Anther culture and pollen culture

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 6 TH 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 A ndrogenesis

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.

15. 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.

16. 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.

17. 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.

18. 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

19. 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.

20. 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.

21. 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.

22. 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.