This document discusses anther culture and pollen culture techniques. It describes the history and development of these techniques, including the first observations of pollen proliferation in culture and embryo development from microspores. It explains the processes of anther culture, pollen culture, androgenesis and different pathways of pollen development. The document outlines the procedure for anther culture and factors that influence culture success. Applications of anther and pollen culture include mutation studies, plant breeding, and genetic engineering.

1. Topic:Anthercultureandpollenculture

2. Anther
culture and
Pollen
culture

3. History:
• W. TULECKE(1953) First observed the mature pollen
grain of zinkgo biloba (a gymnosperm) can be induced
to proliferate in culture to form haploid callus.
• S.Guha and S. C Maheshwari (1964 ) first reported the
direct development of embryos from microspores of
Datura innoxia by the anther culture.
• J. P. BOURGIN AND J. P. NITSCH(1967) Obtained
complete haploid plantlets from anther culture of
nicotiana tabacum

4. Anther culture :
Culturing of anthers obtained from
unopened flower bud in nutrient
medium under aseptic condition.
Callus tissue or embryoids from
anther, that give rise to haploid
plantlets either though organogenesis
or embryogenesis.

5. Pollen
Anther
Pollen culture :
Pollen or microspore culture is an in vitro
technique by which the pollen grains
preferably at the uni-nucleated stage, are
squeezed out aseptically from the intact
anther and then cultured on nutrient medium.
The microspore develop into haploid
embryoids or callus that give rise to haploid
plantlets by embryogenesis or organogenesis.

6. Androgenesis :
Androgenesis is the in vitro development
of haploid plants originating from
totipotent pollen grains through a series
of cell division and differentiation.
It is of two types :
1) Direct androgenesis
2) Indirect androgenesis

7. Direct
androgenesis :
Indirect
androgenesis :
The microspores behaves like a
zygote and undergoes change to
form embryoid which ultimately
give rise to a plantlet.
The microspores divide
repeatedly to form a callus tissue
which differentiates into plantlets.

8. Principle of anther and pollen culture :
 The production of haploid plants is to exploit
the totipotency of microspore.
 In the process the normal development and
function of the pollen cell to become a male
gamete is stopped and is diverted forcely to a new
metabolic pathway for vegetative cell division

9. Pathways of pollen development :
Pathway I
The microspore divide by an equal division and
two identical daughter cells developed .
Vegetative and generative cells are not distinctly
formed in the pathway.
Example : Datura innoxia

10. Pathway II
 The division of uninucleate microspores is unusual ,
resulting in the formation of vegetative and generative
cell.
 The sporophyte arises through further division in the
vegetative cell and generative cell does not divide.
 Examples: Nicotiana tabacum, Hordeum vulgare,
triticum aestivum.

11. Pathway III
 The uninucleate microspore undergoes
pollen embryos are formed from generative
cell alone.
 The vegetative cell does not divide .
 Examples : Hyoscyamus niger

12. Pathway IV
 Both generative and vegetative cell
divide further to the development of
sporophyte.
 Examples: Datura metal, Atropa
belladona, Datura innoxia.
Pathway V
In Brassica napus 1st division is symmetric and
the pollen embryos develop the vegetative cell.

13. Slide Title
Product A
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Product B
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14. Procedure of Anther Culture
First, select the unopened buds of size about 17-22mm in
length.
Buds are washed under running tap water to remove the dirt.
Then, transfer the selected buds to the laminar airflow
chamber.
After that surface sterilization of buds is carried out in the
chamber to maintain the aseptic conditions. Firstly, the buds are
surface sterilized by 70% ethanol for 10 seconds and then 20% of
sodium hypochlorite for 10 minutes.
Then, wash the buds three times by the distilled water.

15. Continue……..
After washing, transfer the buds to the sterilized Petri plate.
Then, separate the stamen from the bud through a scalpel.
Remove the filaments from an anther.
After that, transfer an anther onto the solid or liquid
nutrient medium and incubate it for 3-4 weeks at 24-28
degree C in the dark.
Then either by embryogenesis and organogenesis, haploid
plantlets would appear from the anther culture within 3-4
weeks. During this stage, incubate the culture at 24-28 ֯C for
12-18 hours in light and 6-12 hours in the dark.

16. continue,…..
At last, about 50mm tall plantlets will appear
that are then transferred into the pot containing
bio compost followed by washing.

18. Factors Influencing Anther Culture
Anther wall: It provides nourishment during the developmental stages of the anther.
Stage of microspore : Anthers are more productive when cultured at uninucleate stage.
Bud size: Its preferable size is up to the length of 17-22 mm.
Age of plant: The anther culture prefers the buds from the younger plants.
Culture medium: In the culture medium sucrose, iron, vitamins, coconut milk,
hormones and growth regulators (auxins or cytokinins) play an essential role in the
induction of haploid plant.
Temperature: It varies with the different plant species. Example: In Datura stramonium,
the optimum temperature is 20˚C for the formation of embryoids. In Nicotiana tabacum,
the optimum temperature is 25˚C for the formation of embryoids. In Brassica campestris,
the optimum temperature is much higher, i.e. 35˚C for the formation of embryoids.

19. Advantages
• Simple
• Less time consuming
• Responsive
Disadvantages
• 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.

20. Applications:
• For mutation study .
• For plant breeding and crop improvement.
• Haploids are used in molecular biology and
genetic engineering. ( Haploids tissue of
Arabidopsis and Lycopersicon have been used
for transfer and expression of three genes
from Escherchia coli

21. •
Thankyou