Dr. R.

Divya Bandhavi
SOAS, MRU
 Definition
• Anther Culture: It is an artificial technique by which the
developing anthers at a precise and critical stage are excised
aseptically from unopened flower bud and are cultured on a
nutrient medium where the microspores within the cultured
anther develop into callus tissue or embryoids that give rise
to haploid plantlets (formation of haploid plants) either
through organogenesis or embryogenesis.
• The first report of haploid tissue from anther culture was
in 1964-1966 in pollen grains of Datura by Maheshwari
and Guha.
• Production of reported in about 250 species,
Solanaceae,
haploids gramineae/Poaceae are most
cruciferae,
• common
Anther/pollen culture is referred as ANDROGENESIS (the
male gametophyte (microspore or immature pollen)
produces haploid plant)
 SHORT

 HISTORY
First haploid callus developed by Yamada 1963- Tradescantia
Bourgin and Nitch -1967, mature haploid plants in Nicotiana
Anther/Pollen
culture
Homozygous diploid(BB &
bb)
 The procedure to establish the anther culture of the
plant is as follows:
Select buds- surface sterilize them using a disinfectant.
Excise anthers along with their filaments
Crush an anther and stain it with acetocarmine to test the pollen development
stage.
If the correct stage is found, gently detach anthers from filaments and place
them horizontally in the culture medium
Maintain anther cultures in alternating periods of light(12-18 h; 5000-10 000
lx m2) at 28℃ and darkness (12-6 h) at 22 ℃.
As the anthers respond, wall tissues turn brown and after 3-8 weeks, callus
form
The embryos will germinate on the same media they were cultured or
required to be transferred to another culture media.
When the plantlets attain a height of 3-5 cm, transfer them to a rooting
medium.
After well-developed rooting is observed, transfer the plants to sterilize
potting mix in small pots or seed
A) Swollen anther (after 2 weeks in culture).
B) Cristal, after 6 weeks in culture, producing callus masses at both sides.
C) After 6 weeks in culture, producing both callus masses and embryos
(arrowhead).
D) After 6 weeks in culture, producing only embryos (arrowheads).
E) Regenerating in vitro plantlet
F) fully acclimated plant
G) Shifted to a pot
Pollen culture
1) Genotype of donor plant :
• Determine the frequency of pollen plant
production.
Eg. In Hordeum vulgare each genotype differs
with respect to androgenic response in
anther culture.
• Japonica respond better than Indica.
• High responsive anthers should be taken.
2) Anther wall factor :
•Act as conditioning factors and promote culture
growth.
3) Stage of pollen:
•Development stage of pollen varies with species.
•Before/after 1st pollen mitosis- Datura, tobacco,etc.
4) Physiological status of donor plant:
a) Grown under best environmental
conditions with good anthers.
b) Flowers obtained at the beginning
of flowering season are highly responsive.
5) Pretreatment of anthers :
•Appropriate treatment required for good success
of haploid production(depend on donor plant
species).
6) Temperature influence:
a)Induction of androgenesis is better if stored at
low temperature prior to culture, e.g. maize , rye.
b)Pre-treatment of anthers at higher temperature
stimulates androgenesis
6) Effect of light:
•Pre-treatment of anthers at elevated
temperatures( 3°C) stimulate androgenesis in some
Brassica and Capsicum.
7) Culture medium:
•It vary with the genotype and age of the anther.
•Culture maintained on an auxin medium for longer
period develop a friable callus.
•A compound related to auxin namely 2,3,5-
triiodobenzoic acid (TIBA) gives +ve result at low
concentration.
•Incorporation of activated charcoal/2-chloroethyl-
phosphate stimulates androgenesis in some
systems.
 Applications

• Utility of Anther and Pollen Culture for Basic

Research
• Simple
• Less time consuming
• Responsive
• Mutation Study
• Use of Haploids for Cryogenic Study
• For Plant Breeding and Crop Improvement
• Application of Haploid Culture
for Horticultural Plants
• For study of Secondary metabolites Content
 Applications of haploids in crop improvement
 Dihaploid production through bulbosum technique is successful in
barley.
 Can obtain mutations, selection of mutants resistant to disease.
 Haploids provides relatively easiest system for the induction of
mutations. Therefore they can be employed in rapid selection of
mutants having traits for disease resistance. a. Eg:- Tobacco mutants
resistant to Black shank disease and wheat lines resistant to scar.
Fusarium graminorium
 Developing asexual lines of trees (or) perennial species.
 Pollen derived rubber tree is taller by 6m which could be multiplied by
asexual propagation
 Transfer of desired alien genes/ genetic transformation.
 Haploids are used for the development of substitutional and additional
lines.
 Double haploids are used for QTL mapping and construction of genetic
linkage maps
 Requires skill to remove anthers
without causing damage.
 Not much successful in case of cereal crop.
 Risk of chimera and callus
formation from anther wall.
 Double haploid segregation
 Gameto clonal variations
 Due to callus- dele. Mutations will come
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YOU