and stably inherited
through future generations.
Various genetic transfer techniques are grouped into two main
categories.
1) Vector mediated or indirect gene transfer.
2) Vectorless or Direct gene transfer.
1. Vector Mediated
In this approach the transgene is combined with a vector which takes it
to the target cells for integration. The vector mediated transfer is strongly
linked to regeneration capabilities of the host plant.
Agrobacterium Mediated Transformation
The Agrobacterium system was historically the first successful plant
transformation system, marking the breakthrough in plant genetic
engineering in 1983. The Agrobacterium is naturally occurring gram
negative soil bacterium with two common species A. tumefaciens and A.
rhizogenes. Large plasmids in these bacteria are called tumor inducing (Ti
plasmid) and root inducing (Ri plasmid) respectively. The Ti plasmid has
two major segments of interest in transformation that is T-DNA and virus
region. The T-DNA region of the Ti plasmid is the part which is transferred
to plant cell and incorporated into nuclear genome of cells. The transfer of T-
DNA is mediated by genes in the region of Ti plasmid called virus genes
(virulence genes). Modified Ti plasmid are constructed that lack of
undesirable Ti genes but contain a foreign gene (resistant to a disease) and a
closely linked selectable marker gene (E.g.: for antibiotic resistance). The T-
DNA is generally integrated in low copy number per cell.Transfer of gene
Page | 12
through to wounded plant organs A. tumefaciens has limited range of host. It
can invest about 60% gymnosperms and angiosperms. Hence Agrobacterium
mediated transformation is the method of choice in dicotyledonous plant
species, where plant regeneration system are well established.
Structure representation of the Agrobacterium Ti-plasmid and integrated

T-DNA molecules. (a) The T-DNA section includes left border (LB), right
border (RB) and the virulence (vir) region. (b) The borders of T-DNA are
25-bp repeats and works as targets for the VirD1 - VirD2 endonuclease
complex (c) As single-stranded DNA molecule, the (T-strand) is released
and T-DNA typically take part into the host genome (d) In various
orientations as a single full-length or (e) truncated molecule in addition to (f)
multiple molecules joined to each other.
2. Co-Integrate pTi Vector
Co-integrative vector produced by integration of recombinant
intermediate vector in to a disarmed pTi. Transformed gene is initially
cloned in E. coli for easy in cloning procedure. A suitably modified E.
coli plasmid is used to initiate cloning of gene.The subsequent gene transfer
into plants is obtained by co-integrative vectors. Co-integration of the two
plasmids is achieved with in Agrobacterium by homologous recombination.
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Fig 23.1: Diagrammatic representation of homologous recombination between
disarmed pTi and recombinant IV (intermediate vector) containing the desired DNA
insert to produce a co-integrative vector. (LB & RB – left and right borders of T-
DNA; neo-neomycin phosphotransferase; kan r-Kanamycin resistance; ampr-
ampicillin resistance)
2.1 Binary Vector

A binary vector consists of a pair of plasmids of which one
contain vir region and other contains disarmed T-DNA sequence with right
and left border sequences. The plasmid contain disarmed T-DNA are called
micro-Ti or mini-Ti for.E.g. Bin 19.
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Fig 23.2: Binary vectors Bin19 and PAL 4404 of pTi
2.2 Expression Vector

An expression vector, otherwise known as an expression construct, is
generally a plasmid that is used to introduce a specific gene into a target
cell.The goal of a well-designed expression vector is the production of large
amounts of stable messenger RNA, and in extension, proteins.Expression
vectors are used for molecular biology techniques such as site-directed
mutagenesis. Cloning vectors, which are very similar to expression vectors,
involve the same process of introducing a new gene into a plasmid, but the
plasmid is then added into bacteria for replication purposes.An expression
vector must have elements include a promoter, the correct translation
initiation sequence such as a ribosomal binding site and start codon,
a termination codon, and a transcription termination sequence. For example,
prokaryotes expression vectors would have a Shine-Dalgarno sequence at its
translation initiation site for the binding of ribosomes, while eukaryotes
expression vectors would contain the Kozak consensus sequence.
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2.3 Shuttle Vector
A DNA plasmid with both bacterial and eukaryotic replicative machiner
y, which allows it to propagate within bacteria and yeasts. Recombinant
DNA in shuttle vectors can be amplified in bacteria and expressed in
eukaryotes. One of the most common types of shuttle vectors is the yeast
shuttle vector. Almost all commonly used S. cerevisiae vectors are shuttle
vectors. Yeast shuttle vectors have components that allow for replication and
selection in both E. coli cells and yeast cells. The yeast component of a yeast
shuttle vector includes an Autonomously Replicating Sequence (ARS),
Centromere (CEN) and Selectable marker.
Advantages
It is a natural means of gene transfer. Integration of T-DNA is a relative
precise process.The stability of gene transferred is excellent.Agrobacterium
is capable of infecting intact plant cells and tissue and organs.Agrobacterium
is capable of transfer of large fragments of DNA very efficiently.
Limitations
Host specificity, Soma clonal variation, slow regeneration and Inability
to transfer multiple genes.
2.4 Plant Virus Vector
Viruses have Following Features as a Vector
Infect cells of adult plant (dicotyledonous and monocotyledonous both).
They produce large number of copies per cell which facilitate gene
amplification and produce large quantities of recombinant protein.Mostly
plant viruses have RNA genome; two such viruses have great potential for
vectors are brome mosaic virus (BMV) and tobacco mosaic virus (TMV).
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But maximum processes have been made with two DNA genome containing
viruses as a vector.
2.4.1 Cauliflower Mosaic Virus (CaMV)
The Cauliflower Mosaic Virus (CaMV) is a double-stranded DNA virus
which infects a wide range of crucifers, especially Brassicas, such as
cabbage, cauliflower.In order to get itself and its DNA replicated
(multiplied) within a plant cell. The CaMV 35S well known promoter is
being used in almost all GM crops currently grown or tested, especially GM
maize. It is the promoter of selection for plant genetic engineering, as it is a
strong and constitutive promoter.
2.4.2 Gemini Viruses
Gemini viruses are small circular DNA viruses that replicate in plant
nuclei. The Gemini virus vectors lack a coat protein gene, they are not
transmissible by insect vectors, which are required for plant-to-plant spread.
Viruses from the gemini virus family normally infects a wide range of crop
plants, including maize, cotton, wheat, bean and cassava and are, therefore,
an ideal system of choice for VIGS-based gene function analyses in a broad
range of crop plants. These VIGS virus vectors have been used in a range of
studies to silence single or multiple genes, including the meristematic gene,
Proliferating Cell Nuclear Antigen (PCNA).
2.4.3 Tobacco Mosaic Virus (TMV)
TMV have single-stranded RNA genome which also serves as mRNA.
Viral RNA promoters are successfully manipulated for the synthesis of
recombinant messenger RNAs in whole plants. This vector consist of two
steps: First, is the use of cDNA copy of viral genome for cloning in E. coli.
Second, is in vitro transcription of the recombinant viral genome cDNA to
produce infectious RNA copies to be used for plant infection?
Adenoviral Vectors
Adenoviral vectors are derived from Adenoviruses (AdV), DNA viruses
with a linear double-stranded genome (36 Kbp), a non-enveloped icosahedral
capsid with characteristic morphology replicating in the nucleus and
producing thousands of progeny virions released by cell lysis. The viral
genome encodes about 50 viral proteins, 11 of which are structural and used
to physically build the virion.
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S. No Advantages Disadvantages
1. Transduce non-dividing and dividing cells Highly immunogenic
Carry up to 8 Kbp heterologous DNA The vector genome does not
2.
integrate into the host cell genome
Ensure high levels of transgene expression Transient expression of the
3.
transgene
Well suited as oncolytic vector High levels of pre-existing
4.
immunity
Vector particles produced at high titers
5.
(1010 pfu/ml*)
Adeno-Associated Viral Vectors

Adeno-associated viruses (AAV) are small, icosahedral non-enveloped
viruses that belong to the genus Dependovirus, family Parvoviridae. To
replicate, they necessitate co-infection with a helper virus to complete their
replication cycle. Indeed, rather than a specific, missing viral function, AAV
complete replication only when the cell is activated by co-infecting AdV or
HSV or a genotoxic agent.
Transduce non-dividing and dividing Carry up to 5 Kbp heterologous DNA
1.
cells
2. Parental virus apathogenic High vector titers difficult to achieve
Wide cellular tropism Need co-infection by helper virus
3.
(adenovirus or herpes simplex virus)
4. Potential site-specific integration
5. Low immunogenic
Herpes Virus Vectors

Herpes virus vectors mainly derive from HSV type- 1, a neurotropic
large DNA virus (152 Kbp, double-stranded DNA) that comprises more than
80 genes.Three different classes of vectors are derived from HSV-1:
1) Replication-competent attenuated vectors.
2) Replication-incompetent recombinant vectors.
3) Defective helper-dependent vectors known as amplicons.
1. Wide cellular tropism Possible residual cytotoxicity
Carry up to 50 Kbp heterologous DNA The vector genome does not integrate
2.
into the host cell genome
3. Natural tropism for neuronal (HSV- Transient expression of the transgene
Page | 18
vectors) Transient expression of the
transgene or B lymphoid cells (EBV
vectors)
Well suited as oncolytic vector Risk of recombination with latently
4.
herpes simplex virus-infected cells
Vector particles produced at high titers High levels of pre-existing immunity
5.
(1012 pfu/ml*)
Retroviral Vectors
Retroviruses are enveloped viruses with a capsid enclosing two copies
of a single-stranded, positive sense RNA of 7-11 Kbp. Basically, the
retroviral genome has two Long Terminal Repeats (LTRs) at 5’ and 3’
extremities and encompasses three large open reading frames called gag, pol
and env. The LTRs act as promoters and regulate the expression of gag, pol
and env that encode the capsid proteins, replication enzymes and envelope
glycoproteins, respectively.
The vector genome integrates Transduce only replicating cells
1.
into host cell genome
Carry up to 8 Kbp heterologous Cellular targeting difficult to achieve
2.
DNA
3. Engineering fairly simple Unsuitable for non-replicating cells
4. Wide cellular tropism Random integration of the retroviral genome
5. Low immunogenic High risk of insertional mutagenesis
No (or very low) pre-existing Low stability
6.
immunity
Lentiviral Vectors
Lentiviruses and retroviruses are closely related. However, lentiviruses
can be considered advanced or more complex retroviruses due to the
presence in their genome of several regulatory genes. These regulatory genes
have different and highly specialized functions and act in concert to
neutralize host cell defences, blunt immune responses and regulate viral
replication.
Transduce non-dividing and Possible insertional mutagenesis
1.
dividing cells
The vector genome integrates into Presence of regulatory proteins (tat, rev,
2.
host cell genome and others) in the packaging construct
Carry up to 9 Kbp heterologous Transient expression of the transgene
3.
DNA with integration defective vector
Page | 19
Prolonged expression of the
4.
transgene
5. Integration-defective vectors
Poxvirus and Other Viral Vectors

Thus, besides the major vectors described above that cover
approximately 80% of vector trials, there is a long list of viruses that served
as a platform to develop vectors. Among these, paramyxoviruses,
alphaviruses, flaviviruses, as well as recombinant and artificial viruses have
found a niche or a special application.
Carry up to 30 Kbp heterologous Potentially cytotoxic
1.
DNA
2. Multiple sites of transgene insertion Generation of recombinants complicated
Particularly apt as attenuated Transient expression of the transgene
3.
recombinant vaccine
4. Well suited as oncolytic vectors Highly immunogenic
5. Low levels of pre-existing immunity Heterologous promoters difficult to use
3. Direct methods of Transformation

The various methods of direct gene transfers are
1) Chemical methods
2) Electroporation
3) Particle bombardment
4) Lipofection
5) Micro injection
6) Macro injection
7) Pollen transformation
8) Delivery via growing pollen tubes
9) Laser induced transformation
10) Fibre mediated transformation etc.
General Steps Involved in a Generic Gene Cloning Process
 Gene Isolation and Excision: The DNA or m-Rna is isolated from
an organism that contains the target gene (E.g. a BT toxin (cry)
gene from Bacillus thuringiensis). In the case of DNA it is cut with
a restriction endonuclease.
 Vector Preparation: The chosen DNA cloning vector is cut with
the same restriction endonuclease.
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 Ligation: The two DNA samples are pasted together by a DNA
ligase to produce recombined molecules.
 Transformation with the Vector: E. coli cells are transformed
with the combined DNA molecules from the ligase reaction to
produce cells that carry the target gene vector recombinant
molecules. The vector contains a DNA sequence, origin of
replication (ori), that enables it to be replicated in E. coli, and hence
the recombinant molecules may be replicated into a high number of
copies. Uptake of DNA in E. coli may be facilitated by a number of
procedures, e.g. CaCl2 - heat shock treatment or electroporation.
 Marker and Target Gene Expression: E. coli cells containing the
vector, and hence the target gene, are selected on the basis of an
antibiotic resistance (AR) gene which is an integral part of the
vector. When the corresponding antibiotic (E.g. Ampicillin,
Kanamycin or Neomycin) is added, only cells containing the
recombinant vector molecules will survive the treatment.
Screening
Many general purpose vectors such as pUC19 usually include a system
for detecting the presence of a cloned DNA fragment, based on the loss of an
easily scored phenotype. The most widely used is the gene coding for E. coli
β-galactosidase, whose activity can easily be detected by the ability of the
enzyme it encodes to hydrolyze the soluble, colourless substrate X-gal (5-
bromo 4-chloro 3-indolyl beta galactoside) into an insoluble, blue product
(5,5'dibromo 4,4‘ dichloro indigo). Cloning a fragment of DNA within the
vector based lac-Zα sequence of the β-galactosidase prevents the production
of an active enzyme. If X-gal is included in the selective agar plates,
transformant colonies are generally blue in the case of a vector with no
inserted DNA and white in the case of a vector containing a fragment of
cloned DNA.
Page | 21
Maximum DNA Insert Possible with Different Cloning Vectors
S. No Vector Host Insert Size
1. M13 E. coli 1-4 kb
2. Plasmid E.coli 1-5 kb
3. X phage E.coli 5-25 kb
4. Cosmids E.coli 35-45 kb
5. P1 phage E.coli 70-100 kb
6. PAC's E.coli 100-300 kb
7. BAC's E.coli < 300 kb
8. YAC's S. cerevisiae 200-2000 kb
Denouement
Development of gene transfer systems in plants probably one of the
most challenging aspects of plant research.The development of nanoparticles
for DNA delivery into plant cells is emerging and there are possibilities to
combine the benefits of biolistic and Agrobacterium in the near future.Viral
vectors are used for the delivery of genetic material into cells. Because
viruses have a natural ability to efficiently deliver their genome into host
cells, they are an ideal tool for gene transfer and are often used by molecular
biologists to deliver therapeutic genes.Meanwhile, the approach of plant
transformation using Agrobacterium is likely to continue getting attention,
due to avoidance of a tissue culture step for the regeneration of
transformation events.
References
1. (http://nptel.ac.in/courses/102103016/23)
2. (http://www.chromoresearch.co.jp/e/chromosome/)
3. (http://www.web-books.com/MoBio/Free/Ch9A4.htm)
4. (https://www.boundless.com/microbiology/textbooks/boundless
microbiology textbook /microbial-genetics-7/tools-of-genetic-
engineering-82/plasmids-as-cloning-vectors-4536643/)
5. Engelen FA, Molthoff JW, Conner AJ, Jan-Peter N, Pereira Aand,
Stiekema WJ. pBINPLUS: an improved plant transformation vector
based on pBIN19. Transgenic Research. 1995; 4:288-290.
6. Hamilton CM. A binary-BAC system for plant transformation withhigh
molecular weight DNA. An International Journal on Gene and
Genomics. 1997; 200:107-116.
Page | 22
7. Mitrovic T. Gene transfer systems. Facta Universitatis. 2003; 10(3):101-
105.
8. Primrose SB, Twyman RM. Principles of gene manipulation and
genomics (7th Edition), Blackwell Publishing, Cornwall UK, 2006, 75-
95.
9. Rashid AHA, Lateef DD. Novel techniques for gene delivery into plants
and its applications for disease resistance in crops. American Journal of
Plant Sciences. 2016; 7:181-193.
Page | 23
Page | 24
Chapter - 2
Basic Concepts of Heterosis
Authors
Manav
Ph.D. Research Scholars, Department of Genetics and Plant
Breeding, CCS Haryana Agricultural University, Hisar,
Haryana, India
Jaswant Singh Khokhar
Plant Science Division, Sutton Bonington Campus, University
of Nottingham, LE12 5RD, UK
Bharat Taindu Jain
Ph.D. Research Scholars, Department of Genetics and Plant
Breeding, CCS Haryana Agricultural University, Hisar,
Haryana, India
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Chapter - 2
Basic Concepts of Heterosis
Manav, Jaswant Singh Khokhar and Bharat Taindu Jain
1. Introduction
Heterosis or hybrid vigour is a natural phenomenon in which hybrid
offspring from genetically diverse individuals show increase in size or rate
of growth over their parents. Heterosis in crop species can be accompanied
with increases in growth rate, total biomass, stress resistance, seed yield and
population fitness. In other words, it is the superiority of F1 hybrid over both
the parents in terms of yield and some other characters. A related term
luxuriance which refers to the increase of F1 over parents in terms of
vegetative growth but not in yield and adaptation. Heterosis has apparently
been recognized in one form or another for centuries by various civilizations
(Chen et al., 2008) [1]. There are two theories of heterosis namely,
dominance and over dominance. In dominance theory, recessive alleles at
different loci are complemented in the hybrid whereas, the over dominance
theory states that the interactions between different alleles occur in the
hybrid leading to the increase in vigour. The earliest utilization of heterosis
was in maize (Zea mays), followed by beet (Beta vulgaris), sorghum
(Sorghum bicolor), onion (Allium cepa), eggplant (Solanum melongena),
tomato (Solanum lycopersicum), rice (Oryza sativa), cotton (Gossypium
hirsutum), sunflower (Helianthus annuus) and rapeseed (Brassica napus)
(Melchinger and Gumber, 1998) [2]. East (1908) [3] synthesized data from a
large number of studies involving many species and concluded that, on
average, heterosis increases as the genetic distance between the parental
stocks increases, thereby, interspecific crosses have shown higher heterosis
than intraspecific crosses. For example, hybrids between radish (Raphanus
sativus) and cabbage (Brassica oleracea) exhibit extensive biomass heterosis
(Karpechenko, 1927) [4] as do the hybrids between a wild tomato species
(Solanum pennellii) and cultivated tomato species (Solanum lycopersicum)
as reported by Eshed and Zamir, 1995 [5].
2. Characteristics of Heterosis
2.1 Variability: Heterosis is highly variable. The degree of heterosis
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varies in accordance to the genetic distance of the parents, their
mode of reproduction, the trait under investigation (Zhou et al.,
2012) [6], the developmental stage of the plants (Groszmann et al.,
2013) [7] and the environmental factors (biotic and abiotic) such as
soil-type, topography, climate, solar energy, temperature and water
availability (Munaro et al., 2011; Griffing and Zsiros 1971;
Langridge 1962; Blum 2013) [8, 9, 10, 11].
2.2 Universal: Heterosis is largely universal and can increase crop
yield by 15-50% depending upon the crop type. Many of the major
cereal crops as well as many varieties of vegetable and flower crops
are commercialized using hybrid seeds for increased agricultural
performance (Birchler et al., 2003) [12].
2.3 Confined to F1: Heterosis is confined to Ff1 generation of a cross.
It disappears in F2 and subsequent generation of a cross as a
consequence of segregation and recombination. Further, all F1
crosses do not exhibit desirable heterosis.
3. Types of Heterosis
Heterosis is of Two Type
3.1 On the Basis of Origin and Nature
1) True heterosis/Heterosis
2) Pseudo-heterosis
1) True Heterosis
It is that heterosis which can be transferred from one generation to the
other.
It can be Further Divided into Two Types
a) Mutational True Heterosis
It is simplest type of heterosis. Lethal (mostly), recessive, adaptively
unfavourable mutants are either eliminated or sheltered by their non-lethal,
dominant and adaptively superior alleles in cross pollinated crops. This is
termed as mutational heterosis. Naturally occurring mutants are generally of
recessive and less adaptive to environmental conditions, hence risk of their
elimination by natural selection process is higher.
b) Balanced True Heterosis
The gene combinations which are well balanced, more adaptive to
environmental conditions and useful from the agriculture point of view result
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in balanced heterosis. This type of heterosis found application in hybrid
production.
2) Pseudo-Heterosis
It is also termed as luxuriance. Progeny possess superiority over parents
is in vegetative growth, but not in yield and adaptation, usually sterile or
poorly fertile. This concept cannot be utilized in hybrid varieties production.
3.2 On the Basis of Type of Estimation
3.2.1) Average Heterosis
3.2.2) Superior Heterosis
3.2.3) Standard Heterosis
3.2.1 Average Heterosis
When the heterosis is estimated by taking average of the two parents,
then it is known as mid parent heterosis. It is calculated by using the
formula-
Mid Parent Heterosis = [(F1- MP)/MP] X 100
Where, F1 is the mean of F1 and MP is the mean of two parents
involved in the cross.
3.2.2 Superior Heterosis
When the heterosis is estimated over the better parent, it is known as
superior heterosis. It is also known as heterobeltiosis or better parent
heterosis and calculated by using formula-
Heterobeltiosis = [(F1- BP)/BP] X 100
Where, BP is mean of better parent.
3.2.3 Standard Heterosis
The superiority of F1 over the standard (check) variety. It is also known
as economic heterosis or useful heterosis and calculated by using formula-
Standard Heterosis = [(F1- CC)/CC] X 100
Where, CC is the mean value over replications of the standard cultivar.
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4. Genetic Basis of Heterosis
Genetic Basis of Heterosis can be explained by Three Hypothesis
Namely
4.1) Dominance Hypothesis
4.2) Over Dominance Hypothesis and
4.3) Epistasis
4.1 Dominance Hypothesis
The dominance hypothesis was proposed by Davenport (1908), Bruce
(1910), Keeble and Pellew (1910) [13, 14, 15] and later elaborated by Jones. This
hypothesis assumes that heterosis is due to non-expression of deleterious
recessive alleles in presence of beneficial dominant alleles in the resulting F1
from two parents. Therefore, the F1 produced from such a cross possesses
superior characters because of the contribution of dominant alleles from one
parent (Budak et al., 2002) [16]. Thus, based on the dominance hypothesis,
breeders should be able to fix the inbred lines with favourable alleles and
likely to produce inbreds equivalent to F1 hybrids.
Objections
There are Two Main Objections of this Hypothesis
1) Failure of Inbreds to be Isolated as Vigorous as Hybrids
If the above hypothesis is true, it should be possible to isolate inbreds
with all dominant genes. Such inbred would be as vigorous as the F1 hybrids.
However, such hybrids have not been isolated but in some studies it has been
possible to recombine genes so that inbred lines as good as or superior to the
heterotic hybrids were isolated Jones (1917) [17] in his theory
entitled “Dominance of Linked Genes Hypothesis” provided explanation for
this.
He suggested that there may be a linkage between some favourable
dominant genes and some unfavourable recessive genes, therefore, it is not
possible to obtain true breeding homozygous individual for all the dominant
genes in F2 generation.
2) Symmetrical Distribution in F2
It is already studied that in F2 dominant and recessive characters
segregate in the ratio of 3:1. If heterosis is the result of dominance of
independent factors, then the F2 distribution curve for heterotic character
should be skewed, not smooth and symmetrical.
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But the F2 curve always show smooth and symmetrical distribution
rather than skewed. This objection was removed by Collins (1921) [18]. He
said that trait like yield is governed by many genes or polygenes, which
exhibit continuous variation resulting in symmetrical distribution of genes.
3) Magnitude of Heterosis
The inbred lines have been considerably improved in terms of per se
performance over the decades. If dominance were the main cause of
heterosis, the magnitude of heterosis generated by such inbreds should have
declined. In contrast, it has increased slightly, possibly due to the selection
of allele at the right set of loci that produces the best combination in hybrids
to generate heterosis (Birchler et al., 2003) [12].
4.2 Over-Dominance Hypothesis
The over-dominance hypothesis as major genetic basis of heterosis was
developed independently by East (1908); Shull (1908) [3, 19]. The same
concept was later advocated by Gustafsson (1938); Hull (1945) [20, 21]. Over-
dominance theory is also called as ‘Single gene heterosis’,
‘Superdominance’ or ‘Cumulative action of divergent alleles’. According to
this hypothesis, heterosis is the result of superiority of heterozygote over its
both homozygous parents. Thus, heterosis is directly proportional to the
heterozygosis. The superiority of heterozygote over both homozygotes may
arise either due to (i) Production of superior hybrid substance in
heterozygote which is completely different from either of the homozygous
products or (ii) Greater buffering capacity in the heterozygote resulting from
cumulative action of divergent alleles. Over-dominance has been reported in
barley.
4.3 Epistasis
Gowen (1952) [22] had suggested that influence of one locus on the
expression of another may be involved in heterosis. In many cases, the effect
of a single homozygous recessive allele is epistatic to almost the whole
genetic makeup of an inbred. Epistatic interactions will result in maximum
heterosis when the following two conditions are met (i) First, the epistasis
should be predominantly of complementary type i.e., the estimates of h and i
have the same sign so that they do not cancel each other. Second, the
interacting pairs of genes should be dispersed in both the parents. It has been
suggested that in the absence of over-dominance, dispersion of genes
showing complementary hypothesis seems to be the major cause of
heterosis. In many experiments, multiplicative interaction has been reported
as a cause of heterosis. It was concluded that in such cases, epistatic effects
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are non-linear functions of the one-locus effect on the mean. Obviously, in
case of polygenic traits, such as yield, heterosis would involve several
mutually interacting genes.
The understanding of the phenomenon of heterosis in terms of its
genetic basis is not an easy task. The majority of the earlier studies
speculated dominance and over-dominance as the genetic mechanism of
heterosis but the recent studies have revealed that linkage and epistasis may
also have a role to play (Budak et al., 2002). Therefore, no single hypothesis
holds true for all the experiments and crops. It is, thus, likely that the
heterosis is crop dependant and population dependant.
5. Molecular Mechanism of Heterosis
Molecular Approaches to Study Heterosis
Gene Level
Single locus heterosis examples are sickle cell anemia heterozygote can
more tolerate than homozygous mutant individual, also studied in A.
thaliana, when made cross between A. erecta and A. angustifolia. Here erecta
encode a regulatory protein (receptor like kinase) and angustifolia encode
transcription factor. These regulatory proteins have multiple effects on
phenotypes e.g. early flowering and erect stem.
Genomic Level
5.1) Gene Dosage Balance
5.2) Epigenetic Mechanisms, Including DNA Methylation
5.3) Increased Energy Efficiency Hypotheses
(Kaeppler 2012; Schnable and Springer 2013)
These mechanisms are gene/allele independent and trait-nonspecific,
i.e., would produce heterosis for all the traits to a similar degree.
5.1 Gene Dosage Balance
Aneuploidy reduces vigor, while polyploids are more vigorous than
diploids. Two types of variations in the genomes of different genotypes. i)
Presence/absence variation ii) Copy-number variation (CNV). In case two
inbred lines differ for the genomic regions showing presence/ absence and
CNV, their hybrid would have average copy number of all the genes
involved in these variations. Suppose that one inbred line has three copies
per genome of one genomic region and the other inbred line has four copies
per genome of another genomic region. These inbred lines, therefore, will
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have six and eight copies, respectively, of the concerned genomic regions,
while their hybrid will have only four and five copies, respectively. The
hybrid would show a greater gene dosage balance for the two genomic
regions and would be more vigorous than the two parental inbred lines. The
findings of a study involving diploid and triploid inbred lines of maize
indicate that the main part of heterosis results from dosage-sensitive
mechanisms.
5.2 The Epigenetic Mechanism (DNA Methylation)
Several studies have revealed the presence of epigenetic variation within
plant species, e.g., DNA methylation or histone modification differences in
Arabidopsis, maize, and rice genotypes, that can exhibit stable inheritance.
Genomic DNAs of inbreds are more methylated than those of hybrids and
that DNAs of heterotic hybrids are less methylated than those of less
heterotic and nonheterotic hybrids. In addition, different genomic regions
may be hypermethylated in different inbreds. It is believed that in plants the
level of DNA methylation is negatively associated with gene expression.
Therefore, the reduced level of DNA methylation in hybrids may lead to
increased gene expression and, consequently, heterosis (Baranwal et al.
2012).
5.3 Increased Energy Efficiency Hypotheses
Molecular basis of heterosis based on studies carried out in Arabidopsis,
maize, and rice during vegetative (young and mature) or reproductive stages
of development. Most of these studies reveal a major proportion of
differential expression profiles (DEPs) in younger vegetative and
reproductive stages to be non-additive, although additive DEPs do exist.
During mature stages of vegetative development the differential expression
activity has been reported to be significantly reduced and a major proportion
consists of additive DEPs. The non-additive DEPs have been shown to affect
components of the clock and a significant number of the downstream CCA1
Binding Site (CBS)-/evening element containing genes involved in
photosynthesis and starch biosynthesis, which have been implicated in
biomass heterosis. The additive DEPs may also contribute to biomass
heterosis albeit in a less significant manner. The increase in biomass during
vegetative development may contribute to the yield.
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6. Effects or Manifestations of Heterosis
Heterosis is Being Manifested in the Following Three Ways
6.1 Quantitative Effects
a) Increase in Size and Genetic Vigour
Hybrids are generally more vigorous i.e. larger, healthier and faster
growing than the parents e.g., head size in cabbage, cob size in maize, fruit
size in tomato etc.
b) Increase in Yield
The ultimate goal of any breeding programme is to increase the overall
yield in terms of grain, fruit, seed, leaf tuber or the whole plant. Hybrids are
usually higher yielder than their parents.
c) Better Quality
Hybrids possess better quality than their parents. e.g., hybrids in onion
show better keeping quality.
6.2 Physiological Effects
a) Greater Resistance to Diseases and Pests
Hybrids are usually more resistant to the attack of insects or diseases
than their parents.
b) Early in Flowering and Maturity
Earliness is highly desirable in vegetables. In many cases, hybrids are
early in flowering and attain faster maturity as that of the parents e.g. tomato
hybrids mature earlier than their parents.
c) Greater Adaptability
Hybrids are usually more adaptable to adverse environmental
conditions.
6.3 Biological Effects
Hybrids exhibiting heterosis possesses greater biological efficiency i.e.,
an increase in fertility (reproduction ability) and survival ability than their
parents.
7. Factors Affecting Heterosis
7.1 Geographical and Genetic Diversity
In cotton, there is a close relationship between the genetic diversity of
parental varieties and performance of their hybrids for lint yield. In, inter and
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intra specific hybrids of cotton, the highest heterosis is observed in cross
combinations involving ecologically distant parents.
7.2 Agronomic Performance
High heterosis can be obtained from the crosses involving two low
yielded inbreds but absolute yield of such hybrids is lower than the adapted
varieties. To produce good hybrids, varieties with higher per se performance
must be chosen.
7.3 Adaptability
A close association is observed between the adaptability of the hybrids
and their parents. In India, several hybrids have been developed at intra and
inter specific levels especially in tetraploid cotton.
7.4 Genetic base of parents
Hybrids involving at least one of the parents with broader genetic base
exhibit greater heterosis.
Use of Heterosis in Crop Improvement Programme
 Gains in yield and yield stability offered by heterosis have
prompted use of hybrids in several crops.
 Genetic yielding ability has been increased greatly, and thus total
production has been increased, with minimal dependence on
chemical inputs and maximum use of biological power.
 Enthusiasm and funds have been directed to hybrid breeding, in part
because of the proven efficiency of the inbred/hybrid method for
producing products that farmers need and want, and in part because
private capital was attracted to the profit potential of hybrid
breeding and sales.
 The inbred/hybrid method has given breeders greater precision in
developing, identifying, and multiplying the best hybrid genotypes
in cross-pollinated crops.
New Developments in Heterosis Breeding
 Analysis of heterosis at molecular level e.g. Arabidopsis.
 Analysis of physiological process to understand molecular basis.
 Development of new bioinformatics tools to integrate analysis of
experimental data at biological level.
 New approaches for identification of heterotic groups and
broadening their genetic base.
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 Prediction of heterosis through molecular and bioinformatics tools.
 Analysis of hQTLs e.g. 11 QTL detected in maize for grain yield.
 Heterosis breeding for crop improvement e.g.
i) For disease resistance e.g. Sorghum, Tomato
ii) For abiotic stresses e.g. Maize, Sorghum
iii) For quality e.g. Sunflower- Oil quality, Tomato- Self life
References
1. Chen X, Li M, Shi J, Fu D, Qian W, Zou J et al. Gene expression
profiles associated with intersubgenomic heterosis in Brassica napus.
Theor. Appl. Genet. 2008; 117:1031-1040.
2. Melchinger AE, Gumber RK. Overview of Heterosis and Heterotic
Groups in Agronomic Crops. In: Concepts and Breeding of Heterosis in
Crop Plants. Crop Science Society of America, USA, 1998, 29-44.
3. East EM. Inbreeding in corn. In report of the Connecticut Agricultural
Experiment Station for the year. 1908; 1907:419-428.
4. Karpechenko GD. Polyploid hybrids of Raphanus sativus L. X Brassica
oleracea L. Bull. Appl. Bot. 1927; 17:305-410.
5. Eshed Y, Zamir D. An introgression line population of Lycopersicon
pennellii in the cultivated tomato enables the identification and fine
mapping of yield-associated QTL. Genetics. 1995; 141:1147-1162.
6. Zhou G, Chen Y, Yao W, Zhang C, Xie W, Hua J et al. Genetic
composition of yield heterosis in an elite rice hybrid. Proc Natl Acad
Sci. USA. 2012; 109(39):15847-15852.
7. Groszmann M, Greaves IK, Fujimoto R, James Peacock W, Dennis ES.
The role of epigenetics in hybrid vigour. Trends Genet. 2013;
29(12):684-690.
8. Munaro EM, Eyhe´rabide GH, D’Andrea KE, Cirilo AG, Otegui ME.
Heterosis X environment interaction in maize: what drives heterosis for
grain yield? Field Crops Res. 2011; 124(3):441-449.
9. Griffing B, Zsiros E. Heterosis associated with genotype environment
interactions. Genetics. 1971; 68:443-455.
10. Langridge J. A genetic and molecular basis for heterosis in Arabidopsis
and Drosophila. Am Nat. 1962; 96:5-27.
11. Blum A. Heterosis, stress, and the environment: a possible road map
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towards the general improvement of crop yield. J Exp. Bot. 2013;
64(16):4829-4837.
12. Birchler JA, Auger DL, Riddle NC. In search of the molecular basis of
heterosis. Plant Cell. 2003; 15(10)-2236-2239.
13. Davenport CB. Degeneration, albinism and inbreeding. Science. 1908;
28:454-455.
14. Bruce AB. The Mendelian theory of heredity and the augmentation of
vigor. Science. 1910; 32:627-628.
15. Keeble F, Pellew C. The mode of inheritance of stature and time of
flowering in pea. Genetics. 1910; 1:47-56
16. Budak HL, Cesurer Y, Bolek T, Dokuyuku T, Akaya A. Understanding
of heterosis. Journal of Science and Engineering. 2002; 5(2):68-75.
17. Jones DF. Dominance of linked factors as a means of accounting for
heterosis. Genetics. 1917; 2:466-479.
18. Collins GN. Dominance and vigour of first generation hybrids.
American Naturalist. 1921; 55:-116-133.
19. Shull GH. The composition of a field of maize. Am. Breeders Assoc.
Rep. 1908; 4:296-301.
20. Gustafsson A. Studies on genetic basis of chlorophyll formation and
mechanism of induced mutations. Hereditas. 1938; 24:33-93.
21. Hull FH. Recurrent selection for specific combining ability in corn.
Journal of the American Society of Agronomy. 1945; 37:134-145.
22. Gowen JW. (Ed.). Heterosis. Iowa State College Press, Ames, 1952.
23. Schnable CPS, Springer NM. Progress toward understanding heterosis
in crop plants. Annu Rev Plant Biol. 2013; 64:71-88.
24. Kaeppler. Heterosis: Many Genes, Many Mechanisms-End the Search
for an Undiscovered Unifying Theory. International Scholarly Research
Notices, 2012. Article ID 682824, 12.
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Chapter - 3
Introduction of Plant Breeding
Authors
Madhuri Grace Minz
Department of Genetic & Plant Breeding, BTCARS (IGKVV),
Sarkanda, Bilaspur, Chhattisgarh, India
Anjum Ahmad
Department of Genetic & Plant Breeding, BTCARS (IGKVV),
Sarkanda, Bilaspur, Chhattisgarh, India
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Chapter - 3
Introduction of Plant Breeding
Madhuri Grace Minz and Anjum Ahmad
Introduction
Plant breeding is a resolute rearrangement of plant species in series to
create desired genotypes & Phenotypes for specific purposes. This
rearrangement involves through controlled pollination and by uses of
different plant breeding methods according to their pollination behavior i.e.
self, cross and often cross pollination crop. Plant breeding has been practiced
for many of years, since near the beginning of human civilization. Many
international and national government development organization believe that
breeding noble crops is important for ensuring food security by developing a
noble crop varieties which are higher yield potential, resistant to many
insect-pests and diseases, drought & cold resistant or adapted to different
agroclimatic environment and growing conditions.
Definition of Plant Breeding
Plant breeding has been defined in different ways by various authors.
Some definitions of plant breeding are given below-
 Plant breeding is the art and the science of improving the heredity
of plants for the benefit of human kind. J.M. Poehlman (1959).
 Plant breeding is the genetic adjustment of plants to the social,
cultural, economic and technological aspects of the environment.
Frankel (1968).
 Plant breeding is a technology of developing superior crop plants
for various purpose. Riley (1978).
 Plant breeding is the current phase of crop evolution. N.W.
Simmonds (1979).
Purpose of Plant Breeding
 The primary purpose of plant breeding is to obtain or develop a crop
varieties or hybrids that are effective to take all important plant nutrients
that give the greatest return of high quality products per acre or unit area
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in relation to cost and more production and that are adapted according to
the needs of the grower and consumers.
 The secondary purpose of plant breeding is to obtain or produce
varieties that are able to withstand extreme conditions of frost and
drought or that have resistance to pathogenic organisms or insect pests
such qualities helps materially to stabilize yields by controlling extreme
natural undulation or instability.
Activities in Plant Breeding
To want desired changes in genotypes of plant species and the
consequent profits to farmers are brought about by a series of interrelated
and largely interdependent activities. This activity includes:
1) To create variation
2) Genetic Selection
3) Evaluation
4) Multiplication
5) Distribution
To Create Variation
In any plant breeding programme, it is the first steps unless variation
preexist. Creation of genetic variation is essential for any crop improvement.
It is created by domestication germplasm collection, Plant introduction,
Hybridization (its includes intervarietal, distant & somatic), polyploidy,
somaclonal variation, genetic engineering and mutation.
Genetic Selection
Selection or genetic selection is the process by which certain traits or
characters become more prevalent in a species than other traits and it have to
capacity of growing their progeny. These traits seen in an organism are due
to the genes found on their chromosomes. Each genes are coded for the
specific characters or traits that we are able to observe. The efficiency of this
activity determines the success of a breeding program. A numerous breeding
methods according to different plant species for example mass selection,
pure line selection, pedigree selection, bulk selection etc. have been designed
to increase the efficiency of selection may be natural and artificial.
Evaluation
The recently selected lines/genotypes/strains/populations are tested for
yield and other quantitative and qualitative traits and their performance is
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compared with the existing best or popular varieties called checks.
Evaluation is a proceeding steps process which conducted at different
multiplications. For three or more years under the concerned All India
Coordinated Crop Improvement Project. If the new genotype/line/strain/
population are superior to the checks, it is released and notified as a new
variety or hybrid and its seed can be multiplied and most importantly,
certified by a seed certification agency for quality seed production.
Multiplication
Multiplication means to production of certified seed of a newly released
and notified variety in a large and huge amount. Seed production is usually
done by the seed production agencies in a step wise manner, and the seed is
certified by a seed certification agency.
Distribution
The certified seed of newly released variety or hybrid is ultimately sold
to the farmers who can use it for commercial crop cultivation. This activity
alone makes it possible to reap the economic benefits from the above
activities in form of an enhanced and stable production of superior produce
often at cheapest or less input cost. For an effective crop improvement
programme, the above activities have to be properly coordinated and well
geared to maximize the outputs from a programme. The quality of being
loose in any one of these steps will definitely reduce the efficiency of the
programme. This will lead to a destruction of valuable recourses.
Science Related to Plant Breeding
A Plant breeders has to acquaint himself with knowledge from many
disciplines of agricultural and basic sciences to develop superior plants
which are successful and acceptable as commercial crops. The following
branches of science have a direct application in plant breeding-
1. Genetics
It’s important to understanding of the mechanism of heredity in plants as
modern plant breeding methods are based on knowledge of the gene and its
inheritance. With the advances in molecular genetics, the knowledge of
genes has been extended to the molecular level.
2. Botany
Plant breeders should be accomplished biologists with a broad
understanding of the taxonomic classification, anatomy, morphology,
reproductive mechanism, and cellular structure of the crop plants with which
they work.
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3. Plant Physiology
Cultivar adaptation is influenced by the response of plants to
environmental stresses, such as extremes in temperature, light, soil moisture
and soil nutrients. The plant breeder strives to modify the plant’s
physiological processes, which will enables it to function more efficiently in
the environment in which it is grown.
4. Plant Pathology
Healthy plans are essential for good crop performance. The plant
breeder cooperates with the plant pathologist in identification of genes for
resistance to plant disease pathogens. In corporation of genes for resistance
to disease into cultivars improves plant performance and reduces the need
for chemical disease control.
5. Entomology
Breeding for insect resistance is an economical and an environmentally
sound means for avoiding insect damage while reducing the use of pest
control chemicals in field and horticultural crops.
6. Plant Biotechnology
The inherent nutritional value of a crop cultivar for food or for livestock
feed, or for utilization by industry, often may be improved by plant breeding.
7. Statistics
Analytical statistical procedures provide a better understanding of
quantitative genetics and its utilization in breeding for improved plant
performance.
8. Computer Science
The computer has become an essential tool for systematic planning of
the breeding nursery, recording observation and rapid analysis and
interpretation of the data.
9. Agronomy/Horticulture
Breeders need to know crops and how to produce them. They should
understand the grower’s needs in new cultivars of field or horticultural crops
to evaluate available breeding materials, plan efficient breeding procedures
and direct breeding efforts toward important breeding goals.
Strategy of Plant Breeding
According to David Allen Sleper and John Milton Poehlman strategy of
plant breeding are following-
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 To identify the morphological, physiological and pathological traits
in a cultivated plant species that contributes to its adaptation, health,
productivity, and suitability for food, fiber or industrial products.
 To search out new genes which are encode for desired characters or
traits in different strains of the cultivated species and their close
relatives.
 To combine genes for the interest or desired trains into an improved
cultivar through traditional breeding or new biotechnology
procedures.
 To ass’s performance of the improved breeding lines in the local
environment in comparison with present cultivars.
 To distribute as new cultivars breeding lines superior to cultivars
currently grown.
Agencies Engaged in Plant Breeding
The improvement of inherent potential of crop plants through plant
breeding is essential for increasing agricultural production to a level
matching the ever increasing human population. A relatively slow progress
in food production especially in the developing countries was the deciding
factor for establishing International Agricultural Research Centers (IARCs)
with main emphasis on the evolution of new high yielding varieties of
particular crops. The International Rice Research Institute (IRRI),
Philippines; and Centro Internacional de Mejoramiento de Maiz y Trigo
(CIMMYT), Mexico, established during 1960s made phenomenal
contributions for food production that attracted Rockefeller and Ford
Foundations to initiate two more international research centers viz.,
International Institute of Tropical Agriculture (IITA) in Nigeria; and Centro
International de Agricultura Tropical (CIAT) at cali in Colombia with the
basic theme of engaging and helping the local plant breeders in the
developing countries for solving their own food problem. It is encouraged by
the astonishing success of these four centers the World Bank; the FAO; the
United Nations Development Programme (UNDP) and 12 other donors
agreed to support the idea of establishing a Consultative Group to support a
network of International Agricultural Research Centers (CGIAR) in 1971.
The CGIAR now has sixteen International Research Centers, out of which
eight concentrate on specific agricultural crop plants and one on genetic
resources with a mission to contribute towards sustainable agriculture for
food security especially in developing countries. These centers have strong
crop breeding programmes including the collection, conservation, utilization
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and supply of germplasm resources on specific crops. The CGIAR controls
world’s largest collection of genetic resources. The International
Agricultural Research Centers work in close coordination with national
networks of agricultural research in each country. The Plant breeding
research in India is the responsibility of state Agricultural Universities and
institutes of the Indian Council of Agricultural Research (ICAR). In
Addition to the financial support by the governments, the ICAR provides
funds and coordinates research of important crops under the coordinated
crop improvement Research Projects. At present coordinated crop
Improvement Projects on all the important crops are operative under the
supervision of ICAR to integrate research on varietal improvement and
management practices in a multidisciplinary approach.
International Agricultural Research centers under CGIAR
Year of Crop/Research
S. No Acronym Name Location
Establishment Focus
Los Banos,
The International Rice
1 IRRI 1960 Laguna Rice
Research Institute
Philippines
Mexico Wheat,
Centro Internacional de
2 CIMMYT 1966 city, Triticale,
Mejoramiento de Maizy Trigo
Mexico Maize
International Institute of Ibadan Legumes, Roots,
3 IITA 1967
Tropical Agriculture Nigeria Tubers
Centro International de Cali Beans,
4 CIAT 1968
Agricultura Tropical Colombia Cassava
Lima
5 CIP 1971 Centro Internacional de la Papa Potato
Peru
West African Rice Monrovia
6 WARDA 1971 Rice
Development Association Liberia
Millets,
International Crops Research Hyderabad
7 ICRISAT 1972 Sorghum,
Institute for Semi-Arid Tropics India
Legumes
International Board of Plant
Gene conversion
IBPGR 1974 Genetic Resources (Now Rome
8 & genetic
(IPGRI) (1992) International Plant Genetic Italy
resources
Resources Institute)
International Center for
Aleppo
9 ICARDA 1976 Agricultural Research in the Barley
Syria
Dry Areas
References
1. Allard RW. Principles of Plant Breeding. John Wiley & Sons, 1981.
2. Chopra VL. Breeding Field Crops. Oxford & IBH, 2001.
3. Chopra VL. Plant Breeding. Oxford & IBH, 2004.
Page | 46
4. Gupta SK. Practical Plant Breeding. Agribios, 2005.
5. Pohlman JM, Bothakur DN. Breeding Asian Field Crops. Oxford &
IBH, 1972.
6. Sharm JR. Principles and Practice of Plant Breeding. Tata McGraw-Hill,
2001.
7. David Allen Sleper, John Milton Poehlman. Breeding Field Crops.
Blackwell Publishing, 2006.
8. Simmonds NW. Principles of Crop Improvement. English language
Book Society, 1990.
9. Singh BD. Plant Breeding. Kalyani, 2006.
10. Singh S, Pawar IS. Genetic Bases and Method of Plant Breeding. CBS,
2006.
Page | 47
Page | 48
Chapter - 4
Conventional vs. Marker Assisted Backcross
Breeding
Author
Dr. Sree Latha Edpuganti
Assistant Director-PHM, National Institute of Plant Health
Management, Rajendra Nagar, Hyderabad, Telangana, India
Page | 49
Page | 50
Chapter - 4
Conventional vs. Marker Assisted Backcross Breeding
Dr. Sree Latha Edpuganti
Abstract
Traditional plant breeding methods like conventional backcross
breeding played crucial role in transfer of genes from specific line (Donor
Parent) to elite line (Recurrent Parent) to develop desirable varieties and
hybrids. Most of the procedures of conventional breeding were well
documented and utilized in practical plat breeding successfully. In this
chapter the detailed procedure of conventional back cross breeding for single
dominant, single recessive gene, two dominant and two recessive genes
along with the segregation ratios and appropriate number of plants to be
raised in each season for the selection of desirable plants were represented in
schematic diagrams. Marker-assisted back crossing (MABC) is a novel
method for the improvement of back cross breeding and genetic studies. In
MABC markers were used during backcrossing to select for the presence of
the target gene (foreground selection), to select for recurrent parent genome
recovery (background selection) and for recombinant selection to reduce
potential linkage drag. MABC is becoming popular with the availability of
advanced technologies in marker utilization, ease of adoption, quick
conversion period and accurate results. In this chapter, MABC was
compared with conventional plant breeding and advantages of MABC in
practical back cross breeding were discussed.
Keywords: Convectional back cross breeding, Marker assisted back
crossing, dominant gene transfer, recessive gene transfer
Introduction
Backcrossing is breeding method for introgression of a transgene/s or
replacement of an undesired gene/s of recipient parent with a desired gene/s
of donor parent. It is generally used to transfer one or a few traits of interest
into an adapted/elite/desired variety if it is deficient in only one or few
characteristics. The deficient characters can be transferred from other variety
which possesses desirable characters which are heritable and controlled by
Page | 51
one or more genes. The process involves transfer of one or more genes
which control desirable trait from donor parent “DP” into the recurrent
parent “RP”. The efficiency of back cross breeding depends quick recovery
of the RP genome by eliminating all other genes of DP. If any unwanted
genes from DP are transferred, they may cause problems such as changed
agronomic characters or low yield/quality or susceptibility to biotic and
abiotic stress etc., at the end. [1, 2, 3].
The main objective of either conventional back cross or MABC is to
integrate a targeted gene from the donor parent (DP) into a Recurrent parent
(RP). At the end, the converted line contains targeted gene from the DP, and
all other genes from RP. Marker-Assisted Backcrossing (MABC) is a
recently evolved as quick and effective method compared to conventional
back crossing to introgress dominant or recessive alleles with simultaneous
recovery of recurrent parent genome. MABC is precise for genes or
quantitative trait loci (QTLs) with well-defined differences in phenotypic
expression. The number of backcrosses required to recover the RP genome is
less in MABC compared to conventional back crossing so it takes less time
compared to conventional back crossing.
Chapter Content
Conventional recurrent backcrossing is a commonly used breeding
technique to transfer desirable traits from a donor parent (DP) to recurrent
parent (RP) through screening. The main purpose is to insert targeted trait
from DP into RP with maximum RP characters recovery as quick as possible
by selection.
There is no fixed number for how many backcrosses need to be done but
generally between five to eight backcross generations are required. If RP and
DP are genetically and phenotypically close it may require less generations
and if they are distant it may need more generations. After the final
backcross generation, selected individuals are self-pollinated so that selected
lines will be homozygous for the target trait.
Essential Factors for the Success of a Backcross Breeding Programme
1) Recurrent Parent: With good agronomic performance, highly
adaptable, recommended, popular and preferred by farmers which is
lacking a specific trait of interest
2) Donor Parent: With specific desirable trait which is heritable
3) Effective Screening: Phenotypic screening plays crucial role in
conventional back crossing. The difference in plants which possess
Page | 52
the desirable trait and not should be remarkable and easily
identifiable.
4) Backcross Scheme: The dominant back cross scheme is simple and
easy but the recessive backcross scheme is comparatively difficult
as it involves phenotypic selection on single plants.
5) Number of Backcrosses: Until recurrent parent is reconstituted
completely the back crosses are to be continued. Visual selection
plays crucial role in the early backcross generations as some plants
are close to RP and some are like DP and many are intermediate. In
later generations almost all the plants look like recurrent parent so
difficult to select. So recurrent backcrossing until BC6 will restore
the RP as much as possible.
The end product of a backcrossing programme is to obtain lines that are
as identical as possible to the RP with the target traits. The rate at which RP
genes are recovered during the back cross can be calculated using the
number of recurrent backcross made. Simple principle used in this
calculation is the contribution of the donor parent genome is reduced by half
with each generation of backcrossing [4, 5, 6].
Fig 1: RP genome recovery % in Conventional Back cross program
Page | 53
Fig 2: RP genome recovery % in MABC (BC1F1 to BC4F1 it is range)
Conventional Method of Back Cross Breeding

The scheme of back cross method is to be based on desirable character
being transferred is governed by dominant or recessive gene. The plan for
transfer of a dominant gene is simple, easy and quick compared to recessive
gene. Apart from dominant and recessive nature of a gene, number of genes
involved in governing the trait is also important. When the resistance is
controlled by single gene, it is monogenic resistance. If resistance is
governed by few genes it is oligogenic resistance. Back cross breeding is
highly successful for this type of clear vertical resistance which is easily
transferable for one line to other.
For example if host plant resistance is governed by dominant allele it
masks the expression of the recessive trait of susceptibility in heterozygous
condition. For convenience of understanding dominant genes are indicated
by capital letters, recessives by small letters. For example, "A" stands for
dominant trait and "a" stands for the corresponding recessive trait. In
monogenic dominant resistance both dominant homozygous plant with AA
and heterozygous plant with Aa allele pair shows resistance. But the
objective of back cross breeding is complete replacement of “aa” in RP with
“AA” from DP. If the resistance is governed by recessive gene only “aa”
plants are resistant and both AA and Aa plants are susceptible so the
Page | 54
objective is replacement of “AA” in RP with “aa” from “DP”. The entire
process involves series of steps of hybridization, back crossing and selfing.
Transfer of Single Dominant Gene
Let us suppose that a high yielding, preferred by farmers for cultivation
and widely adapted variety ‘RP’ is susceptible to rust (rr) and another variety
‘DP’ is not adapted but resistant to rust (RR) governed by dominant gene. In
this back cross programme rust resistance trait is to be transferred from
donor parent (DP) into a recurrent parent (RP) through back crossing without
changing genetic back ground of recurrent parent.
The sequence of steps followed by considering rust resistance is
governed by single dominant gene ‘RR’ which is present in DP and RP is
recessive for it ‘rr’ so rust susceptible. The heterozygote of it ‘Rr’ is also
resistant.
Step I. Hybridization
Variety ‘RP’ is to be crossed with variety ‘DP’. Recurrent variety ‘RP’
is used as female parent and donor parent ‘DP’ is used as male. When RP is
used as female parent cytoplasm comes from female parent and it retains in
further back crosses.
Step II. F1 Generation
All F1 plants contain 50% RP and 50% DP genome. Selection for rust
resistance is not necessary as all are resistant ‘Rr’ heterozygotes. Any F1
plant can be crossed to RP, since all the F1 plants will be heterozygous “Rr”
for rust resistance. During the second generation F1 plants are to be
backcrossed to variety ‘RP’.
Step III First Back Cross BC1 Generation
The harvested seed is BC1F1 seed. BC1F1 contains 75% RP and 25% DP
genome with 50% plants (Rr) would be resistant and remaining 50%
susceptible (rr) to rust, select rust resistant plants and backcross to ‘RP’.
Step IV, V VI and VII BC2, BC3 BC4 and BC5 Generations
Seed harvested from BC1F1 plants is BC2F1 seed. BC2F1 contains 87.5%
RP genome with 50% susceptible and 50% resistant plants. The procedure is
same for next 3 generations. In each backcross generation from BC2 to BC5,
segregation in the ratio of 50:50 Rr: rr occurs for rust resistance. Select rust
resistant plants through screening and backcross to ‘RP’. From BC2 to BC5
the plants selected for back crossing must have resistance and also selection
for plant type of ‘RP’ is important. Selecting plants closer to RP in each back
Page | 55
cross generation from BC2 to BC5 helps in quick and true to type conversion.
BC5 generation contains 98.44% RP genome and used to produce BC6F1.
Step VIII BC6 Generation
In BC6F1 on an average all the plants contains 99.22% genes from RP
and among them 50% plants will have rust resistant gene from DP in
heterozygous ‘Rr’ condition. 50% ‘rr’ plants which are susceptible are to be
rejected and removed preferably to avoid cross pollination/contamination.
Best rust resistant ‘Rr’ plants with ‘R’ gene from DP and true to type to RP
are to be selected and selfed and their seeds from individual plants are to be
harvested carefully. Appropriate self-pollination technique is to be followed
especially in cross pollinated crops to avoid cross pollination.
Step IX BC6F2 Generation
Individual plant progenies are to be grown from BC6F2 seed harvested
from individual selfed plants. In F2 generation the resistant genes segregate
1:2:1 RR: Rr: rr genotypic ratio. But phenotypically, resistant: susceptible
ratio is 3:1. In conventional back crossing only 25% ‘rr’ plants can be
rejected based on the expression of susceptibility. Rust resistance plants
which are true to type to variety ‘RP’ are to be selfed and harvested
separately from the selected 75% plants 25% will have homozygous ‘RR’
genes which are desirable and remaining 50% with heterozygous ‘Rr’ genes
which are to be rejected in next generation based on the segregation pattern.
Step X BC6F3 Generation
Individual plant progenies are to be grown and homozygous progenies
with 100% plants resistant to rust and true to type of ‘RP’ are to be harvested
in bulk. Several similar progenies can be mixed to constitute the new variety.
If the converted line is a parent of hybrid, produce hybrid seeds on
BC6F3 selected progeny rows, label each progeny row and collect self-seed
from the same progeny rows to use as parent seed. Evaluate hybrid seed
produced from each progeny row with original hybrid. After selection of the
best performing hybrids, the self-seed from those progeny rows is to be
bulked and used for further multiplication.
Step XI Yield Test: As Variety and as Hybrid
The new variety is to be tested in replicated yield trials along with the
variety ‘RP’ as a check. Plant type, branching pattern, leaf characters, date of
flowering, flower characters, date of maturity, yield and product quality are
to be evaluated critically under protected and unprotected conditions to rust.
If it is evaluated under unprotected conditions the results will be biased
Page | 56
because converted resistant variety shows superiority in yield. Under
protected conditions the new variety should be identical to old ‘RP’ in
performance. Under un-protected conditions converted line must prove
resistance to rust over the original. If there is no significant difference in
yield before and after conversion, the resistant variety may be directly
released for cultivation.
If the line is parent in hybridization program make paired crosses of
each progeny row with the other parent of hybrid and evaluate hybrid
performance of these resistant versions in yield tests along with original
hybrid under protected and unprotected conditions. Under unprotected
conditions only check for rust resistance and consider yield data only from
protected conditions trial for selection of superior progeny. Select the
progenies based on combining ability and yield data of hybrid trial for
further multiplication. The other parent of hybrid need not be resistant to rust
as the resistance is governed by dominant gene; heterozygous “Rr” ‘F1’
generation is resistant to rust.
Transfer of Single Dominant Gene-Conventional Plant Breeding
Female Male
Susceptible rr-RP x RR-DP Resistant
F1 -Making Season - 1
Recovery 50% Rr (100% Plants) 

Rr- F1 x rr-RP
Season - 2
Resistant BC1-Making
Recovery 75% & Rr (50% Plants)  rr (50% Plants) X
Rr-BC1F1 x rr-RP
Season - 3
BC2-Making
Recovery 87.5% & Rr (50% Plants)  rr (50% Plants) X
Rr-BC2F1 x rr-RP
Season - 4
BC3-Making
Rr-BC3F1 x rr-RP
Season - 5
BC4-Making
Rr-BC4F1 x rr-RP
Season - 6
BC5-Making
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Rr-BC5F1 x rr-RP
Season - 7
BC6-Making
Rr-BC6F1 x rr-RP
Season - 8
Selfing
BC6F2
RR (25% Plants)  Rr (50% Plants)  rr (25% Plants) X
Selfing
BC6F3 Season - 9
Progeny rows
RR RR RR
RR Rr Rr
RR Rr Rr
RR rr rr
 X X Season - 10
Yield test with RR plants

Transfer of Singe Recessive Gene-Conventional Plant Breeding
Female Male
Susceptible RR-RP x rr-DP Resistant
Season - 1
F1 -Making
Recovery 50% Rr (100% Plants) 
Rr- F1 x RR-RP
Season - 2
Susceptible BC1-Making
Recovery 75% & RR (50% Plants) Rr (50% Plants)
@
RR & Rr-BC1F1
Season - 3
BC1F2-Making
RR (62.5% Plants) X Rr (25% Plants) X rr (12.5% Plants) 
rr- BC1F2 x RR-RP
Season - 4
BC2F1-Making
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Recovery 87.5% & Rr (100% Plants) 
Rr-BC2F1 x RR-RP
Season - 5
BC3F1-Making
Recovery 93.75% & RR (50% Plants)  Rr (50% Plants) 
RR & Rr-BC3F1 @
Season - 6
BC3F2-Making
RR (62.5% Plants) X Rr (25% pLants) X rr (12.5% Plants) 
rr- BC3F2 x RR-RP
Season - 7
BC4F1-Making
Recovery 96.88% & Rr 100% Plants 
Rr-BC4F1 x RR-RP
Season - 8
BC5F1-Making
Recovery 98.44% RR (50% Plants)  Rr (50% Plants) 
BC5F1
Season - 9
Selfing
RR (62.5% Plants) X Rr (25% Plants) X rr (12.5% Plants) 
rr- BC5F2
Season - 10
Selfing
100% rr BC5F3 progeny
Season - 11
Yield test with rr plants
Transfer of Single Recessive Gene
Transfer of resistance governed by recessive gene through conventional
back crossing is complicated and lengthy compared to the transfer of
resistance governed by single dominant gene. For example smut resistance is
governed by a recessive gene; all the backcross cannot make one after other
to transfer it. After the first backcross and after every two backcrosses F2
must be grown to screen and identity the smut resistant plants. The F1 and
the other back cross progenies are not be screened for smut because all of
them will be susceptible to smut. Only F2 is to be screened for smut
resistance.
Page | 59
The recurrent parent ‘RP’ with dominant ‘RR’ gene susceptible to smut
is crossed with smut resistant donor parent ‘DP’ having resistant recessive
gene ‘rr’. The recurrent parent is used as female and donor parent as male i.e.
pollen source (RRXrr).
All F1 plants are susceptible to smut as they are heterozygous ‘Rr’ for
resistance. If resistance is governed by recessive genes, it expresses only
under homozygous recessive condition i.e. ‘rr’. F1 plants with ‘Rr’ alleles are
backcrossed to the recurrent parent with ‘RR’ alleles to produce BC1F1
plants.
Note: In general RP is female in F1 production. For subsequent back
crosses two options available.
1) RP as male parent against F1/selected back cross plants as female.
2) RP as female in F1 and all subsequent back crosses.
Step III BC1 (F1) Generation
The BC1F1 plants with 75% recovery and 50% ‘Rr’ and 50% plants with
‘RR’ genome, all the plant are smut susceptible. Therefore, there is no need
to test for smut resistance. All the plants are self- pollinated to produce
BC1F2 seed.
Step IV BC1 (F2) Generation
F2 generations are made for screening to identify resistant plants. In
BC1F2 generation the self-seed from 2 types of genetic back ground “RR &
Rr” with respect to smut resistance is bulked. Plants from ‘Rr’ segregates
1:2:1 ratio of RR: Rr: rr and plants from ‘RR’ will not segregate. From this
bulk ‘rr’ plants are to be selected through screening for smut resistance. The
composition of population is 87.5% susceptible plants i.e., RR 62.5% and Rr
25% and only 12.5% plants with ‘rr’ alleles expresses resistance. Along with
smut resistance selection is to be made for the plant type and other
characteristics of the ‘RP’. Smut resistance plants with RP plant type are
selected and backcrossed with recurrent parent to produce BC2F1 plants.
Step V BC2 (F1) Generation
No smut resistance test as all plants contain ‘Rr’ alleles and susceptible
to smut, select plants of recurrent parent type and back cross with the
recurrent parent to produce BC3F1 generation.
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Step VI BC3 (F1) Generation
No smut resistance test as plants are 50% ‘Rr’ and 50% ‘RR’ and all are
susceptible to smut. Selection is made for the only plant type identical to RP.
Self-pollinate the plants to harvest BC3F2 seed.
Step VII BC3 (F2) Generation
Screen plants for rust resistance. Assume that equal proportion of RR
and Rr plants are selected from BC3F1 to produce self-seed, smut resistant
plants with ‘rr’ recessive resistant genes are approximately 12.5% of total
population.
(Otherwise F2 progeny rows may be raised from seed collected from
individual plants. The progenies producing 100% susceptible plants are to be
rejected. Remaining progenies segregate in the ratio of 1:2:1 ratio RR: Rr: rr
from which resistant plants with ‘rr’ alleles need to be selected. Select
resistant “rr” plants and backcross to ‘RP’ to produce BC4F1 seed.
Step VIII BC4 (F1) Generation
Smut resistance tests are not required in BC4F1 generation. RP type “Rr”
plants are backcrossed to RP to produce BC5F1 seed
Step IX BC5 (F1) Generation
Smut resistance tests are not required in BC5F1 generation. Self-pollinate
all “RR” and “Rr” plants to produce F2 seed.
Step X BC5 (F2) Generation
BC5F2 plants are subjected to smut screening to select plants having
homozygous recessive allele ‘rr’ which are 12.5% of total plants. If progeny
rows are raised, reject progenies with 100% susceptible plants and select
25% of resistant plants from progenies segregating for smut resistance.
Among the resistant plants the ones having similar characteristic of RP are to
be selected and harvest self-seed separately.
Step XI BC5 (F3)
Grow individual plant progenies and subject to smut epiphytotic
selection. All the progenies should be 100% resistant, select the progenies
with similar characteristics of RP. Harvest seeds from several similar smut
resistant homozygous progenies and mix to constitute new variety.
If the converted line is to be used for hybrid making individual
progenies may be tagged, numbered and used for hybrid seed production and
selfing. Self-seed is to be harvested and maintained for further utilization and
hybrid seed will be used for yield tests.
Page | 61
If all the progenies are similar to RP then selfing and crossing can be
done on all the plants. It is the breeder’s decision to bulk the seed from all
progenies or carry yield tests from individual progenies to select best
performing ones based on yield data from hybrid trials.
Based on the crop it is breeder’s decision to maintain selfing and
crossing plots separately or same plot can be used for both selfing and
crossing operations to produce self-seed and hybrid seed from the same plot.
If resistance governed by recessive gene, in hybrid trials both the parents
used for hybrid seed production must possess homozygous recessive alleles
conferring resistance to the trait. Then only F1 with homozygous recessive
alleles for resistance expresses its character.
12) Yield Test
Yield tests will be common like dominant gene transfer, once either
dominant or recessive genes are fixed as long as they are maintained with
proper selfing or with proper isolations they will not segregate further.
Transfer of Two Dominant Genes
Let us suppose recurrent parent ‘RP’ is susceptible to blight which is
governed by 2 dominant genes. A donor parent is having 2 resistant genes in
homozygous condition ‘AA’ and ‘BB’ and these are to be transferred to RP
replacing ‘aa’ and ‘bb’ alleles in recessive condition. In this back cross
programme blight resistance trait is to be transferred from donor parent (DP)
into a recurrent parent (RP) through conventional back crossing without
changing genetic back ground of recurrent parent.
The Sequence of Steps to be followed
Cross ‘RP’ with ‘DP’ in which variety ‘RP’ is used as female parent
which is recurrent and variety ‘DP’ is used as donor parent.
During the second generation F1 plants are to be backcrossed to variety
‘RP’. Any F1 plant may be crossed to RP, since all the F1 plants are
heterozygous “AaBb’ and resistant. Selection for blight resistance is not
necessary.
Step III First Back Cross BC1 Generation
BC1F1 contains 75% RP and 25% DP genome. Among the total plants
75% (Aabb, aaBb, aabb) plants are susceptible to blight and 25% (AaBb) are
resistant to blight. Select blight resistant plants and backcross to variety
‘RP’.
Page | 62
Step IV, V VI and VII BC2, BC3 BC4 and BC5 Generations
Seed harvested from back crossed BC1F1plants is BC2F1 seed. The
procedure is same for next 4 generations. RP genome increases from 87.5%
in BC2F1 to 98.44% in BC5F1. In each backcross generation from BC2 to
BC5 25% blight resistant plants need to be selected for crossing. From BC2
to BC5 the plants selected for back crossing must have resistance and also
selection for plant type of variety ‘RP’ is important. Selecting plants closer
to RP in each back cross generation from BC2 to BC5 will help in quick and
true to type conversion. After BC5 generation the seed harvested is BC6F1
seed which contains 99.22% RP genome.
Step VIII BC6 Generation
On an average all the plants will have 99.22% genes from RP and
among them 25% plants will have both rust resistant genes from DP in
heterozygous ‘AaBb’ condition. 75% plants which are susceptible ‘Aabb,
aaBb, aabb’ to be rejected and removed preferably to avoid cross
pollination/contamination. Best blight resistant ‘AaBb’ plants true to type to
RP are to be selected selfed and their seeds are to be harvested carefully.
Appropriate self-pollination technique is to be followed especially in cross
pollinated crops to avoid cross pollination. Harvest BC6F2 seed from
individual selfed plants separately.
Step IX BC6F2 Generation
Individual plant progenies are to be grown with seed harvested from
selected plants. If the genes are not linked and independently assorted they
segregate in the ratio of 1:2:2:4:1:2:1:2:1 of AABB: AABb: AaBB: AaBb:
AAbb: Aabb: aaBB: aaBb: aabb respectively-genotypically and when it
comes to phenotypic expression AABB: AABb: AaBB: AaBb i.e. 9 out of 16
expresses resistance in F2 generation. Select these 9/16 (56.25%) plants and
produce self-seed. Reject remaining 7/16 (43.75%) plants which are
susceptible to blight.
(Note: To make an accurate calculation of segregation of a trait
governed by two or more alleles, it is important to know that the genes are
inherited independently or not. That means whether they "stick together" on
a single chromosome and get inherited as a unit or present on different
chromosomes sorted into gametes independent of each other. If genes are
close together on a chromosome, the alleles on the same chromosome inherit
as unit. Such genes do not independently assort and are linked. Probability of
a crossover will decrease if these genes are physically close together-this is
known as linkage and above segregation ratios are not applicable for linked
genes).
Page | 63
Step X BC6F3 Generation
Individual plant progenies are to be grown and homozygous progenies
resistant to rust and true to type of variety ‘RP’ harvest in bulk. One out of
nine progenies with ‘AABB’ genetic background will not segregate and
remaining all 8 progenies segregates at different ratios of resistant and
susceptible plants. The progenies with 100% resistant plants and true to type
to RP are to be selected. Several similar progenies are mixed to constitute the
new variety or grow F4 progenies from selected F3 plants if the results with
respect to resistance are not satisfactory in F3 generation.
If the line is a parent in hybridization program make paired crosses with
the other parent and evaluate best hybrid performance in yield tests and
select the progeny based on yield data for further multiplication. The other
parent need not be resistant to blight as the resistance is governed by
dominant gene even in heterozygous ‘AaBb’ condition the ‘F1’ generation is
resistant.
Yield Test: As variety and as Hybrid
The new variety is tested in replicated yield trials along with the variety
‘RP’ as a check. Plant type, branching pattern, leaf characters, dates of
flowering, flower characters, date of maturity, yield and quality are to be
evaluated critically under protected conditions to blight. If it is evaluated
under unprotected conditions the results will be biased because converted
resistant variety shows superiority in yield. If there is no significant decrease
in yield before and after conversion, the resistant variety may be directly
released for cultivation.
If the converted line is a parent of hybrid, produce hybrid seeds on
BC6F3 selected progeny rows, label each progeny row collect self-seed and
use it as parent. Evaluate hybrid seed produced from each progeny row with
original hybrid under protected conditions to blight and select the best
performing hybrids and the self-seed from those progeny rows is to be
bulked and used for further multiplication.
Transfer of Two Dominant Genes-Conventional Plant Breeding
Female Male
Susceptible aabb-RP x AABB-DP Resistant
Season - 1
F1 -Making
Recovery 50% AaBb (100% Plants) 
Resistant AaBb-F1 x aabb-RP
Season - 2
BC1-Making
Page | 64
Recovery 75% & AaBb (25%)  Aabb (25%) X aaBb (25%) X aabb
(25%) X
AaBb-BC1F1 x aabb-RP
Season - 3
BC2-Making
Recovery 87.5% & AaBb (25%)  Aabb (25%) X aaBb (25%) X aabb
(25%) X
Season - 4
BC3-Making
(25%) X
Season - 5
BC4-Making
(25%) X
Season - 6
BC5-Making
(25%) X
AaBb-BC5F1 x aabb -RP
Season - 7
BC6-Making
Recovery 99.22% & &AaBb (25%)  Aabb (25%) X aaBb (25%) X
aabb (25%) X
AaBb-BC6F1
Selfing Season - 8
BC6F2
1AABB: 2AABb: 2AaBB: 4AaBb (56.25%): 1AAbb: 2Aabb: 1aaBB:
2aaBb: 1aabb (43.75%) X
Selfing
Season - 9
BC6F3Progeny rows
Page | 65
AABB AABB AABB AABB
AABB AABb AaBB AABb
AABB AABb AaBB AABb
AABB AAbb aaBB AaBB
AABB AABB AABB AaBB
AABB AABb AaBB AaBb
AABB AABb AaBB AaBb
AABB AAbb aaBB AaBb
AABB AABB AABB AaBb Season - 10
AABB AABb AaBB AAbb
AABB AABb AaBB Aabb
AABB AAbb aaBB Aabb BC6F4
AABB AABB AABB aaBB
AABB AABb AaBB aaBb Select the progenies which
AABB AABb AaBB aaBb
AABB AAbb aaBB aabb are not segregating and go
 X X X for yield trials
Transfer of Two Recessive Genes-Conventional Plant Breeding

Female Male
Susceptible AABB-RP x aaBB-DP Resistant
Season - 1
F1-Making
Recovery 50% AaBb (100% plants) 
AaBb-BC1F1 x AABB-RP Season - 2
BC1-Making
Recovery 75% & AABB (25%) AABb (25%) AaBB (25%) AaBb (25%)
AABB, AABb, AaBB, AaBb BC1F1 @

Season - 3
BC1F2-Making
A-/B- (98.44% Plats) X aabb (1.56% Plants) 
aabb-BC1F2 x AABB-RP
Season - 4
BC2F1-Making
Recovery 87.5% & AaBb (100% Plants) 
AaBb-BC2F1 x AABB-RP
Season - 5
BC3F1-Making
Recovery 93.75% & AABB (25%) AABb (25%) AaBB (25%) AaBb
(25%)
AABB, AABb, AaBB, AaBb-BC3F1 @

Season - 6
BC3F2-Making
Page | 66
aabb-BC3F2 x AABB-R
Season - 7
BC4F1-Making
Recovery 96.88% AaBb (100% Plants) 
AaBb-BC4F1 x AABB-RP
Season - 8
BC5F1-Making
Recovery 98.44% AABB (25%) AABb (25%) AaBB (25%) AaBb (25%)
BC5F1
Selfing Season - 9

aabb-BC5F2
Season - 10
Selfing
100% aabb BC5F3 progeny rows
Season - 11
Yield test with aabb plants
Transfer of Two Recessive Genes
Transfer of resistance governed by 2 recessive genes through
conventional back crossing is a bit complicated and lengthy compared to the
resistance governed by two dominant genes. For example ‘rot’ disease
resistance is governed by two recessive genes which are independently
assorted; all the backcross cannot make one after other in conventional
method. After the first backcross and after every two backcrosses F2 must be
grown to screen and identity the rot resistant plants. The F1 and the back
cross plants need not screen for rot because all of them will be susceptible to
rot because rot resistance expresses only under homozygous recessive
condition of both the gens. Only F2 is to be screened for rot resistance.
The recurrent parent ‘RP’ with dominant ‘AABB’ genes susceptible to
rot is crossed with rot resistant donor parent ‘DP’ having resistant recessive
genes ‘aabb’. The recurrent parent is generally used as female and donor
parent as pollen source (i.e. AABB X aabb) to produce F1 seed.
All F1 plants are susceptible to rot as they are heterozygous ‘AaBb’ for
resistance, if resistance is governed by recessive genes it expresses only
Page | 67
under homozygous recessive ‘aabb’ condition. F1 plants with ‘AaBb’ alleles
are backcrossed to the recurrent parent with ‘AABB’ alleles to produce
BC1F1 seed.
(Note: In general RP is female in F1 and in subsequent back crosses it
may be used as male parent using F1/selected back cross plants as female).
Step III BC1 Generation
BC1F1 produces 4 distinct genotypes AABB: AABb: AaBB: AaBb each
25% and as rot resistance expresses at homozygous condition, all the plant
will be rot susceptible. Therefore, there is no need to test for rot resistance.
All the plants are self- pollinated to produce BC1F2 seed.
Step IV BC1 (F2) Generation
In BC1F2 generation the self-seed harvested from 4 types of BC1F1
genetic back ground with respect to rot resistance is bulked. The seed
harvested from 75% of plants AABB: AABb: AaBB is 100% susceptible to
rot. Seed harvested from AaBb only will give 6.25% rot resistant plants with
‘aabb’ recessive homozygous alleles for both gens. From the total bulk
‘aabb’ plants are to be selected through screening for rot resistance. The
chances of occurrence of these plants are 1.5 out of 100. So to get reasonable
number of rot resistance plants seed from as many as possible BC1F1 plants
need to be collected, seed collected from 100 plants each with 100 seed may
give 150 resistant plants. Along with rot resistance selection is to be made
for the plant type and other characteristics of the ‘RP’. Rot resistance plants
with RP plant type are selected and backcrossed with recurrent parent to
produce BC2F1 seed.
Step V BC2 (F1) Generation
No rot resistance test because in BC2F1 generation all plants contain
‘AaBb’ alleles and susceptible to rot, select plants of recurrent parent type
and back cross with the recurrent parent to produce BC3 generation.
Step VI BC3 (F1) Generation
No rot resistance test as plants AABB: AABb: AaBB: AaBb each 25%
and all are susceptible to rot. Selection is made for the only plant type
identical to RP. The plants are self-pollinated to harvest BC3F2 seed.
Step VII BC3F2 Generation
BC3F2 generations are made for screening to identify resistant plants.
Only 1.5% plants are resistant to rot and resistant plants similar to ‘RP’ are
selected and backcrossed to variety ‘RP’ to produce BC4F1 seed.
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Step VIII BC4 (F1) Generation
Rot resistance tests are not required in BC4F1 generation; plants are
backcrossed to RP to produce BC5F1 seed
Step IX BC5 (F1) Generation
Rot resistance tests are not required in BC5F1 generation, plants are self-
pollinated to produce BC5F2 seed.
Step X BC5 (F2) Generation
The RP genome recovery is 98.44%. Plants are subjected to rot
screening to select rot resistant plants having homozygous recessive allele
‘aabb’ which are approximately 1.5% of total plants. If progeny rows are
raised, reject progenies 75% of progenies with 100% susceptible plants and
select 6.26% of resistant plants from 25% progenies segregating for rot
resistance. Among the resistant plants the ones having similar characteristic
of RP are to be selected and self-seed are harvested separately.
Step XI BC5 (F3) Generation
Individual plant progenies are grown and subjected to rot epiphytotic
selection to confirm the resistance. Select for characteristics of RP. Seeds
from several similar rot resistant homozygous progenies are mixed to
constitute new variety.
If the line is to be used for hybrid making individual progenies may be
tagged, numbered and used for hybrid seed production. Self-seed is to be
harvested and maintained for further utilization and hybrid seed versions will
be used for yield tests.
If all the progenies are similar to RP then selfing and crossing can be
done on all the plants. It is the breeder’s decision to bulk the seed from all
progenies or carry yield tests from individual progenies to select best
performing version of hybrid based on yield data from trials.
Based on the crop it is breeder’s decision to maintain selfing and
crossing plots separately or same plot can be used for both selfing and
crossing operations to produce self-seed and hybrid seed from the same plot.
If Resistance governed by recessive genes is to be expressed in hybrid
yield trials both the parents used for hybrid seed production must possess
homozygous recessive alleles conferring resistance to the trait.
Step XII Yield Test
Yield tests will be common, once either dominant or recessive genes are
fixed as long as they are maintained with proper selfing or with proper
isolations they will not segregate further.
Page | 69
Tips for Back Crossing
1) In dominant/recessive gene transfer use RP as female in first
hybridization and use it as male in subsequent back crosses.
2) Don’t use same RP seed in all generations. If RP seed is used from
only few plants in all generations the converted line genetic base
will be narrowed down. Every time change RP seed from bulk
maintained initially so that entire genome of line is recovered.
3) Maintain the back cross population as much as possible with the
existing facility so that chances of selecting desired plants similar to
RP increases.
4) Selection of RP type plants in regular crop season is advisable
because in off season the expression may not be good. In off season
only screening for desired trait and generation advancement can be
done.
5) In recessive gene transfer during selfing generations sowing F2
progenies is advisable because the progenies with 100% susceptible
plants can be rejected. Only plants selected from segregating
progenies for resistance can be screened further.
6) Back up seed in back cross program plays crucial role as there are
many chances to lose the crop in any generation or desired plant
may not be available. Initially harvested back cross seed can be
used for sowing to fast track the program and later harvested seed
can be kept as back up.
Constraints in Conventional Back Crossing
1) A minimum of five or six backcross generations are required to
transfer a gene of interest from a DP to RP. The expected recurrent
parent (RP) genome recovery would be 99.2% by six backcrosses.
The proportion of the RP genome is recovered at a rate of 1-
(1/2) t+1 for each of the generations of backcrossing.
2) In BC1, BC2, BC3… population, theoretically the average percentage
of the RP genome is 75%, 87.5%. 93.75%… For the entire
population. But in reality it is not a fixed number, it is a range in the
population so those individuals that contain the highest RP genome
recovery are to be selected phenotypically which is biased.
3) Before starting back cross program the number of genes governing
desirable trait, dominant/recessive, independently assorted/linked
should be known and for the trait phenotypically district/ screening
method should be available.
Page | 70
Marker-Assisted Backcrossing (MABC)
MABC is effective for the transfer of traits that are difficult to
transfer/expensive to screen/governed by recessive genes/multiple genes
governing a trait/ gene of interest linked with negative traits/ expresses late
in development of plant like reproductive traits. Breeding programs based on
DNA markers for improving quantitative traits in plants is increasing
because the cost of genotyping especially with SNPs is reducing while the
cost of phenotyping is increasing particularly in developed countries (where
raising crop and screening manually is labour intensive and expensive).
MABC is attracting attention of breeders and the development of the
technology continues in future.
MABC Involves
1) Fore ground selection which focus on target gene selection.
2) Back ground selection which focus on recovery of recurrent parent
genetic back ground during back cross generations.
3) Recombinant selection which control the linkage drag for accurate
conversion.
Foreground selection is necessary in all backcross generations and back
ground selection could be skipped in some back cross generations. First use
foreground selection for all the plants and select plants having the
marker allele of the donor parent at the target locus. Markers that are tightly
linked to the gene of interest are to be used in fore ground selection. In all
back cross generations gene/s responsible for desired trait are in
heterozygous condition carrying one donor allele and one recurrent parent
allele. At the end of back crossing self-pollinate the selected plants to
identify the plants homozygous for the targeted donor allele.
Background selection, is to calculate the recurrent parent marker alleles
in all genomic regions except the target locus after confirming the target
locus presence through fore ground selection.
Recombinant selection is an added advantage in MABC to eliminate
'linkage drag' of potential deleterious genes from the DP. Inheritance of
unwanted donor alleles which are present in same genomic region of the
target locus can overcome with MABC [7, 8, 9, 10].
Advantages of MABC over Conventional Back Cross
1) Quick as it reduces the number of backcrosses
2) Eliminate phenotyping/screening process so less laborious
Page | 71
3) Track the genetic back ground
4) If target gene is recessive, the steps of selfing generations are
required in conventional back crossing for selection/screening
which are not required in MABC
Foreground Selection
The first step for foreground selection is to associate a molecular marker
with the target trait by some genetic mapping method. The marker should
have functional polymorphism means plant has to express phenotypic
difference between alleles with the change in DNA. Here most important
point is if polymorphism is associated with a particular character, it is
important to know whether that polymorphism is a function of a single gene
or multiple genes. Usually it is called “functional marker/s” if the trait is
native/ conventional otherwise ‘gene’ in the case of a foreign transgene.
These markers for the desirable trait are to be identified first then they are to
be used at each backcross generation to select those plants carrying the
desired allele/gene.
Use of Fore Ground Markers
There is no need of markers for F1 and BC1 making. Foreground
selection starts with first back cross generation. The markers used should be
tightly linked to the genes. Plant BCxF1 seed and extract DNA from leaf
tissue prior to flowering. Screen the markers on DNA of each plant
separately for each marker. The breeder selects plants having the marker
allele of the donor parent at the target locus in a heterozygous state (one
donor allele and one RP allele) until the final backcross is completed. At the
end of back crossing selected plants is self-pollinated and progeny plants
identified that are homozygous for the donor allele.
After selecting the plants that have the desired marker allele for
backcrossing further subject to back ground selection. Remove the plants
which lack desired alleles after fore ground selection and subject only the
plants with desired alleles for back ground selection [11, 12, 13, 14].
Strategic use of Markers for Background Selection
In contrast to foreground selection, background selection is designed to
eliminate donor parent alleles other than the desired target gene. To achieve
perfect back crossing it is important to limit the introgressed region to gene
of interest. Back ground selection mainly focus on elimination of donor
alleles on non-carrier chromosomes and regions that are far from the target
gene on the carrier chromosome. This can be achieved with simple strategy.
Page | 72
First identify the polymorphic markers between donor and recurrent parent.
From the polymorphic markers pick and assay markers which are uniformly
distributed on all chromosomes. Evenly spaced markers at 50 to 100 cM may
be assayed based on need and crop. In the next backcross generations the
markers for which RP genome is already recovered and became homozygous
need not be assayed, just ignore the markers for which RP genome is
recovered in homozygous condition in subsequent back cross generations.
Markers which are heterozygous along with new polymorphic markers
which are near to het markers need to be focussed in later back cross
generations. After BC2 generation selection of markers on non-carrier
chromosomes is critical as donor regions will be smaller, and hence more
markers may be assayed sometimes. Faster recovery of the recurrent parent
genome with marker assisted selection compared to conventional
backcrossing when foreground and background selection are combined
especially in BC1 and BC2 generations [15, 16, 17].
Strategic use of Markers for Recombinant Selection
Selection against donor alleles in the region of the target is recombinant
selection. Try to reduce quickly the size of the donor DNA segment
surrounding the target gene. Identify the recombinants between the target
and closely linked markers. Individuals with a recombination on both
sides of the target need to be identified and selected to complete MABC
program successfully. Recombination between the target gene and a marker
is proportional to the distance the marker is from the target. For markers far
from the target, recombination is likely to occur early in the backcrossing
process, so it becomes homozygous for the recurrent parent quickly. If
marker is closer to the target, only few recombinations occur between the
trait and markers close to the target, so that large populations need to be
screened to find recombinants in this case. To avoid these complexities
identify a recombinant on one side of the target in one generation and on the
other side in the next generation preferably in early back cross generations
i.e. BC1 and BC2.
Recombinant selection aims at reducing the size of the donor
chromosome segment containing the target locus. It reduces linkage drag by
eliminating undesirable genes linked to the target gene from the donor parent
which may interfere with the crop performance. The major advantage of
MABC over conventional back crossing is by using flanking markers on
either side of target gene, linkage drag can be minimized. By using
recombinant selection at least in two BC generations linkage drag can be
minimized [17, 18].
Page | 73
Factors Effecting Successful Marker-Assisted Backcrossing
I) Population size of each backcross generation: More the population
quicker the conversion.
II) Distance of foreground markers from the target locus: 0 cM
distance between trait and marker is ideal which means less the
distance more efficient.
III) Number of background markers: More the back ground markers
used accurately the recovery can be calculated.
IV) Recombinant selection/Linkage Drag: The recurrent parent genome
is recovered more slowly on the chromosome carrying the target
locus than on other chromosomes because of the difficulty in
breaking linkage with the target donor allele. Which is difficult to
manage in conventional back crossing.
References
1) Acquaah G. Principles of plant genetics and breeding. Oxford, UK:
Blackwell Publishing, 2007.
2) Allard RW. Principles of plant breeding. New York, NY: John Wiley &
Sons Inc., 1960.
3) Allard RW. Principles of plant breeding. 2nd ed. New York, NY: Wiley,
1999.
4) Briggs FN, Allard RW. The current status of backcross method of plant
breeding. Agronomy J. 1953; 45:131-138.
5) Hospital F. Selection in backcross programmes. Philosophical Trans
Royal Soc B. 2005; 360:1503-1511.
6) Stoskopf NC, Tomes DT, Christie BR. Plant breeding: theory and
practice. San Francisco, CA; Oxford: Westview Press Inc., 1993.
7) Toenniessen GH, O’Toole JC, DeVries J. Advances in plant
biotechnology and its adoption in developing countries. Curr Opin Plant
Biol. 2003; 6:191-198.
8) Babu R, Nair SK, Prasanna BM, Gupta HS. Integrating marker assisted
selection in crop breeding-prospects and challenges. Curr Sci. 2004;
87:607-619.
9) Collard BCY, Mackill DJ. Marker-assisted selection: an approach for
precision plant breeding in the twenty-first century. Philosophical Trans
Royal Soc B. 2007; 363:557-572.
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10) Frisch M, Melchinger AE. Selection theory for marker assisted
backcrossing. Genetics. 2005; 170:909-917.
11) Frisch M, Bohn M, Melchinger AE. Comparison of selection strategies
for marker-assisted backcrossing of a gene. Crop Sci. 1999; 39:1295-
1301.
12) Charmet G, Robert N, Perretant MR, Gay G, Sourdille P, Groos C et al.
Marker-assisted recurrent selection for cumulating additive and
interactive QTLs in recombinant inbred lines. Theor Appl. Genet. 1999;
99:1143-114
13) Frisch M, Bohn M, Melchinger AE. Minimum sample size and optimal
positioning of flanking markers in marker assisted backcrossing for
transfer of a target gene. Crop Sci. 1999; 39:967-975.
14) Hospital F, Charcosset A. Marker-assisted introgression of quantitative
trait loci. Genetics. 1997; 147:1469-1485.
15) Morris M, Dreher K, Ribaut JM, Khairallah M. Money matters (II): cost
of maize inbred line conversion schemes at CIMMYT using
conventional and marker assisted selection. Mol Breed. 2003; 11:235-
247.
16) Reyes-Valdes MH. A model for marker-based selection in gene
introgression breeding programs. Crop Sci. 2000; 40:91-98.
17) Mackill DJ, Collard BCY, Neeraja CN, Rodriguez RM, Herzog E,
Frisch M. Selection strategies for marker assisted backcrossing with
high-throughput marker systems. Theor Appl. Genet. 2011; 123:251-
260.
18) Hospital F. Size of donor chromosome segments around introgressed
loci and reduction of linkage drag in marker assisted backcross
programs. Genetics. 2011; 158(3):1363-1379.
Page | 75
Page | 76
Chapter - 5
Study of Induced Polygenic Variability in M3
Population of Aromatic Rice
Authors
Sanjeev Singh
Department of Agricultural Botany, Udai Pratap Autonomous
College, Varanasi, Uttar Pradesh, India
Rishi Kumar Sharma
Department of Agricultural Botany, Udai Pratap Autonomous
College, Varanasi, Uttar Pradesh, India
Satish Kumar Chakravarti
Krishi Vigyan Kendra, Basuli, Maharajganj, N.D.U.A & T,
Kumarganj, Faizabad, Uttar Pradesh, India
Prakash Singh
Department of Genetics and Plant Breeding, I. Ag. Sc., B.H.U.,
Varanasi, Uttar Pradesh, India
Page | 77
Page | 78
Chapter - 5
Study of Induced Polygenic Variability in M3 Population of
Aromatic Rice
Sanjeev Singh, Rishi Kumar Sharma, Satish Kumar Chakravarti and Prakash Singh
Abstract
In the present study, most of the quantitative traits were studied to
screen out the useful and desirable mutants in the mutagen treated population
in M3 generation. In general traits like, plant height and panicle length
showed negative shift in mean whereas days to 50% flowering and days to
maturity showed positive shift in mean in M3 generation in both the varieties
of aromatic rice. Number of grains per panicle, number of panicle bearing
tillers per plant and 100-seed weight showed positive as well as negative
shifts in mean in both the varieties of aromatic rice in M3 generation.
Keyword: Aromatic rice, gamma rays, EMS, micromutation
Introduction
The use of physical and chemical mutagens or combination of both has
been an important tool for the increase of variability in agronomic traits in
rice (Agrawal et al., 2000; Baloch et al., 2002; Domingo et al., 2007;
Siddiqui and Singh, 2010, Chakravarti et al., 2013). The potentiality of
ionizing radiations and chemical mutagens is different and their ability to
induce mutations varies from genotype to genotype in rice (Singh and
Sharma, 2013). Therefore, it is desirable to have the appropriate treatment
schedule before undertaking mutagenesis. Since genetic variability is a pre-
requisite for any successful breeding programme, the creation and
management of genetic variability becomes central base for crop
improvement programme. The primary objective of this research work was
to enhance the frequency and spectrum of mutations and also to increase the
incidence of viable mutations.
The present investigation clearly demonstrated the high potentials of
gamma ray, EMS alone or in combination at higher doses and/or
concentrations in generating desirable variability for yield and component
traits in micromutation population of two aromatic rice. In the present study,
Page | 79
most of the quantitative traits were studied to screen out the useful and
desirable mutants in the mutagen treated population in M3 generation.
Materials and Methods
The two aromatic rice varieties, viz., Pusa Basmati 1 and Kalanamak
were employed as experimental materials. Some of the important
characteristics of cultivars are given below which may serve as useful
criteria for judging the relative merits of induced mutants, if any. Pusa
Basmati 1 was derived from a cross of Pusa 150 x Karnal local. It is a semi
dwarf, medium flowering, long size grain, awned panicle, mildly scented,
high gelatinization temperature, soft gel and intermediate amylose.
Kalanamak is a non-basmati scented rice variety grown primarily in the
Tarai area adjoining Nepal particularly in the districts of Siddharthnagar,
Santkabirnagar and Basti and in small pockets in eastern Uttar Pradesh, and
is named because its husk is black. It is a successful adapter to usar soils
characterised by higher salt concentration and high pH. The name itself --
'namak', means salt signifies this quality.
Three hundred pure, uniform, healthy and dry (12% moisture) seeds for
each treatment of aromatic rice Pusa Basmati 1 and Kalanamak were
irradiated with gamma rays at five doses, viz., 10 kR, 20 kR, 30 kR, 40 kR
and 50 kR at NBRI, Lucknow. Two hundred fifty seeds were sown in the
field and remaining fifty seeds were kept for laboratory observations. EMS
solution with different concentrations, i.e., 0.2%, 0.3%, 0.4% and 0.5% were
prepared by mixing appropriate volume of the chemical (EMS) and
phosphate buffer (pH 7.0). Three hundred pure, uniform, healthy and dry
seeds (12% moisture) were subjected to presoaking in distilled water for 6
hours at room temperature. The soaked seeds were then transferred to EMS
solution of different concentrations in research lab of the department. The
seeds were kept in EMS solution for 6 hours and seeds were given
intermittent shaking throughout the period of treatment to maintain
uniformity. The mutagen solution was drained out. The treated seeds were
then washed in running tap water for 1 hour to remove residual chemical
from the seeds, if any.
The combination treatment of gamma rays and ethyl methane
sulphonate was also done as described earlier. For this, the gamma ray
treated seeds were soaked in distilled water for six hours and then treated
with EMS solution of 0.2% concentration followed by washing in running
tap water for 1 hour. For each combination treatment, 300 seeds were used.
The 250 washed seeds of each treatment were then sown in experimental
Page | 80
field at Research Farm, UP College to raise M1 generation along with control
in three replications and the remaining 50 seeds were used in laboratory for
observing germination and seedling height reduction. The M1 plants were
studied critically and carefully. Any deviations in the characters of treated
plants from the control (parent) plants were screened and marked. The
morphological variations, if any, were noticed and recorded. The M1 seeds
were harvested from each suspected plants and were sown following plant-
to-progeny method. There was likelihood of getting huge number of
qualitative and quantitative mutations.
The field experiment was conducted at Research Farm, U P College,
Varanasi. The field experiment was conducted at Research Farm, Udai
Pratap Autonomous College, Varanasi. The M1, M2 and M3 generations were
sown on 19th July, 2011; 3rd July, 2012, 26th and 13th June, 2013, respectively
and transplanted after 21 days in the field in three replications along with the
control. The distance between and within the rows was kept as 20 cm and 15
cm, respectively. The cultural practices were performed to maintain a good
plant stand. The cultural practices were performed to maintain a good plant
stand.
Results and Discussion
The analysis of variance for all the characters revealed a significant
difference between the treatments in M3 generation in both the varieties. The
values of mean, range and coefficient of variation (CV) for the two varieties
are presented in Tables 1-4.
Page | 81
Table 1: Mean, range and coefficient of variation (CV) for 100-seed weight and grain yield per plant in aromatic rice in M2 generation
Plant Height Days to Flowering

Treatment Pusa Basmati 1 Kalanamak Pusa Basmati 1 Kalanamak
Mean Range CV Mean Range CV Mean Range CV Mean Range CV
Control 102.00 92-109 5.0 168.33 160-180 6.0 118.33 106-130 5.0 147.66 140-158 5.1
Gamma Rays
10 kR 101.33 89-124 6.1 167.00 152-184 7.1 118.33 102-127 7.6 146.33 139-150 7.4
20 kR 102.33 86-116 6.2 168.33 154-180 10.6 120.00 106-130 6.8 148.00 137-153 8.5
30 kR 100.33 83-118 6.7 166.00* 150-181 11.1 118.66 108-128 5.1 146.66 134-153 10.4
40 kR 103.66 80-116 6.0 169.33 148-177 16.4 117.66 105-127 5.0 146.66 133-155 11.4
50 kR 100.00* 79-115 7.0 166.00* 140-175 15.7 121.00* 104-126 6.9 149.00 132-153 12.6
Ethyl Methane Sulphonate (EMS)
0.2% 101.66 86-122 6.2 167.66 150-180 10.1 118.00 102-130 7.1 147.33 138-154 6.5
0.3% 101.66 85-121 6.1 167.76 155-179 9.7 118.66 104-129 6.0 146.66 140-153 7.3
0.4% 100.00* 72-117 7.2 166.66 153-176 10,2 120.66* 103-127 7.9 147.66 139-152 7.8
0.5% 98.66** 79-116 7.4 164.76** 149-170 10.0 119.10 102-129 6.9 147.10 137-153 9.0
Gamma Rays + EMS
10 kR + 0.2% 101.33 84-125 7.0 168.00 157-180 11.4 119.20 104-130 7.0 147.20 133-152 7.0
20 kR + 0.2% 104.33* 80-116 7.9 168.00 150-176 12.0 119.00 105-129 6.7 147.00 134-155 9.7
30 kR + 0.2% 101.00 86-122 6.0 167.00 156-175 14.0 120.00 103-128 7.0 148.00 132-152 10.4
40 kR + 0.2% 102.66 81-120 7.0 168.66 145-188 17.8 118.33 103-125 7.5 146.33 130-151 12.6
50 kR + 0.2% 99.33** 79-120 8.1 165.33** 146-181 18.4 122.33** 102-124 7.2 151.00** 131-150 12.3
SE± 0.83 0.93 0.97 0.86
Page | 82
Days to Maturity Panicle Length

Control 146.66 131-156 5.0 168.66 160-173 4.8 27.66 24-35 6.0 26.00 23-30 6.4
Gamma Rays
10 kR 146.33 128-160 7.0 168.33 157-180 6.2 26.83* 24-37 8.0 25.50 24-31 7.0
20 kR 148.33* 129-161 5.0 170.00 150-180 6.3 27.66 22-35 7.5 26.16 22-31 10.0
30 kR 145.66 133-160 4.3 168.66 150-181 10.0 26.50** 21-38 10.4 25.00* 22-30 8.0
40 kR 145.66 130-161 4.3 167.66 146-177 11.2 28.00 22-37 9.2 26.50 20-31 10.3
50 kR 149.33** 128-159 5.5 171.00** 144-180 10.0 27.83 22-35 9.0 26.33 19-30 12.4
0.2% 147.33 126-155 6.0 169.33 155-181 6.4 27.33 23-36 7.2 25.83 24-29 8.0
0.3% 146.66 125-154 5.0 168.66 155-182 7.7 27.33 22-35 7.3 25.83 25-29 8.1
0.4% 147.66 127-158 6.1 169.66 154-185 8.0 26.83* 24-35 8.2 25.33 24-30 8.8
0.5% 147.20 126-160 6.0 169.20 150-181 9.2 27.50 20-34 8.0 27.33** 24-30 9.2
Gamma Rays + EMS
10 kR + 0.2% 147.33 127-158 5.6 169.33 153-183 9.0 27.66 21-34 8.1 26.00 22-30 9.0
20 kR + 0.2% 147.00 129-159 5.8 169.00 143-182 10.2 28.83* 23-34 7.5 26.00 21-31 10.0
30 kR+ 0.2% 148.00 127-162 6.0 170.00 147-185 12.0 27.33 24-36 8.2 25.50 21-32 11.2
40 kR + 0.2% 146.33 129-161 5.8 168.33 143-186 16.2 27.83 22-34 8.1 26.33 20-32 15.3
50 kR + 0.2% 149.33** 128-158 5.6 171.66** 140-183 16.8 27.50 23-35 8.2 26.00 19-30 15.1
SE± 0.71 0.77 0.36 0.41
Page | 83
Number of Panicle Bearing Tillers Per Plant Number of Grains Per Panicle
Control 8.00 7-16 21.0 6.30 4-10 15.0 139.83 48-148 23.0 130.00 124-180 17.2
Gamma Rays
10 kR 8.16 6-13 21.6 5.80 4-12 20.0 139.00 55-144 22.1 129.00 85-215 24.1
20 kR 9.00* 4-13 23.5 6.46 3-14 20.3 140.30 54-150 25.1 130.33 76-222 27.4
30 kR 7.50 4-13 24.3 5.30* 4-15 24.4 138.00 48-161 24.2 129.33 74-230 31.0
40 kR 9.33** 4-14 21.1 6.80 3-14 28.1 141.00 58-143 25.0 131.00 71-230 32.0
50 kR 8.66 4-15 20.0 6.13 4-15 30.2 137.66* 50-160 27.0 127.66* 70-225 35.1
Ethyl methane Sulphonate (EMS)
0.2% 9.16** 5-16 25.4 6.13 4-13 22.4 139.66 49-145 23.0 129.66 74-223 25.1
0.3% 8.33 4-18 24.3 6.13 4-11 24.0 142.66** 45-140 26.4 129.66 74-220 26.3
0.4% 7.83 4-16 24.2 5.63 4-13 26.1 138.00 45-141 28.0 128.00* 70-230 27.2
0.5% 8.50 4-17 21.0 6.30 4-12 27.5 137.66* 44-160 30.1 127.66* 69-237 28.5
Gamma Rays + EMS
10 kR + 0.2% 8.83* 3-16 26.3 6.63 3-15 25.0 140.00 46-143 28.1 132.66* 75-220 26.2
20 kR + 0.2% 8.50 5-17 25.4 7.63** 3-16 26.1 139.33 52-142 27.2 129.33 75-230 34.2
30 kR + 0.2% 8.16 5-14 24.6 5.96 3-14 31.0 139.66 54-140 25.3 129.66 69-240 33.3
40 kR + 0.2% 8.66 6-15 25.6 6.63 3-17 32.1 140.66 46-140 24.9 130.66 65-241 34.0
50 kR + 0.2% 8.50 5-16 23.5 6.30 3-16 32.0 137.66* 52-141 27.4 128.66 61-240 35.4
SE± 0.37 0.37 0.90 0.87
Page | 84
100-seed Weight Grain Yield per Plant

Control 2.16 2.0-2.4 4.00 1.60 1.7-2.0 6.6 12.20 6.5-16.5 24.0 10.33 11.0-14.0 15.1
Gamma Rays
10 kR 1.90 1.8-2.4 5.00 1.50 1.7-2.0 7.0 10.20* 5.7-20.1 27.3 9.00* 6.9-17.0 26.0
20 kR 2.03 1.6-2.4 6.01 1.63 1.7-2.1 8.1 11.53 7.5-19.0 33.0 10.33 5.9-19.0 26.5
30 kR 1.80** 1.7-2.4 6.02 1.40* 1.5-2.1 11.0 9.20** 6.9-23.5 32.0 9.33 5.6-19.0 30.1
40 kR 2.20 1.7-2.5 7.02 1.80* 1.6-2.1 10.2 13.33 7.2-19.3 33.5 11.00 5.0-19.5 34.3
50 kR 2.16 1.6-2.3 6.29 1.36* 1.6-2.1 11.4 9.53** 5.4-20.0 34.0 8.33** 5.5-20.0 37.5
0.2% 2.26 1.7-2.4 4.36 1.60 1.6-2.0 8.0 10.53 6.3-18.5 23.0 9.33 6.4-17.1 30.0
0.3% 2.10 1.6-2.4 5.52 1.56 1.6-2.1 8.2 10.86 6.9-19.4 32.2 9.66 5.9-17.0 31.2
0.4% 1.93* 1.8-2.4 6.03 1.53 1.6-2.0 9.3 9.86* 6.4-17.1 31.1 8.66* 5.7-16.1 32.3
0.5% 1.76** 1.6-2.5 6.01 1.36* 1.6-2.1 9.5 9.53** 5.3-15.0 33.3 10.00 5.4-16.0 35.4
Gamma Rays + EMS
10 kR + 0.2% 2.10 1.6-2.4 7.19 1.60 1.6-2.0 9.0 11.20 6.2-18.3 27.0 9.66 6.3-17.0 30.1
20 kR + 0.2% 2.00 1.8-2.4 6.90 1.86* 1.6-2.1 10.0 11.20 6.7-18.0 29.0 10.16 5.4-17.1 33.4
30 kR + 0.2% 1.86** 1.8-2.4 6.89 1.46 1.6-2.0 11.1 9,80* 7.4-17.1 22.4 9.50 5.3-19.0 34.4
40 kR + 0.2% 2.06 1.8-2.4 5.60 1.66 1.5-2.1 11.9 10.66 7.0-19.5 25.2 10.50 5.4-19.0 39.0
50 kR + 0.2% 1.90* 1.7-2.4 7.81 1.50 1.5-2.0 12.5 9.00** 5.2-17.1 28.0 8.00** 5.4-18.4 39.1
SE± 0.10 0.07 0.90 0.64
Page | 85
Plant Height
The data pertaining to mean, range and coefficient of variation for plant
height in treated as well as control populations of Pusa Basmati 1 and
Kalanamak are presented in Table 1 (Fig. 1). It is obvious from the results
that there was shift in mean in negative direction over the control. There was
wider range in the treated populations as compared to the untreated
population. Plant height significantly decreased in both the varieties in many
treatment doses. The shift towards reduced height was found maximum as
98.66 cm in Pusa Basmati 1 and 164.76 cm in Kalanamak at 0.5% EMS in
M3 generation
Fig 1: Plant Height
Days to Flowering
The values of mean, range and coefficient of variation for days to
flowering of rice cultivars Pusa Basmati 1 and Kalanamak are presented in
Table 1 (Fig. 2). It was evident from the table that in Pusa Basmati 1, the
shift towards late flowering was observed at 50 kR gamma rays + 0.2% EMS
in M3 generation. The coefficient of variation also increased in all the
treatments as compared to the control in both the varieties. Days to flowering
was significantly increased in Kalanamak in few treatment doses. A
maximum positive shift towards late flowering was observed in Kalanamak
at 50 kR gamma rays + 0.2% EMS. In general, there was a wider range for
days to flowering in treated populations than the control in both the varieties.
Page | 86
Fig 2: Days to flowering
Days to Maturity
The data for mean, range and coefficient of variation for days to
maturity in treated as well as control populations of rice cvs. Pusa Basmati 1
and Kalanamak in M3 generation are presented in Table 3 (Fig. 3). As
evident from the data recorded from Pusa Basmati 1, the maximum shift
towards late maturity was observed maximally at 50 kR gamma rays and
combination treatment of 50 kR gamma rays + 0.2% EMS. The coefficients
of variation for all the treatments increased over control in M3 generation.
There was a wider range in the treated population as compared to the control.
In Kalanamak days to maturity was significantly increased in few treatment
doses and was found maximum at 50 kR gamma rays + 0.2% EMS in M3
generation.
Fig 3: Days to Maturity
Page | 87
Panicle Length
The data on mean, range and coefficient of variation for panicle length
in Pusa Basmati 1 and Kalanamak in M3 generation are presented in Table 3
(Fig. 4). It was apparent from the results that there was shift in mean towards
negative sides over the control in both the varieties. The coefficient of
variation for panicle length was invariably higher in treated population. All
the treatments, except few have shown negative shift in mean panicle length
over the control. The maximum negative shifts in mean panicle length over
the control was observed at 30 kR gamma rays and highest coefficients of
variation were observed at 30 kR gamma rays in M3 generation in Pusa
Basmati 1. Kalanamak also showed negative shifts in mean performance
over the control in M3 generation. The coefficient of variation for panicle
length was invariably higher in treated populations, irrespective decrease in
mean values. The treatment 30 kR gamma rays had shown negative shift in
mean panicle length over the control. The maximum range was observed at
40 kR gamma rays+ 0.2% EMS in M3 generation. The highest coefficient of
variation was observed at 40 kR gamma rays + 0.2% EMS in M3 generation.
Fig 4: Panicle length
Number of Panicle Bearing Tillers Per Plant

The values of mean, range and coefficient of variation for number of
panicle bearing tillers per plant in Pusa Basmati 1 and Kalanamak in M2
generation are presented in Table 3 (Fig. 5). The results revealed that there
was slight change in the magnitude of range for productive tillers in both the
varieties in M3 generation. Negative shifts in mean over control were
observed in both the varieties. In case of Pusa Basmati 1, positive shift for
number of panicle bearing tillers per plant was found maximum at 40 kR
gamma rays in M3 generation. The higher doses of mutagenic treatments
Page | 88
exhibited wider range for this trait. The maximum coefficient of variation
was observed at 10 kR gamma rays +0.2 % EMS in M3 generation. In case
of Kalanamak, a maximum significant increase in number of panicle bearing
tillers per plant was observed at 20 kR gamma rays+ 0.2% EMS in M3
generations. In general, the coefficient of variation among treated population
was higher than the control in M3 generation.
Fig 5: Panicle Bearing Tillers per Plant
Number of Grains per Panicle

The M3 data pertaining to mean, range and coefficient of variation for
number of grains per panicle in both the varieties of aromatic rice are
presented in Table 3 (Fig. 6). In case of Pusa Basmati 1, the positive as well
as negative shifts in mean for number of grains per panicle were observed.
The lower doses of mutagenic treatments had least effect on number of
grains per panicle, whereas higher doses of treatments exhibited reduction in
the number of grains per panicle. The maximum increase in this trait was
observed at 0.3% EMS. The maximum coefficient of variations was
observed at 0.5% EMS. Alike Pusa Basmati 1, the range and coefficient of
variation in Kalanamak were significantly higher in treated population than
the control. A reduction in number of grains per panicle was observed at
many treatment doses. It was maximally increased at 10 kR gamma rays +
0.2% EMS. The maximum value of coefficient of variation was observed at
50 kR gamma rays + 0.2% EMS treatment in M3 generation.
Page | 89
Fig 6: No. of grains per panicle
100-Seed Weight
The data on mean, range and coefficient of variation for 100-seed
weight of Pusa Basmati 1 and Kalanamak are depicted in Table 4 (Fig. 7). A
significant decrease in the mean values for 100-seed weight was observed in
both the varieties at many treatment doses. In few cases, an increased 100-
seed weight was obtained. In case of Pusa Basmati 1, the results showed that
increase and decrease in the mean 100-seed weight values in treated
population over the control were observed and it was reduced maximally at
0.5% EMS. The highest coefficient of variation was observed at 50 kR
gamma rays+ 0.2% EMS. In case of Kalanamak, the coefficient of variation
was observed maximum at 50 kR gamma rays + 0.2% EMS. Reductions in
mean seed weight was found maximum at 50 kR gamma rays and 0.5% EMS
in M3 generation.
Fig 7: 100-seed weight
Page | 90
Grain Yield Per Plant
The data related to mean, range and coefficient of variation for grain
yield in treated as well as control populations of Pusa Basmati 1 and
Kalanamak in M3 generation are presented in Table 4 (Fig. 8). The results
obtained clearly indicate that the mutagenic treatments reduced the yield
significantly at many treatment doses in both the varieties in M3 generation.
In Pusa Basmati 1, the maximum decrease in yield was observed at 50 kR
gamma rays + 0.2% EMS. The maximum coefficient of variation was
obtained at 50 kR gamma rays. The maximum range in M3 generation was
obtained at 30 kR gamma rays. In Kalanamak, the maximum decrease in
yield was observed at 50 kR gamma rays+0.2% EMS and the coefficient of
variation was observed maximum at 50 kR gamma rays + 0.2% EMS,
whereas the value of range was maximum at 50 kR gamma rays in M3
generation.
Fig 8: Grain yield per plant

In general, mutagenic treatments had resulted in decreased mean
coupled with enhanced variability in both genotypes and generations as
compared to their respective control, though the magnitude of shift in mean
varied with the treatment, genotype and trait. The decrease in means of
mutagen treated population might be due to greater frequency of mutations
with detrimental effects or due to difference in magnitude of induced
individual change. The shift in mean was not found to be unidirectional nor
equally in both directions in all the treatments. For days to flowering and
maturity and plant height shifts in mean toward negative direction, as also
noted in the present case, were of great significance as they yielded high
frequency of mutants with early flowering and maturity and short stature.
The induction and isolation of short statured with early maturing mutants
Page | 91
having other desirable traits of the parental genotypes would be quite
rewarding. In rice several workers (Singh and Singh, 2003; Siddiqui and
Singh, 2010) also found induced variability in desired directions for earliness
and short stature.
In general traits like, plant height and panicle length showed negative
shift in mean whereas days to 50% flowering and days to maturity showed
positive shift in mean in M3 generation in both the varieties of aromatic rice.
Number of grains per panicle, number of panicle bearing tillers per plant and
100-seed weight showed positive as well as negative shifts in mean in both
the varieties of aromatic rice in M3 generation. These traits, as expected
because of their positive correlation with grain yield, contributed
significantly towards grain yield. Similar results were also noticed in several
crops, such as, rice (Elayaraja et al., 2005; Bughio et al., 2007, Siddiqui and
Singh, 2010, Chakravarti et al., 2013).
Acknowledgement
The first author is grateful to the University Grants Commission, New
Delhi for sanctioning a major research project.
References
1. Agrawal PK, Sidhu GS, Gosal SS. Induced mutation for bacterial blight
resistance and other morphological characters in indica rice. Oryza.
2000; 37(4):277-280.
2. Baloch AW, Soomro AM, Javed MA, Bhugio HR, Bhugio HR,
Mohammad T et al. Development of high yielding rice mutant variety
through gamma rays irradiation. The Nucleus, 2002; 39(3-4).
3. Bughio HR, Odhano IA, Asad MA, Bughio MS. Improvement of grain
yield in rice variety Basmati-370 (Oryza sativa L.) through mutagenesis.
Pak. J Bot. 2007; 39(7):2463-2466.
4. Chakravarti SK, Singh S, Kumar H, Lal JP, Vishwakarma MK. Study of
induced polygenic variability in M1 and chlorophyll mutations in M2
generation in aromatic rice. The Bioscan. 2013; 8(1):49-53.
5. Domingo C, Andres F, Talon M. Rice cv. Bahia mutagenized
population; a new resource for rice breeding in the Mediterranean Basin.
Spanish J Agric. Res. 2007; 5(3)341-347.
6. Elayaraja Prakash K, Kumar M, Saravanan BS, Ganesan KJ. Effects of
physical and chemical mutagens on certain quantitative characters in
rice (Oryza sativa L.). Crop Res. 2005; 30(2).
Page | 92
7. Siddiqui SA, Singh S. Induced genetic variability for yield and yield
traits in Basmati rice. World J Agric. Sci. 2010; 6(3):331-337.
8. Singh S, Singh J. Mutation in Basmati rice induced by gamma rays,
ethyl methane sulphonate and sodium azide. Oryza. 2003; 40(1-2):5-10.
9. Singh S, Sharma RK. Mutagenic effects of gamma rays and EMS in M1
and M2 generations in aromatic rice. The Ecoscan: Special issue. 2013;
4:45-51.
Page | 93
Page | 94
Chapter - 6
Plant Breeding: A Tool for Potential Exploitation
of Genetic Gain in Crop Plants
Authors
Manish Kumar
Department of Genetics and Plant Breeding, I. Ag. Sc., BHU,
Ravi P Singh
Department of Genetics and Plant Breeding, I. Ag. Sc., BHU,
Onkar Nath Singh
Technical Member, PVPAT, IPAB, Chennai, Tamil Nadu,
India
Vineeta Singh
Crop Improvement Division, ICAR- NRRI, Cuttack, Odisha,
India
Prakash Singh
Assistant Professor, Department of Plant Breeding and
Genetics, Veer Kunwar Singh College of Agriculture,
Dumraon, Buxar, Bihar, India
Page | 95
Authors
Dahiphale A.V.
Agronomist (ECF), AICRP-IFS, Regional Agricultural
Research Station, Karjat, Raigad, Maharashtra, India
Debarchana Jena
India
Diptibala Rout
India
J.L. Katara
India
S. Samantaray
India
Ramlakhan Verma
India
Page | 96
Chapter - 6
Plant Breeding: A Tool for Potential Exploitation of Genetic
Gain in Crop Plants
Manish Kumar, Ravi P Singh, Onkar Nath Singh, Vineeta Singh, Prakash Singh,
Dahiphale A.V., Debarchana Jena, Diptibala Rout, J.L. Katara, S. Samantaray and
Ramlakhan Verma
Abstract
Under dynamic demographic and climatic scenario enhancement of
substantial genetic gain is imperative for food security and livelihood of the
farming community. Since civilization, humans are doing some kind of
breeding practices unknowingly to sustain their life. Selection is the basic
practice our ancestor was adopted initially for domestication of desirable
wild plant types into cultivated. Subsequently, potential of hybridization/
recombination were recognized which along with selection practices makes
breeding a great tool. Genetic variation and survivability of the crop plants
are two prerequisite parameters on which plant breeding is based.
Importance of plant breeding was started to be realized with the discovery of
genetics and factors responding variation, survivability in the plants.
Conventional plant breeding has served immensely in uplifting the crop
productivity in all domesticated plant species which resulted first green
revolution. Subsequently, integration of advanced genomic tools with
conventional approaches has facilitates researchers a very precise and quick
breeding tools for selection, robust genotyping and phenotyping which helps
in harnessing genetic gain in quick span of time. In this chapter we are tried
to elaborate some of the very important breeding practices which might be
purposeful for enhancing genetic gain in crop plants to new height.
Keyword: Breeding approached, genetic gain, genomic tool
1. Introduction
Plants are the key to life on earth as they supply 90% of human calorie
intake. To meet the food demand of our ever-increasing population,
production through conventional breeding of plants is insufficient. Again
though there are so many high yielding varieties, they are unable to
overcome these problems, so advance technologies are necessary for plant
breeding through non-conventional way.
Page | 97
2. Historical Remark
Scientist Year Work
Haberlandt 1902 Plant tissue culture.
Laibach 1925 Technique of embryo culture.
Skoog 1941 In vitro adventitious shoot formation in tobacco.
Ball 1950 Regeneration of organs from callus.
Guha and Maheshwari 1964 Product of first haploid plant in Datura.
Carlson 1972 Interspecific hybridization through protoplast fusion.
Kary Mullis 1983 Polymerase chain reaction (PCR).
Baron et al. 1987 Isolation of Bt gene.
P. Vos. et al. 1995 DNA fingerprinting.
3. Different Methods of Non-Conventional Plant Breeding

3.1 Polyploid Breeding
From different type’s polyploids, euploidy are important in plant
breeding. It could be either autopolyploid or allopolyploids. It can also be
produced artificially by colchicine treatment, heat & cold treatment, X-Rays,
gamma rays etc. Triploids are more useful type which increases vigour, fruit
size and produce seedless fruit e.g. banana, watermelon. It includes
following steps.
 Induction of polyploids.
 Detection of different kinds of polyploids.
 Selection of stable polyploids from variable material.
3.2 Mutation Breeding
 Through mutation breeding, high yielding cultivars are produced in
rice and wheat.
 Semi-dwarf mutant has been produced by X-Ray treatment.
 Thick celled groundnuts are produced which can't break easily.
3.3 Tissue Culture
Organ Method
Seed Culture of seeds In vitro.
Embryo Culture of isolated mature or immature embryos.
Callus Culture of a differentiated tissue from explant allowed to dedifferentiated
In vitro
Cell Culture of isolated cell
Protoplast Culture of plant protoplast
Page | 98
Bud Culture of bud with a piece of stem, with the purpose of forming a shoot
by allowing the bud to develop.
Microspore Culture of immature pollen which is produced by haploid plants through
in vitro of male gametophytic cells.
Anther Culture of anthers to produce haploids.
Anther has advantage over microspore culture being very quick for
practice purpose through which haploids are produced.
Conventional Breeding Haploid Breeding
Parent 1 x Parent 2 Parent 1 x Parent 2
 
F1 F1
 
Haploids
F2 2 crop
7 crops Seasons 
Seasons  Doubled haploid plants (DH1)
F3 
(Near homozygous lines) Doubled haploid lines (DH2)
(Period required to develop homozygous lines through conventional and
haploid breeding)
3.4 Somatic Hybridization
It creates cells with new genetic, nuclear as well as cytoplasmic
constitutions. It involves the fusion of protoplasts, selection of hybrid cells
and identification of hybrid plants.
3.5 Cybrid
Somatic hybrids can be obtained where nucleus is derived from one
parent and cytoplasm is derived from both, thus producing cytoplasmic
hybrid, also called as cybrid.
3.6 Potential of Somatic Hybridisation
It overcomes the sexual incompatibility barriers. Efficient cell fusion
can be achieved between sexually compatible and incompatible parents
involving interspecific or intergeneric combination.
Page | 99
3.7 Molecular Approaches to Crop Improvement
Conventional approaches in plant breeding involve reshuffling of genes
among individual falling within sexually crossable groups. But the new
technology of molecular biology have broadened the scope of gene
manipulation at the level of specific DNA segments across wide range of
organisms to produce noble genome, which is accompanied by following
methods:
3.7.1 Probe
The term probe in molecular biology refers to know small nucleotide
sequence of DNA or RNA, which is used to identify the segments of nucleic
acids with complementary nucleotide sequence.
The probes are being used for variety of purposes including the
construction of molecular maps, identification and isolation of genes or
specific sequences for cloning, confirmation of the integration of gene in
transgenic plants and DNA fingerprinting for identification of varieties.
3.7.2 Gel Electrophoresis
Analysis of nucleic acids is performed by cutting them into small
segments through the action of restriction enzyme the segments with
different placement on the gell are detected by radio labeled probe
hybridization and then exposing to X-Ray film. The molecular size of DNA
fragment can be estimated by comparing the migration of bonds with that of
size of standards separated on the same gel. The ability of complementary
strands of nucleic acids to bind with each other was used by E.M Southern to
assay segments by DNA-DNA hybridization in the form of Southern
Blotting where a sample of DNA fragment is denatured and then blotted
from gel to a nylon filter.
The same concept has been used to explore the sequence of mRNA
which after separation into segment through electrophoresis is blotted into
filter support called as Northern Blots. Similarly proteins can be assayed
where the transfer of protein instead of nucleic acid from a gel to support is
called as Western Blotting.
3.7.3 Cloning Vectors
The characterization, modification or transfer of a gene or any segment
of DNA requires its multiplication in large number i.e. its cloning. The
vector in fact is a DNA molecule that has the ability to replicate in an
appropriate host. The DNA segments to be cloned are added to 'vectors'
called as Chimeric Vectors. Which is incorporated into suitable host like
Page | 100
bacteria where it replicates autonomously into multiple copies. The whole
process of combining DNA fragment with vectors to produce recombinant
DNA and its placement in host cell is termed as Recombinant Gene
Technology.
3.7.4 Plasmids
Plasmids are self-replicating duplex circular DNA molecules which
exist in cell as extra chromosome units. The most of the plasmid vector have
genes containing resistance to antibiotics.
3.7.5 Cosmid
Cosmid vectors are modified plasmid particles that contain specific
segments of bacteriophage including those of the cos region. Cosmids can
accommodate large DNA fragments and have capability of autonomous
replication like plasmids.
3.7.6 Shuttle Vectors
Such vectors are created through recombinant techniques to replicate in
cells of two different species.
3.7.7 Gene Cloning
The process of gene cloning started with the discovery of a specific
enzyme reverse transcriptase in virus. Another approach to gene cloning is
through Polymerase Chain Reaction (PCR) where multiple copies of the
targeted segment of DNA are produced artificially.
3.7.8 Gene Isolation
The particular gene of interest or DNA segment i.e. DNA insert can be
isolated from the genome. The technique depends upon nature of the gene
and information available about the gene and its product. It is essential to
construct library of DNA segments, representing the plant genome either in
the form of genomic library or as cDNA library.
3.7.9 Molecular Markers
Most important use of these markers lies in the indirect manipulation of
desirable genes in the form of marker-assisted selection (MAS).
3.7.9.1 Application of Dna-Markers in the Plant Breeding
The molecular markers are expected to have application in plant
breeding for:
a) Construction of high density (saturated) genetic maps.
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b) Comparative gene mapping indifferent plant species to determine
parallelism in the gene order (synteny) and evolutionary
relationship.
c) Marker aided early generation selection of transgressive segregants
to increase the speed and efficiency of developing new varieties.
d) Introgression of genes from wild-germplasm.
e) Mapping of polygenes.
f) Gene pyramiding.
g) DNA finger printing of crop varieties and genetic collections
through a profile of molecular markers helps to characterize genetic
diversity.
h) Map based cloning of gene.
3.7.9.10 Genetic Transformation and Production of Transgenic
Plants
Transgenic plants are those which carry additional stably integrated and
expressed foreign genes, usually transferred from unrelated organisms. The
whole process of introduction, integration and expression of foreign genes in
the host is called genetic transformation. Infact, transgenesis has emerged as
a novel tool for carrying out single gene breeding or transgenic breeding of
crop plants.
3.7.9.10.1 Gene Transfer Method
Depending upon the mode of transfer of foreign gene to plants two types
of methods are there i.e.
a) Vector mediated and
b) Direct gene transfer method.
The systems available under each of these two categories are:
a) Physico-chemical uptake of DNA.
b) Liposome encapsulation.
c) Electroporation of protoplasts.
d) Micro injection.
e) DNA injection into intact plants.
f) Incubation of seeds with DNA.
g) Pollen tube pathway.
h) Laser microbeam.
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i) Electroporation into tissues/embryos.
j) Silicon carbide fiber.
k) Particle bombardment.
4. Transgenics in Crop Improvement
Different resistant varieties are developed through transgenesis
described below.
4.1 Resistance for Biotic Stress
4.1.1 Insect Resistance
Progress in engineering insect resistance in transgenic plants has
achieved through the use of insect control protein genes of Bacillus
thuringiensis. Insect resistance was first reported in tobacco (Vacck et al.
1987) & tomato (Fischhott et al., 1957).
4.1.1.1 Resistance Genes from Micro-Organisms BT Toxin Gene
Bacillus thuringiensis (BT) is an entomocidal bacterium that produces
an insect control protein. BT genes code for the BT toxin, which differ in
their spectrum of insecticidal activity.
4.1.2 Virus Resistance
The approach is to identify those viral genes or gene products, which
when present at an improper time or in the wrong amount will interference
with the normal functions of the infection process and prevent disease
development.
4.1.3 Disease Resistance
A large number of plant defense response genes encoding anti-microbial
proteins have now been cloned. Most of these are transcriptionally activated
in response to infection or exposer to microbial elicitor macromolecules. The
products of defense response may include.
i) Hydrolytic enzymes e.g. chitinase, 1-3 B-D glucanase and other
pathogenesis related (PR) proteins.
ii) Ribosome inactivating proteins (RIPS).
iii) Antifungal proteins (AFPs).
4.1.4 Herbicide Resistance
To develop herbicide resistance plants three approaches have been
followed.
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a) Over production of an herbicide sensitive bio-chemical target.
b) Structural alternation of bio-chemical target.
c) Detoxification and degradation of herbicide before it reaches the
bio-chemical target.
4.2 Resistance To Abiotic Stress Tolerance
Cloned genes have been transferred to produce transgenic even for
resistance to abiotic stresses. A gene coding for glycenol-3-phosphate
acyltransferase from Arabidopsis has been transferred to tobacco for
enhancing cold tolerance. Recent developments in Quantitative Trait Loci
(QTL) mapping may provide new avenues to identify characterize and
transferred useful DNA segment from wild species.
4.3 Transgenic for Quality
4.3.1 Transgenic For Improved Storage
The food processing can be developed by two approaches using
i) Antisense RNA technology
ii) Using gene for 1-amino cyclopropane-1-carboxylic acid (ACC)
deaminase, which degrades ACC to ethylene.
4.3.2 Longer Life Transgenic Flowers
The post-harvest life of many flowers is determined by the onset of
petal senescence. Petal senescence in carnation exhibit a characteristic “in
rolling” behaviour. This “in rolling” is also observed in response to
exogenously supplied ethylene. Using a cDNA clone for ACC Oxidase
(ACO) produced transgenic carnations expressing antisense ACC oxidase
and producing flowers with little detectable ethylene and a marked delay in
petal senescence.
4.3.3 Transgenics for Flower Colour and Shape
Anthocyanins are the major flower pigments in higher plants.
Delphinidin is the anthocyanin that normally leads to blue pigmentation. A
gene from Petunia hybrida encoding a flavonoid 3`5`-hydroxylase (F 3`5`H)
which is required for biosynthesis of delphinidin which restores the blue
pigment.
4.3.4 Transgenics for Male Sterility
Male sterility in plants is inherited both in a nuclear and cytoplasmic
manner. Cytoplasmic male sterility (CMS) is due to defects in the
mitochondrial genome. Generally CMS is associated with detective
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functioning of the tapetum of the anthers that supply nutrients to the
developing pollen grains. The female component of these plants maintains
fertility. Transgenic with male sterility and fertility restoration are produce in
Brassica Napus: In tobacco also male sterility was introduce using
mitochondrial-mutated gene. This was achieved by introducing a
ribonuclease coding sequence and a tapetum specific promoter was
constructed.
4.4 Transgenics for Terminator Seed
The technology that terminates the viability/fertility of seeds after a
given time is known as terminator technology and the gene involved is
popularly known as terminator gene. The terminator technology employs
three stretches of DNA (genes) which carry the necessary genetic
information into the plant as terminator (lethal) gene, recombination gene
and repressible.
5. Future Outlook
Commercial potential of non-conventional plant breeding is immense
and covers the entire spectrum of human life by drawing the capabilities and
resourcefulness of the entire biological world and even beyond modification
of existing gene like crystal protein genes of Bacillus thuringiensis. Non-
conventional plant breeding enhance the process of Green Revolution to
Gene Revolution as its technology of 21st century.
References
1. Chahal GS, Gosal SS. Principle and procedure of plant breeding, 2003.
2. Chawala HS. Introduction to plant Biotechnology, 2002.
3. Chopra VL. Plant breeding theory and practice, 2000.
4. Sharma JR. Principles and practice of plant breeding, 1994.
5. Sneep J et al. Plant breeding perspectives, 1987.
Page | 105
Page | 106
Chapter - 7
Flower Formation, Photoperiodism and Florigen
Concept in Horticultural Crops
Authors
Dr. Sable P.A., Ph.D. Hort. (Fruit Sci.), NET-Fruit Sci., NET-
Veg. Sci., Assistant Professor (Horticulture), Sardarkrushinagar
Dantiwada, Agricultural University, Sardarkrushinagar,
Banaskantha, Gujarat, India
Sushma Sable (Sonpure)
Sr. Ph.D. Scholar (Agronomy), Mahatma Phule Krushi
Vidyapeeth, Rahuri, Maharashtra, India
R.R. Lipane
M.Sc. Agri. (Agril. Botany), NET-Agril. Botany, Assistant
Professor, Department of Agril. Botany, College of
Agriculture, Sonai, Maharashtra, India
Page | 107
Page | 108
Chapter - 7
Flower Formation, Photoperiodism and Florigen Concept in
Horticultural Crops
Dr. Sable P.A., Sushma Sable (Sonpure) and R.R. Lipane
Flowering Biology of Horticultural Crops

Flowering is a cascade reaction consisting of several steps. Flower
formation is transition phase in the life cycle of a plant. Flower initiation is
takes place by transformation of vegetative apex into reproductive structure.
The shoot meristem is reduced to develop sepals, petals, stamens, carpels
instead of leaves. The plant must attain specific stage of ripeness to response
before it flowers. Once the stage is reached, then it can induce flower.
The physiology of flowering involves the changes in the characters of
cells proliferating in the meristematic tissues of the shoot owing to the
specific gene action and change in the phyto- hormones level.
The flowering is closed linked to the diverse environmental conditions
in which each species has evolved. The effect of large of factors that
influence the proportion of buds giving rise to flower have generally been
interpreted in terms of an inbuilt propensity to flowering and interference
with attainment of this. The controlling factor may involves hormonal
balance availability of nutrient, especially carbohydrates and interaction of
these. (Fruit tree physiology by W.S. Dhillon and Z.A. Bhat).
Vegetative (Juvenile) Transition Reproductive Plant maturation (Adult)
Flowering Physiology
The plant comes to flowering after a certain period of vegetative growth.
The process until plants comes to flowering is classified into three stages viz.
differentiation of flower bud, development of flower buds and opening of
flower.
The differentiation of flower buds is the most important turning point in
this process and the plants life cycle switches from vegetative to
reproductive growth. The switching from vegetative to reproductive growth
is called as flowering or transition phase.
Flowering occurs when the flowering genes are activated. The activation
of the flowering genes occurs automatically or is triggered by environmental
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factors. Autonomous flowering occurs when plant reached to a certain age.
The environmental factors triggering the activation of flowering genes are
photoperiod, low temperature and various stresses. Photoperiodic flowering
regulated by photoperiod and vernalization regulated by low temperature
have been well studied. (Fruit tree physiology by W.S. Dhillon and Z.A.
Bhat).
Physiology of Flowering in Horticultural Crops
It can be studied under following heads
i. Events in the Bud Leading to Flowering
Three events were proposed to occur during the transition of bud to
flowering i.e. induction, evocation and initiation.
Induction
The step of flowering at which the flowering stimulus is generated is
called as initiation. Searle (1965) defined induction and induced stage as a
physiological condition initiated in tissues by external influences such as
water stress, chilling temperature or photoperiod, progressively towards
floral expression even under subsequent non initiating conditions. This event
is translated to production of a putative floral stimulus or alternative in the
ratio of florigenic and antiflorigenic components which may be translocated
to target cell in meristem.
Evocation
The step at which the shoot apical meristem has received the flowering
stimulus, and is irreversibly committed to form flower bud primordia is
called floral evocation, so these are those processes occurring in the apex
essential for the formation of lower primordia (Metzger, 1987).
Immunological on electrophonic analysis of evoked apices of photoperiodic
plants demonstrated alteration of protein components after induction and
prior to initiation of flower primordia. Floral evocation involves two
development stages, competent and determined. In general terms, a cell or
group of cells is said to be competent if it can response in the expected
manner when given the appropriate developmental signal e.g. if a vegetative
shoot (scion) is grafted on a flowering root stock and the scion flowers
immediately. It is obviously capable of responding to the level of floral
stimulus present in the stock and is therefore competent. Failure of the scion
to flower would indicate that shoot apical meristem has not yet attained
competence. This is another way of stating that the apical meristem is in the
Juvenile phase.
Flowering involves several processes that occur in different parts of the
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plant. In photoperiodic plants, the inductive photoperiod causes the leaves to
export a floral stimulus to the rest of the plant that alters the developmental
fate of the cells of the shoot apex. Once the meristem becomes committed to
the new development programme (flowering), it is said to be florally
determined. The meristem is determined if it fallows the same
developmental programme even after being removed from its normal
physical and environmental context.
Initiation
In this process the evoked bud becomes recognizable as a flower bud
and is thus committed to reproductive development. This evident the
broadening and flattening of the growing point with concurrently developing
lobes.
ii. Differentiation of Growing Points
The transition from vegetative to reproductive development is marked
by an increase in the frequency of cell division within the central zone of
shoot apical meristems. This leads to increase in the size of meristem and the
shoot meristem in induced to develop sepals, petal, stamens and carpels in
place of leaves. The first detectable change in the growing points of the bud
after induction of flowering is increasing synthesis of DNA and RNA (Faust,
1989). The first visible sign of differentiation is when the flat apical
meristem becomes domed, then central meristem is partitioned and the pitch
meristem develops. The meristem becomes a block-like structure and its
subsequent development is relatively rapid. The stage of flower bud
development at any given time during summer and autumn varies with type
of bud (basal, middle, upper or terminal shoot buds) and with species and
cultivars (Crabbe, 1984).
In temperate fruit, normally 8 to 14 days are required from the
beginning of histological differentiation to the appearance of lower
meristem. The subsequent development of the flower meristem is relatively
rapid. The entire process of flowers and differentiation may take 54 to 112
days depending on the species.
Flowering view
Source: personal collection
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Changes Occurring in Target Tissues
Biochemical Changes
Development of chloroplast inhibitors.
Moving out of floral stimulus from leaves.
Increase in total RNA synthesis and chromatins dependent RNA
synthesis.
DNA synthesis increases.
Mitotic activities increases.
Number of mitochondria increases.
Starch content decreases.
Metabolic Changes
Meristem height increases
Cell size increases
Lengthening of mitotic phase
Entry of cells from photosynthetic to mitotic phase
Flower bud initiation
De-novo synthesis of DNA and rise in mitotic activity leads to
production of cells which are responsible for formation of flower bud
primordia. Morphogenesis occurs when the flower bud primordia develop
also called morphogenic phase.
Mechanisms of Flowering
Various theories have been proposed to explain the mystery of flowering
Phytochrome Theory
Bunnings Hypothesis
Chalakiyan Hypothesis
Phytochrome Theory
Phytochrome is a non-photosynthetic photo-receptor pigments that
exists in to two forms, Pr and Pfr. The former absorbs light of wavelength
P660 nm and latter of P730. The two forms are inter convertible.
It is believe that phytochromes control flowering in plants. P730
suppresses flowering in short day plants and promotes flowering in long day
plants. On other hand P660 promotes flowering in short day plants and
inhibits in long day plants. Sun light functions as red light. At the end of
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light i.e. evening after sun set Pfr form is pre-dominates while at the end of
dark period i.e. before sunrise Pr form is pre-dominates.
During long night Pfr form is converted to Pr form. If long dark period
is interrupted with red light, sufficient amounts of Pr form is not formed
hence flowering is inhibited in short plants. Flowering in short day plants
depends upon Pr/Pfr inhibits flooring in short day plants and promotes
flowering in long day plants. Pr/Pfr ratio favours flower formation in short
day plants and inhibit in long day plants (Takimoto and Saji, 1984).
Inter convertible form of phytochrome

Source: Ph.D. seminar by Sable. P. A.
Fig 1: Structure of the Pr and Pfr forms of the chromophore (phytochromobilin) and
the peptide region bound to the chromophore through a thioether linkage. The
chromophore undergoes a cis-trans isomerization at carbon 15 in response to red and
far-red light
Bunnings Hypothesis
Bunning (1958) assumes the presence of endogenous rhythms
(oscillator) which consist of two half cycle. The first half cycle occurs in day
called photophilous phase. During this, anabolic process predominates
including flowering in plants. The other half cycle is dark, sensitive and
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called as skotophilous phase. In this, catabolic process (dehydration of
starch) predominates.
Short day plants have a critical day length of 9 hours. This period falls
within the photophilous phase. Light during skotophilous phase will inhibit
photo process initiated during photophase. The long day plants have a
critical day length of 15 hours and some light fall in the skotophilous phase.
Under these condition long day plants will flower. In short day plants
oscillator is present close to skotophilous phase, while in long day plants, it
is close to photophilous phase.
Chailakiyans Hypothesis
This hypothesis assumes that flowering hormone-florigen is a complex
of two type of substances gibberellin (plant growth) and anthesis (flower
formation).
According to Chailakiyans (1937), flowering in all annual seed plants
requires two phases (i) floral stem formation (ii) flower formation phase.
Floral stem formation involves increased carbohydrates metabolism and
respiration with increased content of GA in leaves favour long day condition.
Flower formation phase requires intensive nitrogen metabolism, higher
content of anthesis in leaves and nucleic acid metabolites in stem buds. Long
day conditions favours the first phase while, short day condition second
phase. In long day plants gibberellins are critical while, anthesis is critical in
short day plants. However, anthesin is hypothetical; it has not been isolated
yet.
Chailakiyans (1937) reported that vernalin is plants at low temperature.
In long day conditions it is converted into gibberellin. Anthesin is present in
long day plants. Anthesin along with vernalin cause flowering in long day
plants. But in short day conditions, the vernalin is converted to gibberellin,
hence flowering does not occur. Addition of gibberellin to long day
unvernalised plants in long day conditions leads to flower formation as these
plants contain anthesin. Gibberellin is ineffective in producing flowers in
short day plants as they lack anthesin.
LDP Vernalin GA Flowering
Vernalin
SDP No conversion of vernalin to GA No flowering
As postulated by Purvis (1961) the formation of substance A from its
precursor. A is then converted to B after chilling. The substance B is
unstable. At suitable temperature, B is converted to D called Vernalin. At
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high temperature, B is converted to C i.e. devernalisation occurs. At correct
photoperiod, vernalin is converted to substance F (florigen) a flowering
hormone. Florigen induces flower formation in proper photoperiod.
Chilling Low temp.
A B D F flowering
Precursor (unstable product) Vernalin Florigen
Devernalisation (high temp)
C (Degradation product of B)
Photoperiodism
Relative length of daily light and dark periods helps in transforming a
vegetative shoot meristem to floral bud. The induction of flowering in
response to the relative length of daily light and dark period is called as
photoperiodism. It simply means the duration of light. It is a physiological
response of plants in relation to duration of light. There are various
photoperiodic responses in plants; however the most important is the
initiation of flowering. The photoperiodic signal is first received by
phytochrome in the leaf, and the signal from phytochrome starts the
biological clock. After the biological clock measures a certain period of time
(inductive photoperiod), the production of the flowering stimulus in the leaf
starts. The floering stimulus is transmitted from leaf to shoot apex leading to
change in the growth mode of meristem from vegetative to reproductive. The
shoot apical meristem produces primordia of floral organ viz. sepals, petals,
stamens and carpels, thus generating a flower bud.
Based on photoperiodic responses plants may be classified into three
groups.
Short Day Plants (SDP)
The plant can only flower under day length shorter than its critical day
length. The critical day length is 11 to 15 hours they usually flower in
autumn or early spring e.g. pineapple, strawberry etc.
Long Day Plants (LDP)
The plant can only flower under day length longer than its critical day
length (12-14 hrs.). Bellow critical period, these plants continue their
vegetative growth. Most of the temperate zone fruit are long day plants.
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Day Neutral Plants (DNP)
These plants flower after a period of vegetative growth regardless of
photoperiod. Flowering is not affected by day length and it occurs any time
during the year e.g. guava, and citrus.
In the short day plants, when the long night period is interrupted by a
brief exposure to light, the plants fail to flower. Similarly, long day plants
response to nights shorter than the critical dark period.
Photoperiodic Induction
It is define as the initiation of flowering in a plant after exposure to light
for a required period of time. This is calculated for a 24 hours and the time
of specific exposure to light within a day is called photo-inductive cycle. The
number of these cycles may be one, two or many depending on a particular
plant species and together they called photoinduction and shown as bellow.
Photoperiod induction and flowering

White portion (hours)-light, black portion (hours)-darkness.
LDP- long day plant, SDP- short day plant, NF- no flowering and F-
flowering
Source: Ph.D. seminar by Sable. P. A.
Site of Photo-Induction Perception

Chailakiyans (1937) have demonstrated that, the leaves are the site of
photo-induction perception. E.g. no flowering is produced in defoliated
plants even after exposure to correct photoperiod.
Transmission of Stimulus
The stimulus is transmitted from leaves to the target region (in the axis
or at the branch apices or flowering regions) through phloem at 10-24 mm/
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hr. photosynthates, however, travel at 1000 mm. /hr. For LDP the rate of
transmission is 30 cm. hr. and SDP is 2.4 mm. /hr., however, it may vary
from time to time. (Fruit tree physiology by W.S. Dhillon and Z.A. Bhat).
Nature of Stimulus
Stimulus is a hormone and named it florigen, based on flowering
evidences transmission occurs through phloem. Transmission of stimulus
from photo-induced branch to a non-photo-induced branch through grafting.
Initiation of flowering in a non-photo-induced plant by leaf extracts of a
photo-induced plant e.g. xanthium.
However, others like Sachs and Hackett (1983) opposed the florigen
concept and believed that diversion of nutrient through photo induction
towards meristem leads to the floral initiation.
Photoperiodic induction for long day plant needs 12-14hrs. No Flowering in 8-

11hrs.
Source: Ph.D. seminar by Sable. P.A.
Transmission of stimulus from photo-induced branch to a non-photo-induced

branch through grafting. Several plant flowers by grafting, when only one leaf is
induced in SD (8-13hrs.)
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Photoperiod-induced leaf promotes flower by grafting (left) and the non-induced
leaf promote vegetation
Successful transfer of the floral stimulus between the scions (right branch) is the
LDP (Petunia hybrida) and the stock is non vernalization henbane under LDs.
Photoperiodism and Flowering in Fruit Crops
In mango, flower induction is caused by cool temperature and not by
short photoperiods and that flowering is inhibited by warm temperature not
by long photoperiods. Variety like Neelum flowers twice in Kanyakumari
but only once under north Indian conditions and the common observation
that the eastern side of mango tree, which receives longer hours of sunlight
flowers a few days earlier than others, indicate the effect of light on
reproductive process of mango.
Early flowering to eastern side of mango tree

Source: Photo by Dr. Sable. P. A. from Horti. Farm KVK, S.K. SDAU.
Page | 118
In strawberry, flower formation is influenced by the duration of length
of daily light period. A decreased light period induce flower formation at the
expense of runners, while, the longer tends to induce runner rather than
flower formation. Although short days favour flower formation, regardless
of temperature, yet temperature to a certain extent modifies the day length
response. In general, any decrease in temperature shortens the day length,
which will permit the flower formation. (Fruit tree physiology by W.S.
Dhillon and Z.A. Bhat).
Konsin et al., (2001) found that 12 and 13.5 hours photoperiods were
equally efficient in produced yield in Koroana strawberry, but more vigorous
vegetative growth is maintained during a 13.5 hours.
Teaotia and Singh (1967) studied that the effect of seasonal variation on
sex expression of papaya cv. Coorg Honey Dew and reported that the
production of female flower was promoted by long day and high
temperature. Similarly, Storey (1986) observed that long day treatment i.e.
photoperiod cycle of 16 hours light and 8 hours darkness favored femaleness
in Co.1.
In pineapple, neither a diurnal temperature differential not short day are
necessary for natural flowering. Rather, attainment of some minimum size
and cool night temperature, which regulates the natural flowering to occur.
Pineapple plant generally flowers after attainment of certain vegetative
growth 11-12 months’ after planting and formation of at least 40 leaves.
Pineapples produce only one fruit during life time. There is no involvement
of photoperiod in citrus flowering. Similarly guava is a day neutral pant.
(Fruit tree physiology by W.S. Dhillon and Z.A. Bhat).
In fruit crop like apples, peaches, cherries and plums, floral initiation
appears to be unaffected by photoperiod, which nevertheless affect the
vegetative growth of these species. (Fruit tree physiology by W.S. Dhillon
and Z.A. Bhat).
Florigen Concept
Leaf is the site of photoperiodic perception and shoot apex is the site of
floral bud formation this means that some information must be transmitted
from the leaf to shoot apex which is called as flowering stimulus.
The transmission of information in the plant is generally due to
movement of plant hormone like substances florigen. Florigen is generated
in leaves exposed to a suitable photoperiodic cycle, transmitted through
phloem and activates the flowering genes at the shoot apex and as shown
below.
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Fig 2: Hypothesis regarding photoperiodic induction of short day plant. Synthesis of
florigen in dark and its translocation to the apex for the activation of gene for lower
differentiation. (Chailakhyan, 1937)
Source: Fruit tree physiology by W.S. Dhillon and Z.A. Bhat.
The biochemical nature of florigen is still unknown. Currently, the
existence of florigen can be presumed only by grafting experiments.
Florigen May Not be Universal
The florigen concept is now understood in several ways resulting in
several ways resulting in confusion. It has been misunderstood that florigen
has proposed as a single component that is common in the plant kingdom.
The grafting experiment showed that the flowering stimulus could be
transmitted between different plant species suggesting that florigen was
common among species.
Although, gibberellins can induce flowering of some long day plants
under un-inductive condition but gibberellins are not considered as florigen,
because they do not induce flowering in short day plants. However, there is
no reason to consider those florigens need to be ubiquitous. The florigen of
long day plants could be a gibberellins and the florigen of short day plants
might be a chemical other than a gibberellins. The result of grafting
experiment mentioned above should not be generalized, because grafting is
possible only between taxonomically close relatives. (Fruit tree physiology
by W.S. Dhillon and Z.A. Bhat).
A possible role of GA like hormones in the induction of flowering has
postulated by Brain (1959). According to him, Co2 is converted into a
inactive or less active processor under the influence of red light or infrared
light produces a GA like hormone which may produce florigen. The GA like
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hormone can be converted into its precursor with exposure to far red light.
The same can be achieved by dark treatment but the process is very slow.
This hypothesis of Brain is based on the effect of darkness or red and far red
light and the GA treated long day plants maintained in short day condition as
shown under.

Flowering has been induced by growth regulating chemicals in number
of fruit. It is generally believed that high level of gibberellin inhibits
flowering while high level of auxin like substances may promote flowering
either by reducing the effectiveness of GA or by decreasing the permeability
of cell membrane particularly the plasma membrane. (Fruit tree physiology
by W.S. Dhillon and Z.A. Bhat).
Alternative Ideas on Florigen Concepts
Florigen has not been isolated yet. Some plant biologist said that
florigen has not isolated because it does not exist, and do not assume the
existence of florigen to explain the regulating mechanism of the flowering
process. The alternative ideas include the presence of multiple factors, a
flowering inhibitor and electrical signals.
Multiple Factors
Florigen concept assumes one hormonal compound acting specifically to
induce flowering. However, several compounds which are not specific to
regulation of flowering can act as hormonal regulators. In some plant
species, endogenous levels of cytokine, putrescine, sucrose and calcium ion
increase and they move to the shoot apex under a flower-inducing condition.
These facts suggest that flowering occurs when these compounds work
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together in the shoot apex. Therefore, all these compounds could be factors
necessary for the induction of flowering.
The florigen concept does not necessarily assume a single universal
compound. It does not necessarily assume specific compounds. However, it
could be mixture of two or more compounds, and could be known
compounds with multiple functions as usual plant hormones are. In this
sense, the multifactorial concept may be considered as variation of the
florigen concept. (Fruit tree physiology by W. S. Dhillon and Z.A. Bhat).
Vernalization and Flowering
There are environmental factors regulating flowering otrher than night
length. Temperature is also important. Some plants require exposure of low
temperature, 0 to 10 oC, for a few days to few weeks for flowering. Such an
induction of flowering by a low temperature is called as vernalization. It is
observed in many long day plants, but is not correlated with photoperiodism.
The length of chilling may vary from species to species ranging from four
days to few months and the most effective temperature is found to be 4 oC.
(After seed germination (seedling) is treated with low temperature and sown
in the warm field. The plant cans flowers in summer day, which is called
vernalization). (Fruit tree physiology by W.S. Dhillon and Z.A. Bhat).
Site of Vernalization: Active apical meristem and young leaves.
Stimulus of Vernalization
In annuals, embryo axis is the site of stimulus reception while, in
biennials and perennials it is located in apical meristem of the shoot. In
biennials, vegetative growth takes place during first year and flower in the
following summer after vernalization during winter. However, perennials
need vernalization cycle every winter. Meristem is the site of stimulus and
transmission. It has been postulated that vernalin is transmitted from
vernalized plant to non-vernalized through grafting. (Fruit tree physiology by
W.S. Dhillon and Z.A. Bhat).
Mechanism of Vernalization
Two theories have been postulated to explain the mechanism of
vernalization.
Phasic Development Hypothesis
Hormonal Hypothesis
Phasic development Hypothesis, development of annual seed plants
consists of several physiological stages (phases) which occur in pre-
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determined sequence i.e. when one phase terminates the next begins. The
main stages have been identified as thermostage it is a vegetative phase and
low temperature (0.14 oC), moisture and aeration are necessary for the
completion of this stage wherein, the length depends on plant and
environment. Photostage it requires high temperature and is also called
photoperiodism. Here vernalin helps in the synthesis of florigen. Third stage
is necessary for the formation of sex organ and gametes. (Fruit tree
physiology by W.S. Dhillon and Z.A. Bhat).
Hormonal hypothesis: Same as Chalikhyan hypothesis.
Molecular and Genetic Aspects of Vernalization
Seeds contain specific protein and its associated components which bind
to loci that are involved in flower induction and silence the chromatin by
heterochromatization. Heterochromatization is achieved by histone
methylation and histone deacetylation at specific loci. One such protein is
known as flowering locus C (FLC). FLC acts as a repressor. Cold treatment
in fact induces few FLC antagonizing genes and FLC dissociates from loci
and get degraded or remain free. There are several genes that are involved
VRN1, VRN2, VRN3, Even Frigida (FRI) proteins are involved. Even
Frigida (FRI) promotes FLC and FRI antagonizes FLC. Once the chromatin
is free from FLA and its associated protein, they interact with floral
integrators such as SCO, CO, FT and LFY, which in turn activate genes for
floral parts. In certain cases GA can overcome vernalization. (Fruit tree
Translocation of Flowering Stimulus
Flowering stimulus from leaf to apical apices in translocated through
phloem cell. Effect of photoperiodic stimulus induction may be localized or
generalized depending upon nature of plants. Transmission of stimulus also
occurs one plant to another through grafting shown as bellow in flow chart.
Light darkness
Leaves cotyledons temperature
Physiologically active form (PFR) established
Metabolic processes
Changes in levels of growth hormones, metabolites cofactors
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Synthesis of florigen
Transport of florigen to shoot apex
Vernalization
Shoot apex, initiation of flower primordia
(Metabolic activity of shoot apex depend on light and darkness)
Bud development and flowering
Source: Fruit tree physiology by W.S. Dhillon and Z.A. Bhat.
Factors for Induction of Flowering in Fruit Crops
Major factors regulating induction of flowering could be broadly
classified into fallowing groups.
Nutritional Factors
Plant Growth Substances
Other Chemicals
Cultural Practices
Root Stock
Crop Load
Nutritional Factors
According to Kraus and Kraybill (1918), the concentration of sugars
should be greater than that of nitrogenous compounds for flowering.
Limiting factor for flower formation in the off year of alternate bearing
cultivars of Mandarin has been attributed to levels of stored, non-structural
CHO in the form of starch. Reduced flower production occurs when nitrogen
fertilizers application prolongs the period of extension shoot growth and
delays terminal bud formation.
Ringing of the branches in late summer of early winter in mango trees
can induce flowering in off years and increases flowering in on years, it
gives supporting evidence to the fact that nitrogen and CHO reserves play an
important role in floral initiation in mango by increasing C/N ratio of shoots.
(Fruit tree physiology by W.S. Dhillon and Z.A. Bhat).
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Potassium has a positive effect on flowering as it enhance amino acid
formation, which stimulates the formation of IAA oxidase, thereby flower
induction as well as CHO levels. K might be effective through pyruvate
Chinese, which in turn would determine the levels of several amino acids.
However, C /N ratio theory is inadequate to explain floral bud
differentiation in fruit trees. The wood of perennial tree has rich in CHO and
they also stored large quantities of amino acids and amide nitrogen. It was
difficult to conceive that either of these could ever be inefficient for floral
initiation, unless the trees were nitrogen deficient. Normal flower
development requires adequate mineral elements in the proper balance.
Deficiency of nutrients like Zn, Cu, B is determined to flowering e.g. boron
deficiency in pear during anthesis causes withering of flowers. 1-2 percent
NH4NO3 or 2-4 percent KNO3 have been found to be effective for initiation
floral bud in mango. Maximum flowering is obtained by spraying of urea
(2.5%) and ethrel (200 ppm) in guava. (Fruit tree physiology by W.S. Dhillon
and Z.A. Bhat).
The flower as well as the fruit drop in mango is primarily due to the
formation of an abscission layer at the point of attachment of fruit with the
twig. Several factors have been considered responsible for formation of
abscission layer. It has been observed that Nitrogen retards and reduces
abscission. Low concentrations have also been found to lead leaf, flower,
and bud and fruit abscission. Fruit drop has been attributed to many other
causes e.g. abortion of embryo, degeneration of ovules, poor soil, inadequate
irrigation water, attack of insect-pest, diseases, depletion of nutrients,
hormonal imbalance, etc. Spraying of 50 ppm (50 mg./liter of water)
gibberellic acid, immediately at flowering is recommended to minimize
flower and fruit drop (Sable, et al., 2018)
In mango, fruit drop may be controlled to some extent by the spray of 1
percent (10g./liter water) Potassium nitrate with 20 ppm (20 mg./liter of
water) NAA at 15 days interval during flowering. Spraying of 2 percents (20
g./liter of water) Urea is recommended at full bloom stage correct nutritional
deficiency (Article by Sable et al., 2018).
Inadequate water supplies during fruit development have been
considered responsible for fruit drop. The farmers are advised to irrigate
plants as fruit becomes of ‘Pea’ size at 10 days interval. Flowering in Mango
continues in two or three distinct flushes for a period of 6 to 8 weeks with
ber or lime size fruit development of on different branches of trees. Spraying
of 2 percents (20g. /liter of water) 12:61:00 water soluble fertilizer is
recommended to avoid fruit drop (Article by Sable et al., 2018).
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Plant Growth Substances
Gibberellins
GA is primary inhibiters of floral initiation. A specific stage must be
attained for a bud to be induced and begin to initiate flowering. Initiation at
the apex begins only after a critical node number is reached. This number is
20 for apple cv. Cox’s orange pippin and 16 for golden delicious. The time
interval between successive nodes is termed as plastochron. The plastochron
for Cox is one week. GA extends the plastochron and the bud never reaches
the receptive stage, thereby indirectly inhibiting flowering. Moreover, GA
makes tree over vegetative and flowering may be less than desired. GA also
influences sex expression in papaya. Application of GA either once or twice
before flowering @ 50, 100 mg./liter for two consecutive years in mango
revealed that treated tree had two flowering dates (Jan.-Mar. and Apr.-May)
and two harvesting dates (June and July) while control trees showed only
one flowering date (January to March) and one harvesting date (June).
Treated trees about 16 percents while untreated trees could not produce
mixed panicles. GA delayed flowering and harvesting dates by 90 and 42
days, respectively. However, fruit yield was not affected by GA3
applications. Application of GA on citrus consistently reduced flower
formation, but had a variable effect on the amount of first grade fruit in the
early harvest of Clausellina Satsuma (C. unshiu).
Application of GA during flower bud induction significantly reduced
flowering in peaches and nectarine. Flowering was higher sensitive to GA in
comparison with nectarine. Flowering was reduced by 50 percents in both
which reduces cost of thinning by 50 percents without affection yield when
GA3 had applied at 0.5-1 mg. /tree (Fruit tree physiology by W.S. Dhillon
and Z.A. Bhat).
Spraying of 50 ppm (50 mg./liter of water) gibberellic acid, immediately
at flowering is recommended to minimize flower and fruit drop.
Auxins
Auxins is mainly used for floral induction in pineapple. The induction of
flowering in pineapple with auxin has first reported as early. Among the
Auxins NAA and NAA based compounds like planofix have been reported
to effective in induction of flowering @ 10-20 ppm. Auxins stimulate
ethylene biogenesis to induce flowering in pineapple. Increased production
of flowers has obtained by increase concentration of NAA from 0.25 to 0.50
gram per liter and 0.50 to 0.75 gram per liter. However, increased B-
hydroxyethyl hydrazine from 0.50 to 0.75 gram per liter lead to 51.1 percent
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drop in flower production and 34.1 per cent drop in fruit production.
Uniform fruit of a selected size can be produced by applying the auxin to
plans having appropriate number of leaves. Flowering in pineapple can be
increased by pouting NAA @ 10 ppm (1 g. /1000 l. water) + urea 2% (20 g.
/liter water) @ 50 ml per plant into the crown. (Fruit tree physiology by W.S.
Dhillon and Z.A. Bhat).
Increased ratio of perfect to staminate flowers has been obtained in
mango with the application of NAA @ 100-200 ppm (1-2 g. NAA/ 10 L.
water) about a week following auxin treatment. Fruit drop may be controlled
to some extent by the spray of 1 percent (10 g./liter water) potassium nitrate
with 20 ppm (20 mg./liter of water) NAA at 15 days interval during
flowering. Spraying of 2 percents (20 g./liter of water) Urea is recommended
at full bloom stage correct nutritional deficiency (Sable et al., 2018).
Cytokinins
The precocious bud break and flowering in mango shots in response to
an early October application of 100 mg or 6- BA has described. It also
resulted in full flowering in one month after application as compared with 3
months later on non-fruited trees. Highest level of putative Tran’s zeatin and
its ribosides has measured during the early flowering and full bloom stage
while, the lowest level has translocated from roots during the vegetative
growth and resting stage.
The evolved cytokinins level found prior to and during flowering and
the flowering response to applied BA leads to the conclusion that cytokinins
are involved in flowering in mango. (Fruit tree physiology by W.S. Dhillon
and Z.A. Bhat).
Application of 6 BA 10 ppm + Uracil 50 ppm after 40-50 days of April
pruning in grape recorded higher fruitful cane percentage in both the
seasons. Fruit bud formation is the consequence of transformation of
vegetative primordium into reproductive primordium in a bud. Applications
of 6 BA to has found to prevent the formation of tendrils primordial and
favour the formation of inflorescence and also recorded higher fruitful canes.
(Sable et al., 2017).
Ethylene
Flowering is induced during off year in mango cv. Alphonso by
spraying ethrel @ 200 ppm. Ethephon @ 0.25 ml/L accelerates the rate of
flowering on both ringed and non-ringed mango trees in cv. Haden. Ethylene
is commercially used as a flowering inducing agents in pineapple cultivation
and acts as a florigen of the Annonaceae.
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More than 90 percents flowering is induced in pineapple by ethrel or
ethephon @ 25 ppm in combination with urea 2 percents and Ca CO3
(0.04%) after 50 days of application. The flower and number of fruit is
increased by spraying ethrel @ 1ml per liter due to greater ethylene forming
activity in leaves leading to maximum number of flowers and fruit in guava.
Other Chemicals
Plant growth regulators like Alar, CCC, and triazoles like paclobutrazol,
uniconazole are used to induce flowering in fruit crops. Cycocel @ 1000
ppm (1 g./l) and SADH @ 500-1000 ppm (0.5-1 g./l) favours floral initiation
in pear and apple, respectively. CCC and TIBA advanced flowering by 9 to 4
days, respectively in Co. 2 papaya.
Among triazoles, paclobutrazol is commonly used to stimulate
flowering. PBZ @ 2.5 to 15 g.a.i./tree significantly increases the percentage
of reproductive shoots in mango and also causes extension of the flowering
period. PBZ @ 10 g. per tree had a dual effect i.e. reduction of tree size and
induction of early flowering and cauliflory. It is suggested that under rain fed
conditions, a single application of 10 g. PBZ/ tree should be followed by
further application every two years. Foliar spray of 100 ppm TIBA or 2000
ppm PBZ or soil application of 10 g. PBZ in mango promoted flowering
directly on fruited shoots in the ‘off’ year by passing the need for vegetative
phase. Soil application of PBZ @ 10 G.A.I. per tree significantly increased
the ratio of perfect to staminate flower in mango. Biennial bearing in apple,
pear and cherry can be overcome by compounds like SADH and TIBA
through maintaining the hormonal balance rather than C: N ratio.
Application of CCC @ 500 ppm resulted in earliest flowering and maximum
number of flowers in guava. PBZ @ 2 g. per plant increased flower bud
number in mango. (Fruit tree physiology by W.S. Dhillon and Z.A. Bhat).
Cultural Practices
Several cultural practices like spreading of branches, trunk or branch
girdling, defoliation and Deblossoming, chemical treatments and pruning are
used to provide vigour control and initiate blossom bud formation in fruit
crops.
Spreading, Orientation, Bending and Shoot Type
Spreading of branches in apple towards more horizontal positions
reduces growth and promotes flower bud formation. The major factor
controlling flower initiation on long shoot of pear appeared to pattern of
growth. The number of fruit buds which developed per shoot is inversely
correlated with the relative growth rate of shoot before harvest.
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Training, bending or placing apple shoots horizontally checks their
growth and increases the proportion of bud that become floral. Shoot
bending led to an increase in the sorbitol, amino acid, IAA and cytokine
content of lateral buds and a decrease in these in shoot tips. Bending is
accompanied by a decrease in the gibberellins content of both shoot tips and
axillary buds. The shoot that placed horizontal after cessation of their growth
produced more flower than when left vertical. The bending of branches of
young trees at an angle 45o from main trunk may induce flower earlier.
Orientation of apple of 45o from soil surface reduces vegetative growth, tree
canopy volume and promotes spur formation. The physiology behind it
restricts CHO movement and auxin from the upper portion of the limb
towards the roots. An accumulation of CHOs and slowing down of the
growth beyond the bend is, therefore, assumed to be favorable to flower bud
formation. (Fruit tree physiology by W.S. Dhillon and Z.A. Bhat).
Girdling
Trunk girdling has been used in mango to promote flowering. The actual
case of flowering by girdling cannot solely be attributed to CHO
accumulation but ethylene produced may also play an important role as it
produced under stress condition. It has been found to increase flowering in
‘off’ year of alternate bearing cultivars, although it either has no effect or is
marginally beneficial in the ‘on’ year. Flower initiation in apple is also
achieved through girdling or scoring of trunk. Girdling increased the bud
sprouting, flower formation and reduced the number of vegetative shoots in
the early and late harvested trees in Satsuma mandarin. Tree response is
depends on width of the girdle. Narrow cut result in either short term or n
response, whereas, too wide girdles can kill trees through root starvation. It
is observed that in apple ringing and scoring (making two or more parallel
cut to sapwood around limb without removing the bark) can make the tree
more susceptible to winter injury during the following winter. Girdling in the
main branches in litchi induced higher flowering, without any effect on
flower panicle characteristics and there has no differences in the fruit set, but
with an increase in the flowering, the production increased. (Fruit tree
Defoliation
Defoliation alters the cytokinin to auxin ratio in the buds and stimulates
flowering. Because apical leaves are the primary source of auxin, their
removal results in initiation of new shoots, perhaps due to increase in
cytokinin to auxin ratio. Growth of dormant auxiliary buds is stimulated by
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de-blossoming and produce inflorescence if initiation occurs under
conditions conductive to floral morphogenesis. When leaves in apple are
stripped one month after harvest, permits the tree to initiate flowers before
the next cycle begins. Defoliation experiments have shown that leave
promote flower bud formation. No flower form on apple spurs that had been
defoliated within 6-10 weeks after full bloom but the number of flower buds
formed on spurs defoliated after this period gradually decreased. The effect
of leaves has been attributed to their effects on CHO supply and on the flow
of cytokinins. (Fruit tree physiology by W.S. Dhillon and Z.A. Bhat).
Use of Chemicals
Flower induction has been achieved by several chemicals like dinitro
compounds, KNO3, thiourea, cyanamides and mixture of cytokinin and
gibberellins in fruit tree. Application of KNO3 @ 5-7 percents prior to oil
and DNOC spray improved flower bud development and reduced the number
of abnormal flowers in certain peach cultivars. HCN is found to be useful for
enhancing bloom and synchronizing time of bloom for varieties and their
pollinizers in several temperate fruit. Promalin, a combination of BA and
GA 4+7 is used to cause lateral bud break in sweet cherry, a species with
strong apical dominance.
Imitation of flower buds in mango has been achieved by spraying KNO3
@ 2-4 percents or NH4NO3 @ 1-2 percent. CaNO3 resulted in advanced
flowering in mango cv. Irwin by one month. Plants treated with 0.4 percent
Zn in combination with Fe and B @ 0.1 percent produce more flowers in
mango cv. Fazli. (Fruit tree physiology by W.S. Dhillon and Z.A. Bhat).
Pruning and Pinching
Pruning has been found to be beneficial to promote flowering in many
fruit crops. Increasing severity of pruning usually decreases flowering and
cropping and heading back methods of pruning may convert potential
fruiting spurs into shoots. Inadequate pruning may, however, result in
excessive vegetative growth within three shades and inhibit flowering.
Pruning had no significant effect on time of floral bud formation, but it
enhanced the leaf ethylene level and percentage at flowering in CVS.
Mulgoa, Neelam and Bangalore. Pruning to a length of 30 cm resulted in the
greatest flowering in mango. One leaf pair pruning in guava cv. Sardar has
been found to be superior to initiate flower bud formation. In ber, blossom
initiation can be regulated by time of pruning. Flowering is advanced by
about 3 months i.e. during summer by pruning during spring instead of at the
normal time i.e. 2-3 months later.
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Pinching out of the shoot tip in apple at right time help in development
off bud reported. In Japanese pear, most flower buds are produced on young
wood, especially in cultivars which commonly produce flowers from lateral
buds, and pruning systems are designed to maximize the proportion. (Fruit
tree physiology by W.S. Dhillon and Z.A. Bhat).
Root Stock
Flowering is also influenced by the choice of root stock. Young trees on
selected clonal/dwarfing root stocks often flower earlier than those on
seedling root stocks e.g. apple trees on vigorous M1 root stock flower earlier
and heavier than trees on vigorous seedling root stock. It has been postulated
that root stocks influence flowering through their ability to reduce scion
chilling requirements as observed in pear and apple. When pear cv. Bartlett
grafted on low chilling Pyrus calleryana root stock is required only 850 hrs.
of chilling as compared when grafted on P. communis which required 1500
hrs. Pyrus calleryana have been found to show a much higher rate of
development during autumn, when temperatures are declining, than buds of
P. communis. This is attributed to the relatively higher rate of alternate
pathway respiration under low temperature conditions in Pyrus calleryana
which require more energy for growth and development. Apple cv. Rome
Beauty required less chilling and flowered earlier on M26 rootstock as
compared to MM104 or MM106, both of which are derived from then long
chilling Northern spy apple. Apple root stocks have a major effect on the
proportion of buds that become floral on M9. 48, 38, 28 and 32 percents of
all Cox buds to be floral on M9, M26, M7 and MM106 respectively in mature
apple trees. (Fruit tree physiology by W.S. Dhillon and Z.A. Bhat).
Crop Load
Heavy cropping in one year can inhibit flower bud initiation and so
reduces flowering in following year. Some cultivars consequently tend to
become biennial in cropping. In the year of a heavy crop, the demand on
CHO supply is such that few flower buds are formed for next year in case of
mango and apple and hence leading to an off year. Cultivars effects on the
pattern of flower and fruit development may be related to differences in the
pattern of floral morphogenesis within developing floral buds in previous
season in apple. (Fruit tree physiology by W.S. Dhillon and Z.A. Bhat).
References
1. Brain. The role of gibberellin like hormones in regulation of plant
growth and flowering. Nature. 1959; 181:1122.
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2. Bunning E. Die physiologische. Uhr I Aufl. Berlin: Springer, 1958.
3. Chailakhyan MC. Concerning the hormonal nature of plant development
processes. Comptes Rendus del’ Academic des Sci. del’ USSR. 1937;
16:227-230.
4. Crabbe JJ. Vegetative vigour control over location and fate of flower
buds in fruit trees. Acta Horti. 1984; 149:55-63.
5. Faust M. Physiology of temperate zone fruit tre. New York John Wiley
and Sons, 1989.
6. Konsin M, V Opio I, Palonen P. Influence of photoperiod and duration
of short day treatment on vegetative growth and floering of strawberry. J
Hort. Sci. Biotech. 2001; 76:77-82.
7. Kraus EJ, Kraybill HR. Vegetation and reproduction with reference
Oregon Agril. Exp. Station No. 146, 1998.
8. Metzger JD. Hormone and reproductive development. Plant hormone
and their role in plant growth and development. The Hague, 1987, 442-
441.
9. Purvis ON. The physiological analysis of vernalization. Springer Verlag,
Berlin, 1961, 76-122.
10. Sable PA, Sheikh AS, Sushma Sonpure. Enhanced mango production.
Krishijagram, 06, December, 2018.
11. Sach RM, Hackett WP. Source sink relationship and flowering in
Beltsville. Symposia in Agricultural research. Strategies of plant
reproduction, 1983, 263-272.
12. Searle NE. Physiology of flowering. Ann. Rev. Plt. Physiol. 1965;
16:97-118.
13. Storey WB. Carica Papay. CRC, Handbook of flowering (12). CRC,
Press, Inc. Boca Raton, Florida, 1986.
14. Takimoto A, Saji H. A. role of phytochrome in photoperiodic induction,
two phytochrome theory. Physiology plantarum. 1984; 61(4):675-82.
15. Teaotia SS, Singh RN. Effect of seasonal variation on sex expressions of
papaya cv. Coorg Honey Dew. Ind. Agri. 1967; 11:45-49.
Books Referred
1. Book chapter-High density planting and management of senile orchard
by Sable PA, Kulkarni SS. (Book: Horticultural technology
management).
Page | 132
2. Fruit tree physiology by (Dhillon WS, Bhat ZA.)
3. Newspaper, Agrowon of date 28 March, 2018, Maharashtra.
Seminar Reference
1. Sable PA. Department of Horticulture, MPKV, Rahuri (MS). Academic
Year 2012-15.
Page | 133
Page | 134
Chapter - 8
Unfruitfulness, Pollination and Fertilization
Authors
Dr. Sable P.A.
Ph.D. Hort. (Fruit Sci.), NET-Fruit Sci., NET-Veg. Sci.,
Assistant Professor (Horticulture) Sardarkrushinagar
Dantiwada Agricultural University, Sardarkrushinagar,
Banaskantha, Gujarat, India
Sushma Sable (Sonpure)
Sr. Ph.D. Scholar (Agronomy), Mahatma Phule Krushi
Vidyapeeth, Rahuri, Maharashtra, India
R.R. Lipane
M.Sc. Agri. (Agril. Botany), NET-Agril. Botany, Assistant
Professor, Department Agril. Botany, College of Agriculture,
Sonai, Maharashtra, India
Page | 135
Page | 136
Chapter - 8
Unfruitfulness, Pollination and Fertilization
Dr. Sable P.A., Sushma Sable (Sonpure) and R.R. Lipane
Fruit trees fail to bear fruit untoward expectation is known as

‘Unfruitfulness’ or ‘Barrenness’. It is a major problem in many fruit crops
and their varieties reflects into huge loss to growers and make fruit
production less profitable. In spite of adequate flowering, low fruit yields in
orchards have been observed due to low initial fruit set and subsequently
higher fruit-lets abscission. In fruit orchard, all the trees do not bear fruit
equally or regularly and sometimes fail to flower and bear fruit under similar
conditions where another fruit tree bears heavily. This failure to fruit may be
attributed to unfruitfulness. Any interference with the development of sex
cells and organs leads to unfruitfulness.
Thus, unfruitfulness is one of the serious problems in fruit cultivation
and its causes need to be understood viz. Evolutionary tendencies, genetic
influence and physiological factors.
Source: Basic Horticulture, by Jitendra Singh.
Page | 137
Flower Biology
Evolutionary Tendencies
Due to evolutionary tendencies, cross fertilization must be done in order
to maintain the vigour of the species. In these species, self-fertilization is
difficult or impossible. These factors help in maintaining these species, but
in cultivation or from the growers’ point of view, they limit its usefulness
and range. The evolutionary factors leading to unfruitfulness are as narrated
below
Sex Forms
Staminate and pistillate forms constitute basic sex forms in plants. The
plants in which both the sexes fail to form zygote, Unfruitfulness is noted. A
perfect flower consist androecium and gynoecium, whereas an imperfect
flower may be either androecium or gynoecium. In hermaphrodite flowers,
chances of unfruitfulness are restricted provided sex organs are compatible.
If staminate and pistillate flowers are borne on the same plant but in different
locations, the species is termed as ‘monoecious’ and condition monoecy for
example, coconut, arecanut, oil palm, aonla, walnut (Juglans regia L.),
pecan nut, and chestnut etc. In contrary to this dioecious plants are also
noted in which both the sex forms are found on two different plants
separately and termed ‘dioecy’ e.g. date palm, pistachio nut, papaya,
kiwifruit, capri fig and mulberry. Farther the chances of conjugation of sexes
lower will be the chances of fruitfulness in plants and vice-versa. Monoecy
favours easy conjugation whereas, dioecy disfavours it. Some fruit trees are
self-fruitful owing to compatibility of sex organs. They get fruit when
pollinated by pollen from their own flowers or by pollen from another tree of
same variety. Most of the peaches, nectarine and sour chery are shelf fruitful.
Page | 138
In contrast, many trees are self-unfruitful. Such trees fail to form fruit when
pollinated by its own pollen or by the pollen of another tree of same variety.
Most of apple, pear, peach and cherry varieties are self-unfruitful.
In the dioecious fruit crops such as date palm, papaya, pistachio or
kiwifruit male plants must be planted in an orchard near females for
pollination. Beside pollination, the male plants are useless since they do not
possess ovaries that will ripen into fruit. Generally in monoecious fruit
plants, there is no or very little problem of pollination, fruit setting and
fruitfulness. A number of sex forms have been reported in papaya. The
evolution of unisexual flowers cuts down chances of self-fertilization
completely of the unisexual plants, the pure staminate flowering ones are
essentially barren (Basic Horticulture by- Jitendra Singh).
Male flower in walnut Female flowers in walnut

Source: Internet: U. C. Davis, 26, Feb., 2013 ppt.
Pistachio and kiwifruit are dioecious

Source: Internet: U.C. Davis, 26, Feb., 2013 ppt.
Page | 139
Heterostyly
Many flowers have structural peculiarities. There form and structure
varies from each other. This is also referred to as Heterostyly or dimorphism.
Short style and long filament term as Thrum type e.g. sapota and
pomegranate. The long style and short filaments term as Pin type
Heterostyly e.g. almond and carambola is dimorphism, a type of Heterostyly.
Such types of peculiarities may prohibit self-pollination and makes cross
pollination obligatory. If cross pollination is constrained, unfruitfulness is
encountered. The occurrence of flowers with variable length of styles is
common in prunus fruit crops. Three types of flowers, namely thrum,
homostylous and pin, were found in pomegranate cv. Ganesh-1 and
Kandhari. The stigma is located above the superior plane of the anthers in
some Apricot cultivars which showed differences greater than 3 mm, and as
such, cause problems in self-pollination. However, heterostyly is a relatively
less important factor in dioecious plants (Basic Horticulture by- Jitendra
Singh).
(a) Pin type heterostyly (b) Thrum type heterostyly

Heterostyly
Source: Basic Horticulture by-Jitendra Singh.
Structural Peculiarities
The sexes of the flowers may have different maturity status. When,
stigmatic receptivity period does not synchronies with pollen viability in
monoecious plants, it is known as ‘Dichogamy’. In Dichogamy, self-
pollination is prevented in perfect flowered plants, due to maturity of two
sex organs at different times. If the stamens are matures before the stigmas
become receptive, the flowers are known as ‘protandrous’ and if stigmas
become receptive before the stamens produce viable pollens, it is known as
‘protogynous’. Protandry is observed in soursop (Annona muricata), sapota,
macadamia nut, walnut and passion fruit and protogyny is noted in
pomegranate, plum and banana. Dichogamy has been reported in avocado,
pistachio nut and chest nut (Basic Horticulture by- Jitendra Singh).
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In Avocado, Protogynous Diurnally Synchronous Dichogamy (PDSD)
has been reported. The flowering synchronies in such a way that each flower
appears on a tree is female at one time and male at another time. Diurnal
synchronization is observed on the tree and the tree flowers behave like
functional female during one part of the day and as functional male during
another part of the day. The dichogamy is protogynous in nature and pistil
matures prior to androecium. Thus, flower appears structurally bisexual but
act like unisexual. Such type of peculiarity contravenes self-pollination and
necessitates cross pollination. If cross pollination fail, plant remains
unfruitful.
Duodichogamy is another sexual mechanism in plant which contravenes
self-pollination and may lead to unfruitfulness. It had discovered in Castanea
of Fagaceae family. In this situation, each plant produces two batches of
male flowers that are temporally separated by a batch of female flowers in
between. Such type of flowering favours synchrony with-within individual
and asynchrony among individual to ensure mating partners. It is an adaptive
mating system in plants which results from competition of males to access
small number of females. The plants flower in the order male  female 
male. The resting period between flowering precludes selfing almost
completely e.g. Bridelia tomentosa (Phyllanthaceae), acer, Dipteronia
(Sapindaceae) and sawgrass (Cladium jamaicense) (Basic Horticulture by-
Jitendra Singh).
In Walnut, occurrence of both protandrous and protogynous cultivars
prevents self-pollination, which warrants cross pollination. In mango, the
duration of stigma receptivity is only 2 to 4 hours. Following anthesis. Since
pollen shedding does not start within few hours of anthesis, the periods of
maximum male and female fertility in bisexual flowers fail to coincide
(Basic Horticulture by- Jitendra Singh).
Stigmatic Receptivity
It is the ability of the stigma to support pollen germination and it limits
the effective pollination period (it is defined as the number of days during
which the pollination is effective in producing a fruit and is determined by
the longevity of ovules minus the time lag between pollination and
fertilization) in fruit crops.
Cessation of stigmatic receptivity has been associated with degeneration
of stigma and rupture of papillar integrity in kiwi fruit. In ‘Agua de
Aranjuez’, pear stigmatic receptivity is a limiting factor for flower
receptivity. The highest initial fruit set was recorded for flowers pollinated at
Page | 141
anthesis and 2 days after anthesis. After 4 to 6 days, fruit set was
significantly reduced. Thus, stigmatic receptivity could be an important
factor limiting pear flower receptivity. Fruit set in kiwi fruit after hand
pollination was high, averaging 80 percents during the first 4 days following
anthesis. However, when flowers were pollinated 5 days after anthesis, fruit
set had decreased to 36 percents followed by 7 days after anthesis where
fruit set was practically nil. Thus, the effective pollination period (EPP) was
limited to the first 4 days and the stigmatic receptive averaged 84percents
and sharply reduced to nil after 7 days (Wani et al., 2010).
Abortive Flowers or Aborted Pistils or Ovules
This occurs in the developing flowers, pistils and stigmas. Interference
either in the development of the flower or in the full development of sex
elements and their function may lead to unfruitfulness. Floral abortion is
more common in indeterminate inflorescence as compared to determinate
inflorescence. Pistil degeneration leads to unfruitfulness in certain cultivars
of plum and ornamental pomegranate, while in strawberry, pistil abortion is
so late that unfruitfulness does not take place. Certain olive varieties have 10
to 60 percents abortive embryos. Abnormal ovules and embryo sac appear to
be the main cause of unfruitfulness of the olive cultivar, ‘Swan Hill’, even
though perfect flowers are present (Wani et al., 2010).
The proportion of aborted pollen grains, varied from 22.5 to 46.8
percents in Cashew nut showing a steady increase with plant age and
reflecting an increase in the genetic load with plant age (Wani et al., 2010).
Non-Viable Pollen
It is due to non-functional pollen or the ovule. Non-viability or
impotence of pollen results in unfruitfulness. Unfruitfulness in the case of
Muscadine Grape is due to defective pollen. In grapes, sterile pollen results
from degeneration processes in the generative nucleus or arrested
development prior to mitosis in microspore nucleus. In general, late
flowering in Apricot genotypes showed lower pollen viability than early
flowering genotypes (Wani et al., 2010).
Male Sterility
Male sterility is characterized by non-functional pollen grains, while
female gametes are functional. Male sterility can be classified into three
groups’ genetic male sterility, cytoplasmic male sterility and cytoplasmic
genetic male sterility.
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Genetic Male Sterility
Like any other morphological traits, particularly mono and oligogenic,
this type of male sterility occurs in plant due to mutation of the fertility
locus, situated on a chromosome within the nucleus. In this case cytoplasm is
not involved in bringing of sterility. There could be three possible genotypes
for this locus and only one of them is male sterile (Fruit breeding by Anil
Kumar Shukla, Arun Kumar Shukla and B.B. Vashishtha).
Fertile (R-line) = RR
Fertile (B-line) = Rr
Sterile (A-line) = rr
Sterility Maintenance
By crossing A x B lines sterile and fertile progenies are produced in
equal proportions as shown bellow
Source: (Fruit breeding by Anil Kumar Shukla, Arun Kumar Shukla and
B.B. Vashishtha).
For the maintenance of sterile line the fertile plant to be quickly
removed in nearly stage of plant growth by using marker gene.
Fertility Restoration
Fertile line can be obtained by crossing a line with R-line as shown
below.
Uses
It can be used in hybrid seed production and genetically studies or for
the preservation of variability.
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Cytoplasmic Male Sterility
It occurs due to the maturation of mitochondria or to some other
Cytoplasmic factors outside the nucleus, resulting in the transformation of
the fertile cytoplasm into a sterile one. Nucleus genes are not involved.
Further, with two types of cytoplasm i.e. sterile and fertile. At the most only
two kinds of genotype. The fertile cytoplasm is denoted by (F- B line) and
sterile cytoplasm is denoted by (f- A line).
Due to different type of genotype Cytoplasmic sterility can be
maintained as shown bellow
B.B. Vashishtha).
Since there is no third type of genotype which can act as R-line, such as
restoration of fertility is not feasible. However, this does not exhaust all the
possibilities of the use of cytoplasmic sterile lines.
Uses
As restoration is not possible, this type is useful only in crops where the
seed is not desired end product. This is important for horticultural crops
where vegetative parts are of economic value.
Cytoplasmic Genetic Male Sterility
Such sterility arises from the interaction of nuclear gene (s) and
conditioning sterility with sterile cytoplasm. The cytoplasmic genetic
sterility is essentially a Cytoplasmic sterility with a provision for restoration
of fertility. The fertility is restored by (R) gene present in the nucleus. The
combination of both nuclear gene (s) and cytoplasmic factors determine the
fertility or sterility in such plants. Based on these combinations, there can be
maximum of six types of genotypes and only one of them is sterile and
shown as below.
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B.B. Vashishtha).
Sterility Maintenance and Fertility Restoration
As visualized by their genetic composition and cytoplasm, only ((rr) f)
genotype can maintain the sterility of A- line.
This is achieved by suitable restorer lines which can give rise to all
fertile progenies on crossing with A- line. Among the possible six (6)
genotypes, only ((RR) F) and ((RR) f) are such restorer or R- line. They
produced all fertile progenies is shown as under.
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B.B. Vashishtha).
Uses
Cytoplasmic-genetic male sterile lines are of immense importance in
exploitation of hybrid vigour in crops where seed is derived an end product.
Some species have genes that prevent development of the pollen or the
ovule. Generally, sterility is due to failure to obtain normal development of
pollen, embryo sac, embryo and endosperm. Morphological sterility is due to
rudimentary pistil or abortion of sex organs ovule degeneration. If one of the
sexes is inactivated, then cross pollination has to take place. Pollen sterility
is common in grape, peach cv. J.H. Hale and Shanghai, Washington Navel
orange and in Tahiti lime. The dropping of grape blossoms or partly
developed berry of vinifera varieties known as ‘coulure’ is ascribed to
sterility. The degeneration occurring in generative or vegetative nucleus or in
both leads to sterility of pollen grains and such plants remains unfruitful.
Pollination from defective pollen leads to abortion of ovary. Growth and
development of fruit are quite dependent on the fertilization process.
Fertilization of ovary with defective pollen leads to its degeneration, it drop-
off and the plant remains barren. Abortion of flowers due to degenerative
pistil in plum, defective embryo in apple, defective embryo sac in oranges
are very common. (Wani et al., 2010).
The proportion of sterile ovules varies between 22 percents in apricot
and 98 percents in avocado. In Apricot a clone of Trevatt variety ovules were
small and retarded in development were present in flower and anthers
contain degenerated microscopes. This is the first report of simultaneous
mutation in both female and male function. A high percentage of ovules in
cv. Swan Hill of Olive contain poorly developed embryo sacs at anthesis and
had not fertilized (Wani et al., 2010).
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Genetic Influences
Unfruitfulness Due to Sterile Hybrids
Genetic inheritance mechanism determines fruiting status of the plant.
Hybridity is associated with sterility as well as unfruitfulness. The degree of
sterility increases with wider crossing. peach and plum hybrids known as
‘blackman’ or ‘mule’ have complete sterile and barren flowers and are also
present in ‘Kamdesh’ which is a hybrid between peach and sour cherry.
Pyronia a hybrid between pear and quince, flowers and sets fruit, but, there is
only few viable seeds. Citrange a hybrid between Poncirus trifoliata and
sweet orange is fruitful but fails to produce fertile gametes. The popular
tangelo is a hybrid produced by crossing a grapefruit (Citrus paradisi) with a
tangerine (Citrus reticulata) are seedless or they produce seeds only with
nuclear embryos (Wani et al., 2010).
Walnut Cvs. Royal (Persian x California) and paradox (Persian x
Eastern Black), raspberry hybrids, grape hybrids Vitis rotundifolia and
Uveitis all are sterile. Hybridity is attributed to sterility in all these cases.
(Wani et al., 2010).
Incompatibility
It is the inability of a functional male and female gametes of the
hermaphrodite flowers to set seeds on self-pollination.
Genetic Control of Self-Incompatibility
Incompatibility is generally controlled by a special gene at S-locus
represented by multiple allelic series in the population, each of these alleles
control the formation of a specific substance that determines the
incompatibility reactions, both in the pistil and pollen. Identical substances
specified by identical genes in pollen and pistil favours to prevent
fertilization. Based on the timing and mode of S-gene activity, the
incompatibility reaction among homomorphic angiosperm is characterised
into two group. (Fruit breeding by Anil Kumar Shukla, Arun Kumar Shukla
and B.B. Vashishtha).
Gametophytic Control of Pollen Reaction
Sporophytic Control of Pollen Reaction
Gametophytic Control of Pollen Reaction
In this type pollen is binucleate and pollen behavior is determined by the
S allele present in each pollen and in this type stigma is wet type. It means
the incompatibility reaction of pollen is determined by its own genotype, and
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not by the genotype of the plant on which it is produced. Generally,
incompatibility reaction is determined by a single gene having multiple
alleles. Sometimes, polyploidy may lead to the loss of incompatibility due to
competition between the two S alleles present in diploid pollen. Important
examples are pineapple, loquat, apple, pear, plum, cherry, almond, apricot,
some citrus and members of Solanaceae family.
Sporophytic Control of Pollen Reaction
The incompatibility reaction of pollen is governed by the genotype of
plant on which the pollen is produced, not by the genotype of the pollen. It
means the incompatibility is imposed by the maternal genotype, due to that
all the pollen grains from a given plant behave similarly. Incompatibility
occurs at the stigmatic surface resulting in the inhibition of pollen
germination. Pollen are trinucleate and the stigmatic surface is dry e.g.
mango.
Mechanism
Based on the various phenomenon observed in self-incompatible mating
it can be grouped into…
Pollen Stigma Interaction
Pollen Tube Style Interaction
Pollen Tube Ovule Interaction
Pollen Stigma Interaction
This interaction occurs just after the pollen grains reach the stigma and
generally prevent pollen germination. In the gametophytic system stigma
surface is plumose having elongated receptive cells and is commonly known
as wet stigma. Incompatibility reaction occurs at later stage. There are clear
cut serological differences among the pollen grains with different S
genotype, such difference have not been observed in sporophytic system.
In sporophytic system, stigma is papillate and dry cover with a hydrated
layer of protein known as pellicle. There is evidence that the pellicle is
involved in incompatibility reaction. There are striking difference in the
stigma antigens related to the S allele composition. Within few minutes of
reaching the stigmatic surface, the pollen releases an exine exudates which is
either protein or glycoprotein in nature. This exudate induces immediate
callose formation in papillae and which directly in contact with pollen of
incompatible stigma. Often callose is also deposited on young protruding
pollen tubes preventing any further germination of pollen. Thus, in the
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sporophytic system, stigma is the site of incompatibility reaction. The
incompatibility reaction of pollen is probably due to deposition of some
compounds from another tapetum on to the pollen exine (Fruit breeding by
Anil Kumar Shukla, Arun Kumar Shukla and B.B. Vashishtha).
Pollen Tube Style Interaction
In most of the gametophytic system, pollen grains germinate and pollen
tubes penetrate the stigmatic surface. But in the incompatible combinations,
the growth of pollen is retarded within the stigma.
Pollen Tube Ovule Interaction
In some case pollen tube reaches the ovule and affects fertilization.
However, in incompatible combinations, embryo degenerates at early stage
of development.
Method of Overcoming Self-Incompatibility
One of the following methods can be used for bringing partial fertility
by temporarily suppressing the incompatibility reaction (Fruit breeding by
Anil Kumar Shukla, Arun Kumar Shukla and B.B. Vashishtha).
Bud Pollination
Application of mature pollens to immature non receptive stigma i.e. 1-2
days prior to anthesis.
Surgical Technique
Removal of stigmatic surface.
High Temperature
Exposure of pistils to temperature up to 60 oC.
Irradiation
With X-rays or gamma rays for single locus gametophytic
incompatibility.
Double Pollination
Incompatible pollen is applied as mixture with compatible pollen.
Pollination at the end of Season
In case of sporophytic incompatibility system the breakdown is
comparatively easy because the incompatibility reaction takes place between
stigmatic surface and pollen wall in comparison to gametophytic
incompatibility in which reaction starts when the pollen tubes have already
travelled 1/3 to ½ length of styler tissue.
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Role of Self-Incompatibility
Where male sterility is nonexistent, self-incompatibility can
alternatively facilitate the production of F1 hybrid.
Seedless verities, such as in pineapple, grape etc. can be evolved if
incompatibility is present (Fruit breeding by Anil Kumar Shukla, Arun
Kumar Shukla and B.B. Vashishtha).
Incompatibility is a genetically controlled character manifested by the
presence of multiple alleles at a single locus. In fruit production,
incompatibility may create a platform to create variation leaving no scope for
inbreeding. In mango, self-unfruitfulness is reported in Cvs. Dashehari,
Chausa and Langra and Bombay green. Most pear (Pyrus communis L.)
cultivars are impaired to set fruit under self-pollination because self-
fertilization is prevented by gametophytic self-incompatibility system. In
loquat varieties, improved Golden yellow, Tanaka, Late yellow, Pale yellow,
Golden yellow, California Advance and the pollen tube penetrated the stylar
canal up to 1/4 to 1/3rd of its length and did not go further below, even after
72 hours of pollination. As such, this suggests incompatibility in loquat. In
pear cv. ‘Agua de Aranjuez’ 80 percents of the pollen tube reaches the base
of the style and fertilized 70 percents ovules after cross-pollination whereas,
only 10 percents pollen tube reaches the ovule and fertilized only 5 percents
of the ovule which indicates a high degree of incompatibility in pear (Wani
et al., 2010).
Pant Lemon, Kagzi Kalan etc. do not flowers if planted solely as single
cultivar due to self-incompatibility (Wani et al., 2010).
Physiological Influences
Premature or Delayed Pollination
Premature or delayed pollination leads to unfruitfulness and is reported
that premature pollination followed by germination and tube growth causes
fruit drop due to toxicity in pistil. However, in case of oranges, premature
pollination did not have any deleterious effect. Low setting due to premature
pollination was noticed in persimmon, pear, plum and peach. Similarly, if
pollination is delayed, the flower falls without setting (Wani et al., 2010).
Nutritive Condition of Plant
Nutrition of plant controls the percentage of defective pistils. Defective
pistils are formed especially on exhausted or weakened plants caused by
overbearing, drought and poor nutrition. Nutrition also determines the
percentage of flower carried for setting, maturity and also pollen viability
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Fruit Setting of Flowers in Different Positions
Fruit borne on terminal growth have more competition in many fruit
crops and its maturity is set under normal nutritional conditions, but the
percentage of the set is small. This positional competition takes place
between fruits and branches as well as between different fruits influencing
fruitfulness. The apical (king flower) flower in the bud of Apple (Malus
domestica) develops first, followed by the lateral flower which develop in
sequence, beginning at the base of the spur. Some fruits like plum and
cherry, bear on both shoots and spurs and it is to be expected that slightly
different nutritive conditions are obtained in the different tissues and a
distinctly heavier June drop occurs in shoot borne fruits. In apple, old spurs
are less likely to initiate flowers than the young ones (Wani et al., 2010).
External Factors
Any external factor that affects the nutrition of the fruit tree is soil
condition, water supply and cultural practices and is important on behalf of
this.
Temperature
Among the environmental factors, temperature has a great importance. It
affects flowering and fruit set in several ways. It is a common knowledge
that a period of cool, yet frostless, weather is conducive to better
blossoming, fertilization and fruit set. However, the abscission of flower
bud, fruit, etc. is a function of temperature.
High temperature, above 32 °C, desiccation of the stigmatic surface and
more rapid deterioration of embryo sac occurs. As temperature increases,
ovule senescence become faster in ‘Italian’ than in ‘Brooks’ cultivar of
plum. At 15 °C, only one ovule per flower remains viable by 8 days after
fruit bud for Italian, whereas for ‘brooks’ cultivar, higher temperature results
to a decrease in ovule longevity. Low temperature, in plum, cherry, apple,
pear, etc., the temperature of 4.4 °C or lower, completely check the
blooming, fertilization and fruit set. The polythene cage induced a mean
increase in the maximum temperature of 7.6 °C (warm treatment). In the
control treatment, most of the flowers (92%) had morphologically well-
developed pistil, while in warm treatment, 33 percents of the flowers
presented pistils that are not completely developed and 13 percents of the
flowers shows short styles and un-swelled ovaries. Fruit set and production
of fruit in peach Cv. Granda were absent or very low in green house grown
trees in orchards (Wani et al., 2010).
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Humidity
Low atmospheric humidity causes drying of stigmatic secretions. Wet
and humid weather favours anthracnose and poor fruit set in mango. The
poor germination of pollen in almonds is attributed to damp weather during
fruit set (Wani et al., 2010).
Rain
It directly affects fruit setting by distributing the process of pollination
and germination of pollen grains and staminal fertilization. In almond
washing, treatment decreased the number of germinated pollen grains on the
stigma mainly when the flowers were immersed before pollination and it
seems to affect adhesion in forthcoming pollinations. Rains at the time of
blooming period causes unfruitfulness by washing pollen grains, inhibit
pollinators and cause spread of diseases and pests. Every fruit species need a
specific time period without rains at the time of blooming for successful
pollinations (Wani et al., 2010).
Light
Light affects fruitfulness indirectly by its effect on photosynthesis. Light
is a pre-requisite for photosynthesis and low light intensity or its duration
reduces the carbohydrates reserves in the trees. In addition to this, poor light
conditions promote fruit abscission (Wani et al., 2010).
Wind
Wind also affects the fruitfulness indirectly by its effect on the
pollinating agents, that is, it controls pollination either by promoting wind
pollination or by checking insect pollination. It is desirable in wind
pollinated fruit trees like Hazelnut and Walnut, but if its speed is too high, it
is harmful because it results in low fruit set on exposed sides and heavy crop
on other sides. Excessively, speedy winds cause ovary abortion and also
make the stigma dry (Wani et al., 2010).
Chemicals and Pesticides
The use of pesticides can kill bees, therefore reducing pollination. Some
pesticides can also be toxic to delicate flowers causing abortion and loss of
fruit. Pesticides spray effects on receptive stigmatic surface, showed varying
degree of injury and range from minor surface wrinkling to degeneration of
stigma papillae. Controlled pollination for 1 hour after pesticides sprays,
results in an inhibition of pollen germination and tube growth. Commercial
fungicides containing Captan, Dinocarp and Sulphur sprayed on un-dehisced
anthers of several apple cultivars reduced the viability of pollen, impaired
pollen release, kill pollen when sprayed onto dehisced anthers. Pollen
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germination and tube growth were drastically suppressed by the spray and
almost no fruit set was observed on the treated inflorescence and there was a
highly significant difference between fruit set on exposed and protected
cluster (Wani et al., 2010).
Remedial Measures
Balancing Fruiting and Vegetative Growth
Pruning and Thinning
The main techniques for controlling the vigour of fruit trees and
increasing their relative fruitfulness are the use of dwarfing rootstocks,
compact, short-internodes scions and of trees training and pruning system
which give horizontal or wide-angled branches. As such, growth retardants
are also used. The influence of pruning varies with the amount, season and
kind of pruning. Judicious and proper pruning is needed to improve the fruit
set and fruit retention on the trees and also on the removal of dead results in
loss of carbohydrates reserves along with pruned wood. Also, severe pruning
promotes too much vegetative growth and hence reduce the productivity.
Experiments revealed that pear on P. communis and ‘E. M. Quince C’
rootstocks showed early fruit thinning to 1 fruit per cluster and increased the
ultimate percent fruit set of ‘Comic’ girdling and cluster thinning in ‘Anjou’
set beyond either treatment. In mango, the highest number of new flushes per
shoot was achieved with severe pruning and spraying of GA3 at 100 ppm
Control of Pollination
Use of Pollinizers
Pollen transfer may present an application problem in fruits which are
self-incompatible. Many apple varieties are at least partially self-fertile,
especially under warm weather, but in most cases, fruit-sets are improved by
the use of pollinator varieties and bees. Cherries and plums also need cross
pollination and should be inter planted with pollinizer varieties. Breeding
and selection of self-fertile cultivars or clones of cherry and apple may
reduce difficulty or achieve satisfactory pollination, especially in cool
marginal areas of fruit production where temperatures at blossoming time are
sub-optimal both for bee activity and for pollen-tube growth. Until such
improved varieties become available, provision of suitable pollinizer
varieties and bee-hives can be of great help in ensuring satisfactory fruit-set.
There is an increasing tendency for using crab-apples, as pollinizers of these
occupy very little space in orchards. It is advisable to have a number of
pollinizer varieties with widespread flowering dates to ensure cross-
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pollination. One of the basic requirements for setting fruit is an adequate
requirement of compatible pollen. With most tree fruit crops, the need for
cross pollination is recognized. Pollinating insects are necessary for fruit set
on all cultivars, and most cultivars will benefit from crosspollination. Under
general conditions, the closer a tree is to a pollinizer, the better fruit set will
be. Cross pollination necessitates the availability of sufficient quantity of
compatible pollen, as pollinizer cultivars flower synchronously the main
cultivar and suitable agent for the successful and effective transfer of pollen.
Therefore, suitable pollinizer cultivars must be inter planted at the time of
orchard layout (Wani et al., 2010).
Desirable Characteristics of Pollinizers for Delicious Apple
1) The pollen of this variety should have the ability to fertilize the
delicious flowers.
2) It must flower with the main variety.
3) It must come into flower with the same age of delicious apple.
4) It should possibly be a commercial variety.
5) It should also be suitable for place and micro climate.
6) It should also be a long flowering season.
In apple style, receptivity and pollen potency should be considered first
during screening for effective pollinator. ‘Red Sleeves’ is the most
productive and it shows the highest style of receptivity. It gives the highest
pollen germination at 8-10 °C and at 5 °C. As such, it is the most effective
pollinator for Cox. California advance variety was found to be the best
pollinizers for improved golden yellow and pale yellow (Wani et al., 2010).
Introduction of Pollinators
The population of natural pollinators has gone down due to
indiscriminate use of pesticides and deterioration of the ecosystem. The
managed bee pollination is very limited and the available bee hives, during
bloom, hardy meet 2 to 3 percents of the demand. In spadona Pear,
introducing the colonies sequentially (sequential introduction means
introducing half of the number of the recommended number of colonies at
10% full bloom and half at full bloom) increases the number of bees per tree
and their mobility among the rows, and consequently, it increases fruit set
and yield by 50 to 80 per cent. In red delicious apple, it was found that
sequentially increasing the density of colonies increases the amount of cross
pollination and a high proportion of top workers. However, the increased
pollination efficiency results in high fruit set and higher yield (50 to 100%)
in treated plots (Wani et al., 2010).
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There was significant increase in fruit set in the apple orchard where bee
colonies were kept for pollination and increase in fruit was significantly
higher in orchard with sufficient pollinizer more than 15 percents than in
orchards having insufficient pollinizers less than 15 percents (Wani et al.,
2010).
Proper Nutrition
The nutritional requirement of guava after fruiting varies with region to
region, soil type, and age. A dose 50 kg well decomposed organic manure,
978 g urea (450 g. N), 1875 g single supper phosphate (300 g. P) and 500 g.
murate of potash (300 g. K.) to be applied during monsoon season.
Fertilizers are applied in basins by the ring or drip point at a depth 15 to 20
cm. Area around the active root zone is at 30 cm. from the trunk at a depth
30 cm. Split dose of urea @ 978 g. (450 g. N) per plant to be applied in
October to November (Sable and Sonpure, 2018). Spraying of 50 g urea
through 15 liters of water to the plants during March and October are
recommended for adequate growth and development (Fruit crops by K. V.
Peter).
Spraying of zinc sulphate, magnesium sulphate, manganese sulphate and
copper sulphate each @ 0.5 percent (5 g. per liter of water) at the time of
new flush and blooming respectively, to correct zinc, magnesium, copper
and manganese deficiency (Fruit crops by K. V. Peter).
Fruit drop is a serious problem in mango and cause great loss to mango
growers. A tree producing several thousand panicles yields only a few
hundred fruit. Most of the flowers fall down after full bloom or at later stage
of development. Only 0.01 to 0.1 percent flowers develop into mature fruit.
Fruit drop has been divided into three distinct phases e.g. pin head drop,
post-setting drop and June drop.
Probably, nutritional factor have been considered responsible for fruit
drop in mango. The nutritional requirement of mango after 5 years varies
with the region, soil type and age. A dose of 50 kg well decomposed organic
manure, 1630 g urea, 3125 g single super phosphate (SSP) and 833 g murate
of potash (MOP) should be applied during monsoon season. Fertilizers are
applied in basins by ring or at drip at a depth 15-20 cm. Area around the
active root zone is at 30 cm from the trunk at a depth 30 cm. The split dose
of urea @ 1630 g per plant should be applied in September-October
(Sable and Sonpure, 2018). Spraying of zinc sulphate 0.3 percent (3 g./liter
of water) during January, February, March and April is recommended at
monthly interval to correct zinc deficiency. Spraying of Borax 0.5 percent (5
g./liter of water) after fruit set twice at monthly intervals and 0.5 percent (5
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g./liter of water) manganese sulphate after blooming correct manganese and
boron deficiencies respectively (Handbook of Horticulture).
The flower as well as the fruit drop is primarily due to the formation of
an abscission layer at the point of attachment of fruit with the twig. Several
factors have been considered responsible for formation of abscission layer. It
has been observed that Nitrogen retards and reduces abscission. Low
concentrations have also been found to lead leaf, flower, and bud and fruit
abscission. Fruit drop has been attributed to many other causes e.g. abortion
of embryo, degeneration of ovules, poor soil, inadequate irrigation water,
attack of insect-pest, diseases, depletion of nutrients, hormonal imbalance,
etc. Spraying of 50 ppm (50 mg./liter of water) gibberellic acid, immediately
at flowering is recommended to minimize flower and fruit drop.
Fruit drop may be controlled to some extent by the spray of 1 percent
(10 g./liter water) Potassium nitrate with 20 ppm (20 mg./liter of water)
NAA at 15 days interval during flowering. Spraying of 2 percents (20 g./liter
of water) Urea is recommended at full bloom stage correct nutritional
deficiency (Sable and Sonpure, 2018).
Inadequate water supplies during fruit development have been
considered responsible for fruit drop. The farmers are advised to irrigate
plants as fruit becomes of ‘Pea’ size at 10 days interval. Flowering in mango
continues in two or three distinct flushes for a period of 6 to 8 weeks with
ber or lime size fruit development of on different branches of trees. Spraying
of 2 percents (20 g./liter of water) 12:61:00 water soluble fertilizer is
recommended to avoid fruit drop (Sable et al., 2018).
The nutritional requirement of banana varies with region to region, soil
type, and age etc. 30 kg. Well decomposed organic manure or 5 kg.
Vermicompost to be applied per plant before planting. At planting, for better
Nitrogen fixation and Phosphorus viability 25 g. Azospirillum and 25 g. PSB
should be applied per plant through compost.
Nutrient management in banana to be done as per schedule given
bellow, the dose may be varies as per Soil analysis report.
Fertilizer Management in Banana (g./Plant)
S. No Application Time Urea SSP MOP
1 Planting-up to 30 DAP 82 375 83
2 75 DAP 82 -
3 120 DAP 82 -
4 165 DAP 82 83
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5 210 DAP 36 -
6 255 DAP 36 83
7 300 DAP 36 83
Total 435 375 332
DAP-Days after planting. Anon., 2018.
At 2nd and 4th months of plantation, banana plant to be sprayed with
ferrous sulphate and zinc sulphate @ 5 g. per liter of water. On 5th and 7th
month 15 g ferrous sulphate and zinc sulphate to be applied per plant
through 100 to 150 g. of compost to correct the ferrous and zinc deficiency.
(Sable P.A. Agrowon newspaper article).
The nutritional requirement of matured Custard apple plant varies with
the region, soil type and age. A dose of 30 to 40 Kg. well decomposed
organic manure, 271.5 g. Urea (125 g. N), 781 g. Single super phosphate
(125 g. P) and 208 g. Murate of potash (125 g. K.) to be applied per plant
during monsoon season. Fertilizers are applied in basins by ring or at drip at
a depth 15 to 20 cm. Area around the active root zone is at 30 cm from the
trunk at a depth 30 cm. Split dose of Urea @ 271.5 g. (125 g. N) per plant to
be applied in September to October. Twenty five gram each of Azospirillum
and PSB may be applied per plant through 100 to 150 g.
of FYM (Directorate of Extension Education, MPKV, Rahuri- Krushi
darshani, 2018).
Application of Plant Growth Regulators
The unfruitful behavior of several fruit plants can be overcome by the
use of plant growth regulators which may be due to decreased fruit set and
abscission at various developmental stages. Some of the recent findings on
the use of plant growth regulator to overcome unfruitfulness in fruit trees.
Nutrient deficiencies have also been found to lead leaf, flower, and bud
and fruit abscission. Fruit drop has been attributed to many other causes e.g.
abortion of embryo, degeneration of ovules, poor soil, inadequate irrigation
water, attack of insect-pest, diseases, depletion of nutrients, hormonal
imbalance, etc. Spraying of 50 ppm (50 mg./liter of water) gibberellic acid,
immediately at flowering is recommended to minimize flower and fruit drop.
Fruit drop may be controlled to some extent by the spray of 1 percent
(10 g./liter water) Potassium nitrate with 20 ppm (20 mg./liter of water)
NAA at 15 days interval during flowering. Spraying of 2 percents (20 g./liter
of water) Urea is recommended at full bloom stage correct nutritional
deficiency (Dr. Sanjay Patil, FRC, Himayatbagh, Aurangabad, Agrowon
article).
Page | 157
Use of Suitable Rootstocks
There can be as much as 50 percents or more difference in the yield of a
given cultivar grown on different rootstocks. The reasons for such an effect
can be traced to difference in tolerance to adverse soils, in resistance to pests
or in uptake of nutrients. Four rootstocks namely M9, M7, M4 and M1
induced 50 percents or more bloom in the fifth year in Starking delicious
apple and resulted in higher yield efficiency by controlling tree size (Wani et
al., 2010).
Unfruitfulness could be managed by…
Unfruitfulness can be due to lack of balance between growth and
fruiting and lack of flowering and poor fruit-set as the result of various
internal and external factors in different fruits and their cultivars. So, it is
necessary to make necessary corrective measures which should begin from
planning level and extends to an established orchard. The crop/variety
should be chosen on the basis of climate and edaphic factors. Different
varieties should be cultivated and the introduction of effective pollinizers’
varieties and pollinator (Honey bee) is necessary. While, selecting
pollinators for apple styler, receptivity and pollen potency should be
considered first. Therefore, in order to obtain high productivity in apple plant
diploid, self-fruitful and compatible varieties are used to ensure cross
pollination. Also, old orchards should be rejuvenated. Thinning and crop
regulation should be practiced and regular bearing varieties should be
planted. So, it is important to analyze the problem and then corrective
measures could be suggested. Basically, planning should be done, so that the
future will be problem free, and then, adoption of correct package of
practices should be followed (Basic Horticulture by-Jitendra Singh).
Pollination and Fertilization
It is the act of transfer of pollen from the anthers of a flower to the
stigma of the same flower or of another flower. When, pollen from anther is
transfered to the stigma of same flower, the process is referred to as self-
pollination or autogamy. Self-pollination occurs within hermaphrodite
flower or perfect flowers or bisexual flowers or between two monoecious
flowers which are located on the same plant of the same variety or cultivar.
However, transfer of pollen from the anthers of a flower to the stigma of
another flower located on different plant of same cultivar or same/related
species is referred as cross pollination or allogamy. Pollination is a
prerequisite for fertilization.
Upon successful transfer of pollen, produced in the anther sac, to the
pistil, the pollen germinates as pollen tube. The pollen tube further grows via
Page | 158
stigma, style and ovary and enters the ovule (eggs) at micropyle and
fertilizes them.
After fertilization of the ovules, the seed develops and the ovary starts
swelling in and the fruit are developed are shown in fig. bellow (Basic
Horticulture by-Jitendra Singh).
Fertilization
Source: Basic Horticulture by Jitndra Singh.
Pollination
Pollen is released as a dehydrated cell. When, it lands on the stigma it
rapidly hydrates in the fluid that is secreted by the stigma surface.
Pollen germination Pollen tube growth
Page | 159
When, pollen becomes hydrated on the stigma it germinates to form a
tube that penetrates between stigma cells.
Ovules
Female germ cell Fertilization

Source: Internet: U.C. Davis, 26, Feb., 2013 ppt.
The ovules are potential seeds. They contain the female germ cells,
eggs. There is one egg cell in each ovule. Pollen tubes grow to the ovules.
The first pollen tube to arrive enters the ovule and releases its contents. One
sperm cell fuses with the egg. A second sperm cells fuses with two other
female cells to form endosperm. Fertilization triggers fruit set.
Pollinizer
A plant, provides pollen consider as pollenizer. The pollinizer should
bear the good characteristics viz. pollens of pollinizer variety must be
compatible with the variety. A pollinizer variety should produce a good
amount of pollen. The flowering period of pollinizer should coincide with
flowering of main variety. It should also be a long flowering season and the
pollinizer variety must produce a good amount of crop.
Page | 160
Transference of Pollen
Upon maturity, anther sac ruptures and pollen grains are released. The
pollen grains reach to the stigma of flowers by various means/agencies. They
are called pollinators. They may be Insects viz. bees, butterflies, housefly
(pollination in mango), moths, and wasp (pollination in fig) and Weevil
(pollination in oil palm) etc. transfers the pollen grains is known as
entomophily.
Birds. Parrots, bats etc. these refer to ornithophily e.g. banana and
pineapple. Mammals/animals. Elephants, monkeys, squirrels etc., this mode
of pollination is treated as zoophily.
Wind represents Anemophily. Water represents Hydrophily e.g. black
pepper (pollination by rain water droplets).
EPP integrates three factors viz. stigma receptivity i.e. the ability of the
stigma to support pollen germination. Pollen tube growth rate, the time
required for the pollen tubes to grow through the style to the ovule and
Ovule viability, the time that the ovule is capable of being fertilized. Each of
these is temperature dependent.
Factors Favouring Self-Pollination
Self-pollination is the closest form of inbreeding. It leads
to homozygosity. Such species develop homozygous balance and do not
exhibit significant inbreeding depression.
Mechanism Promoting Self-Pollination
Bisexuality
Presence of male and female organs in the same flower is known as
bisexuality. The presence of bisexual flowers is a must for self-pollination.
All the self-pollinated plants have hermaphrodite flowers.
Homogamy
Maturation of anthers and stigma of a flower at the same time is called
homogamy. As a rule, homogamy is essential for self-pollination e.g.
apricot, citrus, peach and dwarf coconut cultivars.
Cleistogamy
When pollination and fertilization occur in unopened flower bud, it is
known as cleistogamy. It ensures self-pollination and prevents cross
pollination. It is common in grape, papaya and sapota.
Page | 161
Chasmogamy
Opening of flowers only after the completion of pollination is known as
chasmogamy. This also promotes self-pollination and is found in tomato.
Geitonogamy
It is a Greek word derived from geiton means neighbor and game in
mean marry. Thus, it is the process interferes with sex function and leads to
reduced out crossing e.g. orchid.
Position of anthers
In some species, stigmas are surrounded by anthers in such a way that
self-pollination is ensured. Such situation is found in tomato and brinjal. In
some legumes, the stamens and stigma are enclosed by the petals in such a
way that self-pollination is ensured e.g. green gram, black gram, soybean,
chickpea and pea.
Factors Favouring Cross-Pollination
Dicliny
It refers to unisexual flowers. This is of two types viz.
i) Monoecy and
ii) Dioecy
When, male and female flowers are separate but present in the same
plants, it is known as monoecy. In some crops, the male and female flowers
are present in the same inflorescence such as in mango, castor and banana. In
some cases, they are on separate inflorescence as in maize. Other examples
are cucurbits, grapes, strawberry, cassava and rubber. When, staminate and
pistillate flowers are present on different plants, it is called dioecy. It
includes papaya, date palm, spinach, hemp and asparagus.
Dichogamy (from the Greek Dikho -Apart and Famous-Marriage)
It refers to maturation of anthers and stigma of the same flowers at
different times. Dichogamy promotes cross pollination even in the
hermaphrodite species. Dichogamy is of two types viz.
i) Protogyny and
ii) Protandry
When, pistil matures before anthers, it is called protogyny e.g. walnut,
coconut, sapota, passion fruit soursop (Annona muricata) etc. When, anthers
mature before pistil, it is known as protandry e.g. Annona sp. except Soursop
(Annona muricata), fig, banana, plum, pomegranate, sugarbeet and avocado
etc.
Page | 162
Heterostyly
When, styles and filaments in a flower are of different lengths, it is
called heterostyly. It promotes cross pollination.
Protogynous Diurnally Synchronous Dichogamy
In Avocado, Protogynous Diurnally Synchronous Dichogamy (PDSD)
has been reported. The flowering synchronies in such a way that each flower
appears on a tree is female at one time and male at another time. Diurnal
synchronization is observed on the tree and the tree flowers behave like
functional female during one part of the day and as functional male during
another part of the day. The dichogamy is protogynous in nature and pistil
matures prior to androecium. Thus, flower appears structurally bisexual but
act like unisexual. Such type of peculiarity contravenes self-pollination and
necessitates cross pollination. If cross pollination fail, plant remains
unfruitful (Basic Horticulture by- Jitendra Singh).
Duodichogamy
Duodichogamy is another sexual mechanism in plant which contravenes
self-pollination and may lead to unfruitfulness. It had discovered in Castanea
of Fagaceae family. In this situation, each plant produces two batches of
male flowers that are temporally separated by a batch of female flowers in
between. Such type of flowering favours synchrony with-within individual
and asynchrony among individual to ensure mating partners. It is an adaptive
mating system in plants which results from competition of males to access
small number of females. The plants flower in the order male  female 
male. The resting period between flowering precludes selfing almost
completely e.g. Bridelia tomentosa (Phyllanthaceae), Acer, Dipteronia
(Sapindaceae) and Sawgrass (Cladium jamaicense) (Basic Horticulture by-
Jitendra Singh).
Herkogamy
Hindrance to self-pollination due to some physical barriers such as
presence of hyline membrane around the anther is known as herkogamy.
Such membrane does not allow the dehiscence of pollen and prevents self-
pollination such as in alfalfa.
Self-Incompatibility
The inability of fertile pollens to fertilize the same flower is referred to
as self-incompatibility. It prevents self-pollination and promotes cross
pollination. Self-incompatibility is found in several crop species like
Page | 163
Brassica, Radish, Nicotiana, and many grass species. It is of two
types sporophytic and gametophytic.
Male sterility
In some species, the pollen grains are non-functional. Such condition is
known as male sterility. It prevents self-pollination and promotes cross
pollination. It is of three types viz. genetic, cytoplasmic and cytoplasmic
genetic. It is a useful tool in hybrid seed production.
Study of floral biology and aforesaid mechanisms is essential for
determining the mode of pollination of various crop species. Moreover, if
selfing has adverse effects on seed setting and general vigour, it indicates
that the species is cross pollinated. If selfing does not have any adverse
effect on these characters, it suggests that the species is self-pollinated. The
percentage of cross pollination can be determined by growing a seed mixture
of two different varieties together. The two varieties should have marker
characters say green and pigmented plants. The seeds are harvested from the
recessive (green) variety and grown next year in separate field. The
proportion of pigmented plants in green variety will indicate the percentage
of outcrossing or cross pollination.
Pollen Viability
Pollen is said to be viable if it is germinates over stigmatic surface
effecting elongation of pollen tube sufficiently to fertilize egg cell. Bearing
in fruit plants depend a lot upon pollination and subsequent fertilization of
egg cell. The viability of pollen is adjudged by acetocarmine test. In this test,
the pollen grains are placed over slide and 1-2 percents acetocarmine
solution is placed over it. The pollen which are viable are stained and non-
viable types fail to get stained. The viable pollens are counted under
microscope to ascertain viability percentage. The germination of viable
pollen is further studied to know the fertilization of egg cells and ultimately
the fruit set. For this purpose sugar solution of 10-25 percents are used.
Pollen grains are incubated in sugar solution for different periods of time and
there germination percentage is studied under microscope. Agar and Boron
add in enhancing germination of pollen grains.
Significance of Pollination
The mode of pollination plays an important role in plant breeding. It has
impact on five important aspects viz. gene action, genetic constitution,
adaptability, genetic purity and transfer of genes.
Classification of crop plants based on mode of pollination and mode of
reproduction.
Page | 164
Mode of
Pollination and Examples of Crop or Plants
Reproduction
A. Self-Pollinated Crop
Seed propagated Rice, wheat, barley, oats, chickpea, pea, cowpea, lentil, green gram,
black gram, soybean, common bean, moth bean, linseed, sesame,
khesari, sunhemp, chillies, brinjal, tomato, okra, peanut, etc.
Vegetatively Potato
propagated
B. Cross Pollinated Crop
Seed propagated Corn, pearl millet, rye, alfalfa, radish, cabbage, sunflower,
sugarbeet, castor, red clover, white clover, safflower, spinach,
onion, garlic, turnip, squash, muskmelon, watermelon, cucumber,
pumpkin, kenaf, oil palm, carrot, coconut, papaya, etc.
Vegetatively Sugarcane, coffee, cocoa, tea, apple, pears, peaches, cherries,
propagated grapes, almond strawberries, pine apple, banana, cashew, irish,
cassava, taro, rubber, etc.
C. Often Cross Pollinated Crop
Sorghum, cotton, triticale, pigeonpea and tobacco.
(Basic Horticulture by- Jitendra Singh)
References
1. Anonymous. Krushidarshani, Directorate of extension education,
MPKV, Rahuri, 2018, 115.
2. Sable P.A, Sushma Sonpure. Banana planting and nutrient management.
Agrowon newspaper (MS), 12, 2018.
3. Sable PA, Shiekh AS, Sushma Sonpur. Enhanced mango production.
Krishijagran, Com/agripedia, 6, December, 2018, 1-3.
4. Imtiyaz Ahmad Wani, M.Y Bhat, Abid Ali Lone, Mohd. Younis Mir.
Unfruitfulness in fruit crops, causes and remedies. African Journal of
Agricultural Research. 2010; 5(25):3581-3589.
Books Referred
1. Basic horticulture by Jitendra Singh
2. Fruit breeding by Anil Kumar Shukla, Arun Kumar Shukla and B.B.
Vashishtha
3. Fruit crops by K.V. Peter
Other Sources
1. Internet: U.C. Davis, 26, Feb, 2013.
Page | 165
2. Article by Dr. Sanjay Patil, Newspaper, Agrowon of date 28 March,
2018, Maharashtra.
3. Article by Dr. Sable. P. A. and Sushma Sonpure, Newspaper, Agrowon
of date 26 July, 2018, Maharashtra.
Page | 166

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Volume - 3

Chief Editor: Dr. Pidigam Saidaiah

The author/publisher has attempted to trace and acknowledge the materials

Chapters Page No.

1. Different Vector Systems and Their Utilization in Gene Transfer of

2. Basic Concepts of Heterosis 25-37

3. Introduction of Plant Breeding 39-47

4. Conventional vs. Marker Assisted Backcross Breeding 49-75

5. Study of Induced Polygenic Variability in M3 Population of

6. Plant Breeding: A Tool for Potential Exploitation of Genetic Gain

7. Flowering Biology of Horticultural Crops 107-133

Properties of a Good Vector

It contains a polylinker which can recognize several different restriction

Genes (III, VI, VII, VIII and IX encode structural proteins

Bacterial Artificial Chromosomes (BAC)

Structure representation of the Agrobacterium Ti-plasmid and integrated

2.1 Binary Vector

2.2 Expression Vector

Adeno-Associated Viral Vectors

Herpes Virus Vectors

Poxvirus and Other Viral Vectors

3. Direct methods of Transformation

Fig 1: RP genome recovery % in Conventional Back cross program

Conventional Method of Back Cross Breeding

Recovery 50% Rr (100% Plants) 

Yield test with RR plants

Transfer of Two Recessive Genes-Conventional Plant Breeding

AABB, AABb, AaBB, AaBb BC1F1 @

AABB, AABb, AaBB, AaBb-BC3F1 @

A-/B- (98.44% Plats) X aabb (1.56% Plants) 

Plant Height Days to Flowering

Days to Maturity Panicle Length

100-seed Weight Grain Yield per Plant

Fig 1: Plant Height

Fig 3: Days to Maturity

Fig 4: Panicle length

Number of Panicle Bearing Tillers Per Plant

Fig 5: Panicle Bearing Tillers per Plant

Number of Grains per Panicle

Fig 7: 100-seed weight

Fig 8: Grain yield per plant

3. Different Methods of Non-Conventional Plant Breeding

Flowering Biology of Horticultural Crops

Inter convertible form of phytochrome

Photoperiod induction and flowering

Site of Photo-Induction Perception

Photoperiodic induction for long day plant needs 12-14hrs. No Flowering in 8-

Transmission of stimulus from photo-induced branch to a non-photo-induced

Early flowering to eastern side of mango tree

Source: Ph.D. seminar by Sable. P.A.

Leaves cotyledons temperature

Physiologically active form (PFR) established

Transport of florigen to shoot apex

Fruit trees fail to bear fruit untoward expectation is known as

Source: Basic Horticulture, by Jitendra Singh.

Male flower in walnut Female flowers in walnut

Pistachio and kiwifruit are dioecious

(a) Pin type heterostyly (b) Thrum type heterostyly

Pollen germination Pollen tube growth

Female germ cell Fertilization

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