production of double haploid plants

production of double haploid plants

1. PRODUCTION OF DOUBLE HAPLOID PLANTS Presented by:- Desai Vruddhi K. Reg. No- 04-AGRMA-01581-2017 Department of Genetics & Plant Breeding C.P.College of Agriculture Sardarkrushinagar , Dantiwada

2. INTRODUCTION A doubled haploid (DH) is a genotype formed when haploid cells undergo chromosome doubling. Artificial production of doubled haploids is important in plant breeding . Haploid cells are produced from pollen or egg cells or from other cells of the gametophyte, then by induced or spontaneous chromosome doubling, a doubled haploid cell is produced, which can be grown into a doubled haploid plant.

3. HISTORY Blakeslee et al. (1922) - Datura stramonium Guha and Maheswari (1964) - Anther culture technique for the production of haploids in the laboratory • Niizeki and Oono (1968)- Production of rice haploids • wide crossing Kasha and Kao, (1970) - Barley Burk et al., (1979) - Tobacco Doubled haploid methodologies have now been applied to over 250 species

4. Why we need double haploids(DH) Development of homozygous lines Fixation of heterosis Mutation studies and easy to induce mutation Production of biotic and abiotic stress resistant plants Cytogenetical research Induction of genetic variability at haploid level Double haploids in genome mapping Evolutionary studies

5. What are uses of double haploids Development of purelines Development of cultivars Development of hybrids as parents Construction of genetic maps Gene tagging/location genes Identification of moleculars markers for trait selection.

6. How to induce haploids 1. wide crossing 2. Irradiation and- chemical treatment, 3. Selection of twins, and 4. By anther and pollen culture.

7. Methods to Induce DHs In vitro Methods Haploids from male gametes a) Anther culture b) Pollen/Microspore culture Haploids from female gametes a) Ovary slice culture b) Ovule culture

8. ANTHER CULTURE 1.collection of unopened flower buds 2.surface sterilized with tween 80 and mercuric chloride 3.Anthers excised from flower buds and kept seperately 4.Anthers in first meiotic division is selected by acetocarmine test 5.Inoculated in the medium containing glutamine, L-serine and inositol 6. Incubated the culture at 25˚C for 15 days. Here, anthers grow in to embryoids . 7. embryoids transfer to rooting medium under 3000 lux illumination after 4-5 weeks the embryoids became plantlets. 8.After aclimatization , transfer to green house

9. POLLEN/MICROSPORE CULTURE 1.Anther collected from flower buds and pollen grains are isolated and inoculated in the nutrients medium with the concentration of pollen 0.5ml. 2.Nitsh (1974) medium is used for pollen culture 3. Anthers are place on the medium. some times nurse culture may used. 4. A paper disc is placed over anther or callus. 5 O.5ml pollen suspension is poured on the disc 6 Petridish covered with lid and incubated at 50˚C for 4 weeks. Pollengrains grown in to individual clones with 60% efficacy. 7 Clones are transferred to callus induction medium to produce calli . 8 Calli transfered to shooting medium and then to rooting medium to produce plantlets

10. Importance of pollen and anther culture 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 factor controlling pollen embryogenesis of higher plants Formation of double haploid that are homozygous and fertile.

11. Ovary Slice Culture Ovary slice culture technique involves culture of transverse sections of unpollinated ovaries on culture media. The following protocol was used to induce in vitro gynogenesis in Tea ( Hazarika and Chaturvedi 2012): a.  For Ovary slice culture in Tea, unopened and unpollinated mature flower-buds (6-10 mm) size were collected early in the morning. Some of the buds were fixed in FAA (5:5:90 v/v/v Formaldehyde: Acetic acid: 70% Ethanol), for 48 h, and then stored in 70% alcohol. Later on, the appropriate developmental stage of the embryo sac was determined by histological analysis.

12. conti …   b. The flower buds were surface sterilized with 0.1% HgCl 2 for 7 minutes, followed by rinsing with sterile distilled water at least thrice. c. Carefully dissected transverse sections of ovaries were cultured on Murashige and Skoog's media supplemented with varying concentrations of Auxins and Cytokinins . d.  Six ovary slices containing unpollinated ovules were cultured in 60 mm X 15 mm pre sterilized disposable Petriplates containing 10 ml MS medium. e.  The sealed Petriplates were subjected to various regimes of temperature and light treatments.

13. Ovule culture The unfertilized ovary is surface sterilized and the ovules were taken and placed into culture. Excision of ovule, followed by culture on specific media may be either extremely easy to accomplish, as in case of large-seeded species in which only a single ovule is present, or time-consuming and intricate, in small-seeded polyovulate species. Two types of ovule support systems have been developed. The filter paper support system involves culturing of the ovules on top of filter paper placed over liquid medium, whereas the vermiculite support technique demands placing the ovules on a sterile vermiculite/liquid media mixture (vermiculite support) with the micropylar side down. Unpollinated ovule culture has been used for haploid production in sugar beets and onions.

14. Factors influencing anther culture 1) Physiological status of donor plant 2) Effect of temperature 3) Culture medium 4) Anther wall factor 5) Stages of microspore 6) Stages of embryo sac 7) Genotype of donor plants 8) Effect of female flower position

15. Genotype of donor plants :- The genotype of the donor plant plays a significant role in determining the frequency of pollen production. Example :- Horedum of each genotype differs with respect to androgenic response in anther culture. 2) 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 myo inositol could replace the anther wall factor for isolated cultures.

16. 3) Culture medium:- For anther culture, medium Requirements vary with genotype and the age of the anther as well as condition under which donor plants are grown. Incorporation of activated charcoal into the medium has stimulated the induction of androgenesis . The iron in the medium plays a very important role for the induction of haploids . Potato extracts, coconut milk and growth regulators like auxin and cytokinin are used for anther and pollen culture.

17. 4) Stages of microspores:- Anther 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 is cultured at first mitosis or later stage Example:- datura , tobacco

18. 5) Effect of temperature:- Temperature enhance the induction frequency of microspore androgensis . The low temperature treatment to anther or flower bud enhance the haploid formation. The low temperature effects the number of factors such as dissolution of microtubules lowering of absicisic acid maintenance of higher ratio of viable pollen capable of embryognesis .

19. 6) Physiological status of donor plant:- Physiological status of donor plant such as water stress nitrogen requirement and age of 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.

20. Advantages of DHs The ability to produce homozygous lines after a single round recombination saves a lot of time for the plant breeders. Studies conclude that random DH’s are comparable to the selected lines in pedigree inbreeding. The other advantages include development of large number of homozygous lines, efficient genetic analysis and development of markers for useful traits in much less time. More specific benefits include the possibility of seed propagation as an alternative to vegetative multiplication in ornamentals, and in species such as trees in which long life cycles and inbreeding depression preclude traditional breeding methods, doubled haploidy provides new alternatives.

21. Disadvantages of DHs The main disadvantage with the DH population is that selection cannot be imposed on the population. But in conventional breeding selection can be practised for several generations: thereby desirable characters can be improved in the population. In haploids produced from anther culture, it is observed that some plants are aneuploids and some are mixed haploid-diploid types. Another disadvantage associated with the double haploidy is the cost involved in establishing tissue culture and growth facilities. The over-usage of doubled haploidy may reduce genetic variation in breeding germplasm . Hence one has to take several factors into consideration before deploying doubled haploidy in breeding programmes.

22. Case study Recent advances and application of doubled haploids in wheat breeding W. Tadesse *, M. Inagaki, S. Tawkaz , M. Baum and M. van Ginkel abstract: Genetic improvement to develop varieties with high yield potential and resistance/tolerance to abiotic and biotic stresses with acceptable end use qualities is the most viable and environment friendly option to increase wheat yield in a sustainable fashion. In vitro haploid production followed by chromosome doubling greatly enhances the production of complete homozygous wheat lines in a single generation and increases the precision and efficiency of selection process in wheat breeding.

23. Conti… It also enables the detection of linkage and gene interactions, estimate genetic variance and the number of genes for quantitative characteristics, produce genetic translocations, substitutions and chromosome addition lines, and facilitate genetic transformation and mutation studies. Wheat cultivars developed from doubled haploids using anther-culture and maize induction systems have been released for cultivation in both developed and developing countries. In this review, the origin and production of haploids, techniques in anther-culture and their application in wheat breeding are summarized.

24. The procedure of anther-culture in wheat is illustrated in Figure a = pre-treatment of the donor plants at 4°C; b = anthers in liquid induction medium; c = developing of embryos in liquid induction medium; d=embryo converting to green on solid regeneration medium; e= green plants in regeneration medium; f = haploid plants at acclimatization stage; g = haploid plants under colchicine treatment (0.2%); h = doubled haploid lines in the field showing uniformity within lines ( Tawkaz , 2011).