HAPLOID

PRODUCTION –
ANDROGENESIS
PRESENTED BY :

Vipul Aggarwal
Ravinandan Goyal
 WHAT IS HAPLOID
PRODUCTION IN PLANTS?

The development of a new plant

from a cell containing haploid
number of chromosomes.
 HAPLOID PLANT
PRODUCTION
•Formation in vivo
–Spontaneous occurrence in low frequency
–Induction by physical and/or chemical treatment
–Chromosome elimination following interspecific
hybridization. Specific for some plants such as barley.
Not widespread.
•In vitro methods:
–Anther culture (androgenesis) - production of haploid
plants from microspores
•Anther culture for production of haploids reported
in about 250 species
•Solanaceae, Cruciferae, Gramineae, Ranunculaceae
most common
–Ovule culture (gynogenesis) - production of haploid
plants from unfertilized egg cell
VALUE OF HAPLOIDS IN
BREEDING
•Haploids are very valuable in plant breeding for
several reasons
–Since they carry only one allele of each gene,
mutations and recessive characteristics are
expressed in the plant.
–Plants with lethal genes are eliminated from
the gene pool.
–Can produce homozygous diploid or polyploid
plants - valuable in breeding
–Shorten the time for inbreeding for
production of superior hybrids genotypes.
 HISTORY
Anther and microspore ( pollen ) culture - haploid
plants are derived from microspores (pollen) cultured
individually or in anthers
•
History :
Tulecke (1953) - haploid callus (but no plants) derived
Ginkgo biloba
Guha and Maheshwari (1964) - haploid plants derived from
cultured Datura anthers

Nitsch , C (1974) - haploid plants derived from cultured

tobacco microspores
 PATHWAYS TO
ANDROGENESIS
 MICRO - SPOROGENESIS /
MICRO - GAMETOGENESIS
LEADING TO HAPLOID EMBRYO
FORMATION
Haploid embryo formation based on continued
divisions of the vegetative or generative cells -
embryos are derived from continued proliferation of
either of these cells rather than pollen formation
•
•
Haploid embryo formation based on symmetric
division of the microspore - rather than asymmetric
division that leads to pollen formation, most common path
to haploid formation , example
FACTORS AFFECTING HAPLOID
EMBRYO FORMATION

–Genotype of donor plants

–Anther wall factors
–Culture medium and culture density
–Stage of microspore or pollen development
–Effect of temperature and/or light
–Physiological status of donor plant
•Genotype
–Response is genotypically determined depending
on the species. In cereals, there is a major
genetic component controlled by many genes.
–In plants such as tobacco, genotype is less
important.
•Anther wall factors
–The specific compounds are not known. Addition
of anther wall extracts, however was
promotive in tobacco.
–In some plants, glutamine alone in in
combination with serine and myo-inositol
replaced the wall factors.
•
Donor plant or anther pretreatment – enhances
haploid embryo formation

•Actively growing plants and the first set of flowers

are most responsive
•
•Cold pretreatment of anthers - either pre- or post-
culture treatment (3 to 5 oC for 2 to 4 days),
symmetric rather than asymmetric division of the
microspore nuclei or division of the vegetative
nucleus, examples
Cold Treatment ( 3 to 5 ° C ) Enhances Symmetric
Division of Microspores or Division of
Vegetative nuclei

3 to 5 ° C

Vegetative Microspore

Similar nuclei

Generati
ve
3 to 5 ° C

Embryo
 Cold Pretreatment of Anthers Enhances the
Embryogenic Response
Cold treatment imposed prior to the first pollen
mitosis increases the frequency of symmetric
divisions of the microspore leading to embryo
formation, control – room temperature.

100 3°C Tobacco

Producing Embryos

80

w / identical nuclei
5 ° C for 72 h
5°C
% Anthers

C
60 10
% Pollen
40 5 Control
C
20
0
0 3 7 12
0
Tobacco Datura Days in Culture
 Effect of culture
medium
•Two hormone groups
•Without hormones - mostly dicots. Most
success with solanaceous species. Do not
want the anther wall to form callus.
•With hormones - most non-solanaceous species.
Many monocots. Require hormones or complex
organics such as coconut milk.
•Medium particularly important in cereals and
rice to be able to produce green plants. A
major difficulty was large number of
albino plants that resulted.
–Sucrose - ranges from 2% (Nicotiana) to 10%
(Brassica)
•Density
•In Brassica napus minimum density
required is 3000 pollen/ml of culture
medium
•
•Atmospheric volume of the vessel
•For embryos 15 ml/anther
•For producing plants 5.5 ml/anther
•Effect may be ethylene
 PRODUCTION OF DOUBLED
HAPLOIDS
•Use solution of colchicine which interferes with
cell division, but DNA is doubled
•For polygenic traits, use two anther-derived plants
–Shortens the breeding cycle considerably by
reducing number of generations required in
normal breeding programs
 PROBLEMS ASSOCIATED
WITH ANTHER CULTURE
•Anthers fail to grow, embryos fail to continue growth
•Developing tissue or callus may be diploid or polyploid
–Chimera of different ploidy may result
•Formation of albinos in cereals (especially rice)
•Low success rate - not commercially viable
•Use of growth regulators for callus production usually
detrimental for haploid production since diploid and
polyploid cells are produced
•Doubled haploids sometimes are not homozygous
–Segregation may be seen in progeny
THANK YOU