Anther and microspore culture

A haploid is a sporophyte with the gametic chromosome number

Methods for producing haploids
I. Conventional methods
1. Parthenogenesis or apogamy
2. Delayed pollination
3. Irradiation of pollen
4. Temperature shocks and
5. Distant hybridization
II. In vitro methods
1. Anther & microspore culture (successful in >200 species)
2. Ovule culture
3. Chromosome elimination after inter-specific crossing or Bulbosum method
Pathways lead to the development of haploids in tissue culture
I.Division unequal.
Normal microspore mitosis giving rise to a bi nucleated microspore with a generative and
vegetative nucleus
1. Continued mitotic division of the vegetative nucleus leads to the formation of callus or
embryoids Triticale, Capsicum, Datura Hordeum
2. Formation of embryoids from the generative nucleus occurs, but is not common.
Hyocyamus niger (black henbane)
3. Asymmetrical as above. Both contribute for embryo/callus development Datura, Atropa
II. Division Equal
1. Mitosis of uni nucleated microspore resulting in formation of two equal and identical cells.
Callus or embryoids formed by continued divisions of these cells. Datura innoxia
2. Two similar nuclei fuse together, results in formation of homozygous diploids-Datura
Patterns of Development
1. Cell division in anther/microspore cultures results in the development of haploid calli or
embryos.
2. In embryogenic cultures, the stages of development are analogous to zygotic
embryogenesis and globular, heart-shaped torpedo and cotyledonary stages can be
observed.

Microspore culture
Culturing pollen grains /microspores to generate haploid plants.
Reasons:
1. Anther wall detrimental to microspores
2. Anther wall is involved in formation of callus
Methods of isolation of microspores
1. Spontaneous
2. Homogenization and filtration
3. Slit techniques
Diploidization/ Dihaploid production
Most of the plants produced by anther/microspore culture are haploid.. In order to obtain fertile
plants, the chromosome number of the haploids must be doubled which is most often
accomplished by treatment with colchicine. The different methods include
1. Placing the roots of the haploid plants in a colchicine solution
2. Applying a colchicine solution directly to the meristems in leaf axils
3. Adding colchicine to the tissue culture medium.
Utilization of Haploids and Doubled Haploids
1. Complete homozygosity in one step, compared to 7-8 generations of selfing by normal
breeding approaches
2. Recessive alleles are uncovered, thus, facilitating mutational breeding
3. Doubled haploid populations have less complex genetic ratios than the corresponding F2
population, hence, analysis of qualitative or quantitative genetics is made easier (genotype
of gamete = genotype of plant)
4. It is possible to obtain pure stands of one sex in dioecious species.
Problems
1. Extreme variability in the response to culture
2. Albinism in the haploids
3. Very low response in some species

4. Colchicine doubling not successful in some species.

Questions
1. How will you generate haploid plants?
2. Explain the different pathways leading to development of haploid plants.
3. Distinguish anther and microspore culture
4.

Explain the culture technique for anther culture.

5. How will you isolate microspores?

6. How will you double the haploids? What is the exact mechanism
involved.
7. What are the applications of anther culture?
8. Enumerate some problems associated with anther culture.