La Aclimatacin

Interspecific hybridisation
Fertilisation barriers
Hybrid abortion problems
Embryo rescue

Hybrid sterility (chromosome

incompatibility)
Lack of chromosomal
recombination
Many backcrosses needed to
eliminate most donor DNA

Making the Intermated B73 X MO17 mapping population

of recombinant inbred lines (RILs)
Mo17

B73

Single F2 plant
was selfed

X
Grow up 200 plants,
random mating

Illustration of the genotype of 6 of those 200 plants.

Recombination during meiosis results in the exchange of chromosome segments

Select 100 ears, pick 5 kernels from each ear. Put in a bag, shake, plant,
more random matings (2nd generation).
Repeat, (repeat.)

From these lines, generate Recombinant Inbred lines by repeated selfing (5x or more)
x
x
x
x
x

x
x
x
x
x

x
x
x
x
x

x
x
x
x
x

x
x
x
x
x

And Tahdah! the Recombinant Inbred mapping population

x
x
x
x
x

Finding quantitative trait loci (QTLs)

You test your population of plants for the presence of a phenotype (e.g. leaf size) and you
determine the statistical likelihood that each molecular marker is associated with the trait. This
gives you a LOD score (logarithm of the odds that they are associated). You plot LOD scores
along the chromosome, and where it is highest you have a QTL locus (one or more genes) that
determines your phenotype.
In the example shown, Antirrhinum majus (snapdragon) has 4 QTLs for leaf size.

How do we get pairing of homoeologous chromosomes?

Wild sex in this example means interspecific crossing or hybridization.
Homologous chromosomes readily
pair during meiosis, but homoeologous
Chromosomes do not. Homoeologous
means partially homologous, usually
indicating some original ancestral
homology. Pairing is prevented by
Certain genes that evolve as part of
speciation. Can those genes be
identified and then knocked out?

What we need is a little more

Breaking the cereal species barrier:

pairing of homoeologous chromosomes
Bread wheat contains three closely related genomes: A, B and D. Chromosome pairing during
meiosis is confined to strict homologues, despite the co-existence in the genome of
homoeologous chromosomes. This control maintains the integrity of the three genomes. The
genetic control of chromosome pairing in wheat is dependent on a series of genes that
suppress and promote homoeologous pairing (Ph genes). The strongest effect on pairing is
associated with a gene (or genes) at the Ph1 locus) that suppresses homoeologous
chromosome pairing within the polyploid wheat genome. In the absence of Ph1, homoeologous
recombination can occur between wheat chromosomes and those from related species or
genera. An X-ray-induced deletion at the Ph2 locus, ph2a, and a chemically induced mutant,
ph2b, have demonstrated that removal of the Ph2 gene induces an intermediate level of
homoeologous chromosome pairing in wheat hybrids with alien species but does not affect
chromosome pairing in wheat itself. These observations suggest that Ph2 will be a valuable
resource for the introgression of alien genes from related species into bread wheat, with
minimum disruption of endogenous homologous chromosome pairing.

Rice flower

Male stamens

Female pistil

1. Put pollen from Oryza glaberrima

on the pistil of Oryza sativa, and
allow embryo to develop.
Collect the seeds that are formed
with very low frequency.

QuickTime and a
TIFF (Uncompressed) decompressor
are needed to see this picture.

2. Backcross the progeny multiple

times to O. sativa so that you end
up with a plant that is 85 % Sativa
and 15 % Glaberrima.

AA X BB A (pollen) + B (egg) --> A (50%)B (50%)

Mostly infertile and very few seeds are formed
After back-crossing --> A(85%)B(15%) highly fertile plant
Such a hybrid does not form in Nature, first because the two species grow on
different continents, but second because the pollination is nearly infertile.
It combines genes from two different species, but is not a GMO.

Inter-specific hybridization:
Nerica, an upland rice for
Africa.
Asian rice

African rice
Nerica rice

Oryza sativa (Asian upland rice): non-shattering,

resistant to lodging, high yield potential
Oryza glaberrima (African rice): drought tolerant,
disease resistant, weed-suppressing
Nerica rice combines the best of both species.

Triticale, a new cereal created in the

lab.
Triticale, a cross between wheat and rye, was
produced by embryo rescue of the product of
fertilization and a chemically induced doubling of
the chromosomes. Embryo rescue becomes
necessary when fertile offspring is never produced
by an interspecific cross.

Triticaleisnowgrownalloverthe
world

Wheat,triticaleandrye

Marker Assisted Selection (MAS)

Marker Assisted Selection

is the use of molecular markers to follow the inheritance of genes, particularly those
genes which cannot be readily identified.
Selection of a marker flanking a gene of interest allows selection for the presence
(or absence) of a gene in a new progeny.

Marker Assisted Selection (MAS)

AFLP-markers of Individuals in a population
1 2 3 4 5 .
Marker bands
at different
marker loci
may
co-segregate
with
certain traits
(e.g.
resistance,
yield, etc.)

Marker identification:
Materials
and Methods

QTL (Quantitative Trait Locus)

Crossing of two diverse lines (differing in quant. res.)

Development of a segregating
mapping population

Resistance evaluation in
several environments

Genotyping with
molecular (DNA)
markers

Construction of a
genetic map (putting
markers in order)

Phenotypic data

QTL Analysis for co-segregation of markers with a trait

Protoplast fusion and plant somatic hybrids

(IHAR, Plant Breeding and Acclimatization Institute, Radzikow, 05-870 Blonie, Poland
http://www.ihar.edu.pl/ZIKiT/ )

Mutation breeding
Artificially induced mutations can be used in plant breeding if
-

a cultivar is to be improved by change of one

specific character controlled by one gene

the character is not available in breeding material,

which can be crossed with the cultivar

the character can not be transferred with molecular

biology methods

Mutation breeding

Spontaneous mutations of a gene occur with

probability 10-6 per meiosis
In general mutations are
-

recessively inherited
negative effects
not directed

Induction of mutations

The mutation rate can be increased by

Radiation
- x-rays
- gamma-rays
- ultraviolet radiation

Chemical mutagens, e.g., sodium azide (N3Na)

Long time cell or tissue culture

Temperature
Mutagen = carcinogen!

Induction of mutations

The mutation rate can be increased by

Radiation
- x-rays
- gamma-rays
- ultraviolet radiation

Chemical mutagens, e.g., sodium azide (N3Na)

Long time cell or tissue culture

Temperature
Mutagen = carcinogen!

Radiation

(From Romagosa, 2004)

Sodium azide (N3Na)

(From Romagosa, 2004)

Mutation breeding
Types of mutations
Genome mutations
- change in the number of chromosomes
- addition or loss of chromosome sets: polyploidy
- addition or loss of chromosomes: aneuploidy
Chromosome mutations
- translocation
- deletion
- duplication
- inversion
Gene (point) mutations
Usually gene mutations
- cause sterility
- are
lethal

Extracellular mutations
- occur in organelles
- show maternal inheritance
Example:
cytoplasmic male sterility (CMS) is
very useful in hybrid breeding
for example in rye

Conclusions: Mutation breeding

Genetic variation can be induced.

New, previously not observed characters can be generated.
Limited applications!
Success can not be calculated!
During the past 70 years, worldwide more than 2250
varieties have been released that have been derived
from mutants or mutant progenies
(Ahloowalia et al. 2004: Global impact of mutation-derived varieties,
Euphytica 135, 187-204)

Gene transfer
Examples

Product

Gene donor

Recipient

Insect resistance
Bt-Protein

Bacillus thuringiensis

Cotton, tomato
maize, rice
Tobacco

Trypsin-Inhibitor Prot. Vigna ung.(cowpea)

Virus resistance
(Cover protein)

Tobacco mosaic virus

Potato leafroll virus
Potato Virus X and Y

Herbicide resistance Streptomyces hygro.

PhosphinotricinacetylTransferase (Basta res.)

Tomato
Potato
Potato
Beta vulgaris
Rape seed
(Ordon & Friedt, 1998)

Gene transfer
Example: Golden Rice
Vitamin A deficiency affects 800 million people.
It impairs vision, immune response, skelettal growth,
fertility (male and female), and embryo-genesis.Despite
intensive traditional interventions it leads, to date, to
500 000 blind children p.a.
Rice as major staple does not contain any provitamin A.
The transgenic concept: introduce, under endospermspecific regulation, all genes necessary to establish the
biochemical pathway for beta carotene.
The required enzymes are:
phytoene synthase and Lycopene cyclase (Narcissus), and
phytoene double-desaturase (Erwinia bacteria)
Potrykus, 2002: http://nutrition.tufts.edu/ppt/agri_biotech/potrykus.ppt

Genetic engineering of golden rice

Inserting pro-vitamin A genes into popular varieties
using Natures genetic engineer Agrobacterium:

Narcissus

Kernel

Genes
Plasmids

Locally
important
varieties

Hull

Agrobacteria
Embryo

Provit. A
producing rice Embryo
(Bennett 2002: http:/www.irri.org/apec/APEC-Bennett-2.pps)

Promises of the Golden rice

- in 2005 on the market

- 300 g of the best lines shall cover
15 -20% of the daily required
Vitamin A of an adult.
- 200 g sufficient to avoid chronic
diseases

Golden Rice: the reality

- Originally, rel. small content of Beta-carotine: 1.6 g/g
- conversion rate: 12 g Beta-carotine 1 g Vitamin A
- Required: Adults: 500 -800 g Vitamin A ; Kids: 300 g
9 kg Rice would have to be eaten daily by adults.
- But 2006: Improved Golden Rice (with different genes from maize)
shows high Beta-Carotine Conc. Of up to 37 g/g
(Coghlan, 2005, New Scientist)

Source

Beta-Carotine Conc.
(g/g Fresh weight)

Red Palmoil
Carot
Sweet potato
Mango
Papaya, Melon
Golden Rice
New Golden Rice

93
46-125
11
6
2-3
1.6
37

however,
1 teaspoon Palmoil,
2 teaspoon sweet potato,
or 1 Mango could cover
the Vit. A requirement of
5 yr old Kids.

Outlook: Future challenges of plant breeding

Food
security

Food
quality

Ecology

Keep pace Reduce mal- Reduce

Protect
with popul. nutrition of pesticide ground
growth
800 million applic.
water
people

Yield,
Product Resistance
yield
quality to insects,
stability
diseases,
weeds

Economy

Adapt to
Conserve Reduce Reduce
changes in biosurproduction
climate/soil diversity pluses and proces(industr. sing costs
countries)

Nutrient
Stress
efficiency, tolerance
N fixation

Industrial /
Special
medical plants characminor / under- ters
utilized crops

Solutions offered by plant breeding: new cultivars and crops

Artropodlar

ELISA
(Enzyme-Linked Immuno Sorbant Assay)

Variacin somaclonal

ITS

(Internal Transcribed Spacer)

Ribosomals
Genes

Highly conserved genes :

18S, 5.8S y 28S

ITS Sequences
Hundreds and thousands copies
in the genome
Appropiate length to
Sequence.
Highly variable regions
among species

Figure 1. Pattern alleles of Argentinean and Peruvian criollas

varieties using VrZAG79 loci.

Cirolla Chica
Criolla Grance
Buredeos
Buredeos Tacna
Uvina
Borgoa
Mollar ICA
Mollar Majes
Torronts Sanjuanino
Torronts Riojano
Torronts Mendocino
Moscatel Mendocino
Moscatel Rosado
Pedro Gimnez
Malbec
Rosada Vitor
Moscatel

Quebtanta

Italia Tacna

Italia Majes

Italia Moquegua

Negra Corriente NCI

Negra Corriente Majes

Negra Corriente Tacna

Salt tolerance
Genetic Transformation
Pre-embryogenics
callus from anthers
co- cultured with
Agrobacterium
tumefaciens LBA
4404

RB

pCaMV 35S-nptII-tNOS

pCaMV35S-AtNHX1-tNOS

LB

Acclimatization
Adaptation of tissue cultured plants
to non-culture environment
Extremely different environments - in
and out of culture
May require special techniques for
hardening

Acclimatization
Most plants cannot be transferred
directly from culture to the greenhouse
or field
Many characteristics of plants grown in
vitro are very different from that in
plants grown in situ

Environments

Tissue culture
Low light
High RH ~98%
[CO2] - 300 ppm
Sucrose present
Sterile

Greenhouse/field
High light
RH ~ 30-80%
[CO2] - low
No sucrose
Pathogens present

Changes in tc plants

Epicuticular wax
Stomatal functioning
Leaf anatomy
Root functioning

Hyperhydric Plants
Most extreme - in maladaption to
outside environment
Glassy, water-soaked appearance
Poorly developed palisade layer
Reduced lignification
Thin cell walls
Large intercellular spaces
Lack of epicuticular wax

Causes of Hyperhydricity
Physical state of the medium
Liquid
Agar
Gelrite

Cytokinins

Normal TC plants
Can TC plants be normal?
Many of hyperhydric symptoms
except less severe
Poorly developed cuticle
May have epicuticular wax
Large intercellular spaces
Minimal palisade development

Water loss

Epicuticular
Wax
Carnation tc
plants
A. Tissue
culture
B. Growth
chamber
C. Growth
chamber
D. Abnormal
E. Greenhouse

Epicuticular
Wax
Carnation
A.
Greenhouse
B.
Greenhouse
C. Tissue
culture
D. Tissue
culture

Epicuticular wax on plants

Leaf Anatomy
Narrow cuticle
Large intercellular air
spaces
Poorly developed palisade
layer

Strawberry - Greenhouse
grown
palisade
layers
air spaces

spongy
mesophyll

Strawberry - Tissue culture

one layer
of palisade
cells

large air
spaces

Changes that Must Occur

Epicuticular wax must be formed
Stomates must respond more rapidly
Photosynthetic activity - increased
Note: the amount of change is not
the same for all plants.

Stomatal Functioning

Conductance of Tissue Cultured

Plants

Techniques for
Acclimatization
Removal of sucrose from roots
Raise RH
fog
intermittent mist

Reduced light
Cheesecloth
Saran cloth

Gradually reduce RH and increase

light

Hood in Commercial Lab

Packing Tissue Cultured Plants

Tissue Cultured Plants in

Greenhouse

Mist Systems for TC Plants

FASE IV: TRANSFERENCIA AL AMBIENTE

NATURAL
El xito conclusivo del cultivo de
explantes depende de la capacidad de
aclimatar plantas en condiciones ex
vitro.

100% humidity in the test tube

no wax
poor stomatal response

What is subculturing?
Transferring plant tissue to a new culture

Tissue Culture Systems

Plantlet
Plantlet formation
formation -- Axillary
Axillary shoot
shoot formation
formation from
from nodes
nodes

Multiple Node culture

Single Node culture

Micropropagation - The Basics

[auxin]

10

[cytokinin] 1

10

30

Micropropagation - The Basics

[auxin] : [cytokinin] regulate
organogenesis
Organogenesis the formation of shoots
or roots

[cytokinin]

0.3

0.3

0.3

[auxin]

0.1

0.3

0.3
1.0

0.3

mg/L

3.0

mg/L

Micropropagation - The Basics

Stage II Multiplication of shoots
Subcultured or transferred to media
with
[cytokinin] = shoot initiation

callus

CIM: callus inducing media [auxin] =

[cytokinin]
SIM: shoot inducing media [cytokinin ]

African violet
leaf explant

Tissue Culture Systems

Plantlet
Plantlet formation
formation -- Adventitious
Adventitious shoot
shoot formation:
formation:

Tissues that can regenerate plants:: cotyledons

cotyledons and
and hypocotyls
hypocotyls

cotyledons

hypocotyl

monocot | dicot

gymnosperm

Tissue Culture Systems

Plantlet
Plantlet formation
formation -- Adventitious
Adventitious shoot
shoot formation:
formation:

Tissues that can regenerate plants: young

young needle
needle fascicles
fascicles
fasciclesheath

fasciclesheath

Tissue Culture Systems

Plantlet
Plantlet formation
formation -- Adventitious
Adventitious shoot
shoot formation:
formation:

Tissues that can regenerate plants:: immature

immature
inflorescence
inflorescence

Gladiolus
Gladiolus
Iris
Iris
Hosta
Hosta
Orchids
Orchids
Mums
Mums
Daylily
Daylily

Tissue Culture Systems

Plantlet
Plantlet formation
formation -- Adventitious
Adventitious shoot
shoot formation:
formation:

Tissues that can regenerate plants:: base

base of
of bulb
bulb scales
scales

Adaptacin de plantas obtenidas in

vitro a condiciones naturales
Una de las etapas que reviste gran importancia
dentro la tcnica de propagacin in vitro es el
transplante de las plntulas al suelo, as como
la adaptacin de estas al medio ambiente, lo
cual se llevado a cabo el xito en varias
especies a travs de numerosos intentos.
Podemos considerar tres fases secuenciales
para lograr una buena respuesta:
1.FASE DE ACLIMATACIN.
2.FASE INVERNADERO.
3.FASE DE CAMPO.

1. FASE DE ACLIMATACIN
Proceso por el cual las plantulas se ajustan fisiolgicamente y
anatmicamente de condiciones de cultivo y medio ambientales in vitro a
condiciones ex vitro.
Dos razones por las cuales las plantas micropropagadas pueden no poder
aclimatarse ex vitro: baja competitividad fotosinttica (nutricin
heterotrfica) y pobre control de la perdida de agua.
Durante la fase de aclimatacin los cultivos debern ser colocados durante
una o dos semanas bajo un ambiente controlado, donde la intensidad
lumnica deber ser de 10 000 lux; tanto la temperatura como el
fotoperiodo sern reguladores de acuerdo con las necesidades de cada
cultivo.
La plntula dentro del tubo de ensayo se encuentran bajo condiciones de
esterilidad y con la alta hmedad relativa la cual debera reducirse
eliminando el papel parafilm (si se usa) unos cinco das antes al transplante
al suelo, lo que dar a la planta mayor tolerancia a la baja hmedad
relativa del medio ambiente, facilitndole su posterior adaptacin a
condiciones auttrofas, donde tendrn que regular adecuadamente su
procesos de absorcin, translocacin y transpiracin del agua.

2. FASE INVERNADERO
Una vez efectuada la fase de aclimatacin las plantas
pueden ser transportadas y trasplantadas en el
invernadero donde sern colocadas en un sustrato,
proporcionndoles un buen sistema de drenaje y
aireacin para evitar el desarrollo de hongos y bacterias.
Antes de efectuar el transplante es recomendable que el
sustrato sea esterilizado en una autoclave a una
temperatura de 121 C por 30 a 60 minutos
(dependiendo de la cantidad), lo cual reducir la
incidencia de patgenos, particularmente los que causas
pudricin de raz.
La etapa de adaptacin a invernadero puede tener una
duracin de cuatro a ocho meses, dependiendo de la
respuesta particular de las plantas.

3. FASE DE CAMPO
El transplante a campo se recomienda
realizarlos en das nublados y teniendo
en suelo una humedad a capacidad de
campo.
La metodologa del trasplante al
campo estar en funcin del tipo de
cultivo con el cual se est trabajando.
Sin embargo, se recomienda que una
vez efectuado el trasplante se aplique
al suelo algn fertilizante comercial.