1. Haploids & their applications

2. Definition  The term haploid refers to those plants which possess a gametophytic number of chromosomes (n) in their sporophytes.

3. History - In1953, Tulecke obtained haploid callus (but no plants) derived from Ginkgo biloba. - In1964, Guha and Maheshwari reported direct development of embryosfrom microspores of Datura innoxia. - In1967, Bourgin & Nitsch obtained complete haploid plants of Nicotiana tabacum.

4. Overview of floral organs

5. Haploid Plant Formation In vitro methods: –Anther culture (androgenesis) - production of haploid plants from microspores Anther culture for production of haploids reported in about 250 species Solanaceae, Cruciferae, Gramineae, Ranunculaceae most common –Ovule culture (gynogenesis) - production of haploid plants from unfertilized egg cell.

6. Androgenesis Haploid plant derived from male gametophyte of an angiosperm plant (most common method in vitro). Haploids can be obtained by the culture of excised anthers & culture of isolated pollen.

7. Reproductive floral organs: male Stamen – male floral organ, consists of: -Anther – A typical anther shows 2 anther lobes & each lobe possesses 2 microsporangia or pollen sacs. -Filament – stalk-like structure that holds anther -Pollen – immature male gametophyte

9. Microsporogenesis/microgametogenesis leading to haploid embryo formation Haploid embryo formation based on continued divisions of the vegetative or generative cells - embryos are derived from continued proliferation of either of these cells rather than pollen formation. Haploid embryo formation based on symmetric division of the microspore - rather than asymmetric division that leads to pollen formation, most common path to haploidy.

10. Normal pollen development Pollen mother cells are in anther primordia First phase - meiosis - pollen mother cell (PMC) A tetrad forms from each PMC Second phase - microspores released from tetrads Third phase - microspores mature into pollen grains - first pollen mitosis Second pollen mitosis, maybe after germination Generative and vegetative cells formed

11. Pathways to Androgenesis

12. Culture medium Anther culture – - essential micro- and macronutrients, sucrose and vitamins; bicellular pollen types require 2 to 4% and tricellular types 6 to 12% sucrose. Microspore/pollen culture – - bi-cellular pollen types only - basal components + glutamine, serine and elevated levels of inositol.

13. Culture medium Hormone dependency as follows: Hormone independent group - embryos directly from the microspore, predominantly bi-cellular pollen types, e.g. tobacco Hormone dependent group - bi- or tri-cellular pollen types and plants are regenerated through a callus intermediary, typically requires auxin and, in some instances cytokinin, e.g. grasses.

14. Growth Regulators Pollen embryogenesis requires low levels of auxins, cytokinins and giberellins. Wheat anthers cultured on a medium having 2,4-D produce callus while those kept on a medium supplemented with coconut milk give rise to embryos.

15. Factors affecting the development of haploid plants in vitro Anther stage - most responsive cells for haploid embryo formation are those between the tetrad stage of microsporogenesis to just past the first pollen mitosis. Donor plant or anther pretreatment – enhances haploid embryo formation. Cold pretreatment of anthers - either pre- or post- culture treatment (3 to 5 o C for 2 to 4 days).

16. Similar nuclei 3 to 5°C Microspore Embryo 3 to 5°C Generative Vegetative Cold Treatment (3 to 5°C) Enhances Symmetric Division of Microspores or Division of Vegetative Nuclei

17. Cold Pretreatment of Anthers Enhances the Embryogenic Response Cold treatment imposed prior to the first pollen mitosis increases the frequency of symmetric divisions of the microspore leading to embryo formation, control – room temperature. TobaccoDatura 0 20 40 60 80 100 5°C5°C 3°C3°C C C % Anthers Producing Embryos

18. Factors influencing androgenesis –Genotype of donor plants –Anther wall factors –Culture medium and culture density –Stage of microspore or pollen development –Effect of temperature and/or light –Physiological status of donor plant

19. In vitro morphogenesis of pollens Direct androgenesis (Normal embryogenesis occurs as in the plants of family Solanaceae and Brassicaceae) Indirect androgenesis (Callus formation occures as in barley, wheat and coffee)

20. Bajaj, Y.P.S. 1983. In D.A. Evans, W.R. Sharp, P.V. Ammirato, and Y. Yamada (eds.), Handbook of Plant Cell Culture. Volume 1. Techniques for Propagation and Breeding. MacMillan, New York. p. 228-287. Production of haploid plants & diploidization

21. Flower buds Anther Microspore mother cells Meiosis Tetrads of microspores (Pollen grains) Uninucleated pollens Culture medium Callus (haploid) Embryogenesis Plantlet (n) Mutagens 0.5% Colchicine For 24-48 hrs Binucleate (n) Fusion (2n) Plantlet (2n)

22. Gynogenic Haploids Haploid plant derived from megaspore or female gametophyte of an angiosperm plant. Haploids can be obtained by the culture of excised ovary and ovule.

23. Gynogenic Haploids In vitro culture of unpollinated ovaries and ovules represents an alternative for the production of haploid plants in species for which anther culture has given unsatisfactory results. Used in plant families that do not respond to androgenesis –Liliaceae –Compositae The first report on the induction of gynogenic haploids was in Barley by San Noeum (1976)

24. Culture medium The normal White or MS or N 6 inorganic salt media supplemented with growth substances are used. Sucrose as a carbon source is essential

25. Factors influencing gynogenesis –Genotype of donor plants –Growth conditions of the donor plant –Stage of harvest of ovule –Embryo-sac stage –Culture medium –Physical factors

26. Value of Haploids in Breeding Haploids are very valuable in plant breeding for several reasons –Since they carry only one allele of each gene, mutations and recessive characteristics are expressed in the plant. –Plants with lethal genes are eliminated from the gene pool. –Can produce homozygous diploid or polyploid plants - valuable in breeding –Shorten the time for inbreeding for production of superior hybrids genotypes.

27. Agricultural applications for haploids Rapid generation of homozygous genotypes after chromosome doubling. Reduce time for variety development, e.g. 10 to 6 years or less. Homozygous recombinant line can be developed in one generation instead of after numerous backcross generations. Selection for recessive traits in recombinant lines is more efficient since these are not masked by the effects of dominant alleles.