BASIC TERMINOLOGY OF FLOWER INTRODUCTION TO ANTHER AND POLLEN CULTURE PROCEDURE FOR CULTURE FACTORS INFLUENCING CULTURE APLLICATIONS HOMOZYGOUS PLANTS HAPLOID PLANTS

1. ANTHER &POLLEN CULTURE
HARI KRISHNA PALEM
6TH SEMISTER
BIOTECHNOLOGY AND BIOINFORMATICS

2. CONTENTS
• Introduction
• History
• Method of anther culture
• Method of pollen culture
• Factors influencing anther & pollen culture
• Advantages of pollen culture over anther
• Importance of pollen & anther culture

3. Introduction
• Basic Terminology of Flower
• Anther: A part of stamen containing pollen
• Pollen : fertilising powder discharged from flowers
anther

4. Principle of anther & pollen culture
• The production of haploid plants is to exploit
the totipotency of microspore.
• In this process the normal development and
function of the pollen cell to become a male
gamete is stopped and diverted forcely to a
new metabolic pathway for vegetative cell
divison

5. Anther culture
• Culturing of anther obtained from unopened
flower bud in the nutrient medium under
aseptic conditions.
• Callus or embryoids that give rise to haploid
plantlets either through organogenesis or
embryogenesis

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

7. Androgenesis
• Androgenesis is the in vitro development of
haploid plants originating from the totipotent
pollen grains through a series of cell divison
and differentiation.
• It is of two types:-
• Direct androgenesis.
• Indirect androgenesis.

8. • Direct androgenesis:- the microspore behaves
like zygote and undergoes chance to form
embryoid which ultimately give rise to a
plantlet.
• Indirect androgenesis:- the microspore divide
repeatedly to form a callus tissue which
differentiates in to haploid plantlets

9. Process of Androgenesis

10. History
• W.TULECKE(1953): first observed that mature
pollen grains of Ginkgo biloba (gymnosperm) can
be induced to proliferate in culture to form haploid
callus.
• G.GUHA AND S.C MAHESWARI(1964): first reported
the direct development of embroys from
microspores of ‘Datura innoxia’ by the culture of
excised anther.
• J.P BOURGIN AND J.P NITSCH(1967): obtained
complete haploid plantlets from anther culture of
‘Nicotiana tabacum’.

11. Procedure for anther culture
• Collection of unopened flower buds
• Surface sterilisation with enthanol and sodium
hypochloride.
• Anthers excised from flower buds and kept separately.
• Anthers in first meiotic divison is selected by acetocarmine
test.
• Inoculated in the medium containing glutamine,
L-serine and inositol.
• Incubate the culture at 25 c for 15 days. Here, the anthers
grow into embryoids.
• Embryoids transfer to the rooting medium under 3000 lux
illumination after 4-5 weeks the embryoids become plantlets.
• for the acclimatization, transfer to green house.

12. process of anther culture

13. Procedure for pollen cultuer
• Anthers are collected from flower buds and pollen
grains are isolated and about 50 anthers are placed
in small sterile beaker containing 20ml of liquid
basal medium (MS or white or Nitsch)
• Anthers are then pressed against the side of a
beaker with sterile glass rod to squeeze the pollen.
• The homogenised anthers are then filtered through
a nylon sieve to remove that the anther tissue
debris.
• The filtrate or pollen suspension is then
centrifuged at low speed for five minutes.

14. • The supernatant containing fine debris are discarded and
pellet of pollen is suspended in fresh liquid medium and
washed twice by repeated centrifugation and resuspended
in fresh liquid medium.
• A 2.5 ml pollen suspension is pipetted off and is spread in
the 5cm petri dishes.
• Pollen are best grown in liquid medium but, if necessary
they can be grown by plating soft agar added medium.
• Petri dishes are incubated at 27-30c under low intensity of
white cool light (500 Lux,16hrs).
• Young embryoids are observed after 30 days.these are
ultimately give rise to haploid plantlets.

16. Advantages of pollen culture over anther
• During anther culture there is always the
possibility that somatic cells of the anther that
are diploid will also respond to the culture
condition and so produce unwanted diploid calli
or plantlets.
• Sometimes the development of microspores
inside the anther may be interrupted due to the
growth inhibiting substances leaking out of the
anther wall in contact with nutrient medium.

17. Factors influencing anther culture
• Genotype of donor plants:- the genotype of the donor
plant plays significant role in the determining the
frequency of the pollen production.
• Example:- ‘Horedum’ of each genotype differs with
respect to the androgenic response in anther culture.
• Anther wall factor:- the anther wall provide the
nourishment in the development of isolated pollen of a
number of species.
• There are reports that glutamine alone or in
combination with serine and myoinositol could replace
the anther wall.

18. • Culture medium:- for anther culture, medium
requirements may vary with ‘genotype and age of
anther ‘as well as the condition under which
donor plants are grown.
• Incorporation of activated charcoal into medium
has stimulate the induction of androgenesis.
• The iron in the medium plays a importantly role
for the induction of haploids.
• Potato extracts, coconut milk and growth
regulators like ‘auxins’ and ‘cytokinins’ are used
for anther and pollen culture.

19. • Stages of microspores :- anthers are most
productive when cultured at the uninucleate
microspore stage.
example :-barley, wheat, rice etc.
Anther of some species give the best
response if pollen cultured at first mitosis or
later stage
example :- Datura ,tobacco

20. • Effect of temperature :-Temperature enhances
the induction frequency of microspore
androgensesis.
• The low temperature treatment to anther or
flower bud enhance the haploid formation.
• The low temperature effects the number of
factors such as the dissolution of microtubules
lowering of abscisic acid maintenance of higher
ratio of viable pollen capable of embryogenesis.

21. • Physiological status of donor plant :-
physiological status of donor plant such as the
water stress nitrogen requirement and age of
the donor plant highly affect the pollen
embryogenesis.
• Plants starved of nitrogen may give more
responsive anthers compared to those that
are well fed with nitrogenous fertilizers.

22. Advantage of pollen culture and anther
• Utility of anther and pollen culture for basic research
cytogenetic studies.
• Study of genetic recombination in higher plants.
• Study of mode of differentiation from single cell to
whole organism.
• Study of factors controlling the pollen
embryogenesis of higher plants.
• Formation of double haploid that are homozygous
and fertile.

23. • For mutation study.
• For plant breeding and crop improvement.
• To obtain the secondary metabolites.
• Example:- ‘Hyoscyamus nigrer’ obtain by anther
culture having higher alkaloid content.
• Haploids are used in molecular biology and genetic
engineering.