1. 1.Terminology and Background 2.Processes Leading to Production of Haploid Plants 3.Production of Haploids through Chromosome Elimination and Embryo Rescue 4. Production of Haploids In Vitro through Anther and Microspore Culture Plant Cell, Tissue and Organ Culture HORT 515 Haploids In Vitro

2. 1. Terminology and Background Haploid - gametic number of chromosomes, n which may not be equivalent to x Monoploid - haploid derived from a diploid, x is one genomic complement Polyhaploid - haploid from a polyploid (n  x), prefix indicates genome complement number, e.g. tobacco is a dihaploid

3. 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 Agricultural applications for haploids - Rapid generation of homozygous genotypes after chromosome doubling

4. 2. Processes Leading to Production of Haploid Plants Androgenesis – haploid plant derived from male gamete, most common method in vitro Parthenogenesis - from unfertilized egg Apogamy - from other cells of the mega-gametophyte, example Chromosome elimination - chromosome elimination in somatic cells, most common method used with plant breeding

5. Parthenogenesis and Apogamy

6. Androgenesis – haploid plant derived from male gamete, most common method in vitro Parthenogenesis - from unfertilized egg Apogamy - from other cells of the mega-gametophyte Chromosome elimination - chromosome elimination in somatic cells, most common method used with plant breeding

7. 3. Production Haploids through Chromosome Elimination and Embryo Rescue Production of haploids by chromosome elimination - There are numerous examples, primarily achieved by wide crosses and embryo culture The barley example - Achieved by an interspecific cross between barley (Hordeum vulgare, 2n = 2x = 14, VV, female) x H. bulbosum (2n = 2x = 14, BB, male), see examples

8. Day 0 - emasculation Day 2 - pollination with H. bulbosum pollen Day 3 (to 5) - 40% of the embryonic cells are haploid, endosperm abortion occurs, GA 3 treatment enhances retention of florets Day 11 - 94% of the embryonic cells are haploid Day 14 (to 16) - embryos are dissected and cultured in the dark at 18 to 22 C, embryos develop in vitro Day 22 (to 28) - embryos are transferred to light for seedling development Day 50 - plants Cross (VV x BB) Progeny:VVVVBVBB n =(7)(14)(14)(21) 1517 0 26 0 Monoploid Production of Barley (H. vulgare)

9. Barley Monoploid Production H. vulgare (n=7) H. vulgare H. bulbosum (n=7) H. bulbosum X Chromosome Elimination Embryo Culture and Haploid Plant Production Hybrid Zygote

10. Production of Barley Haploids through Chromosome Elimination and Embryo Rescue Possible mechanisms for chromosome elimination: Asynchrony of mitotic cycle times - H. bulbosum cell cycle is much longer Spindle or centriole abnormalities - spindle formation or centriole attachment of H. bulbosum chromosomes is defective in the H. vulgare nucleus

11. 4. Production of Haploids In Vitro through Anther and Microspore Culture Definition, History and Background Anther and microspore (pollen) culture - haploid plants are derived from microspores (pollen) cultured individually or in anthers History: Tulecke (1953) - haploid callus (but no plants) derived Ginkgo biloba Guha and Maheshwari (1964) - haploid plants derived from cultured Datura anthers Nitsch, C (1974) - haploid plants derived from cultured tobacco microspores Background – micro-sporogenesis and micro-gametogenesis leading to pollen development, example

13. 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, example

14. Vegetative Generative Similar Nuclei Germination Haploid Proembryo Haploid Embryo First Mitosis Microspore Tetrad Microspore Mother Cell

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, example Donor plant or anther pretreatment – enhances haploid embryo formation Actively growing plants and the first set of flowers are most responsive Cold pretreatment of anthers - either pre- or post-culture treatment (3 to 5 o C for 2 to 4 days), symmetric rather than asymmetric division of the microspore nuclei or division of the vegetative nucleus

17. 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, example Donor plant or anther pretreatment – enhances haploid embryo formation Actively growing plants and the first set of flowers are most responsive Cold pretreatment of anthers - either pre- or post-culture treatment (3 to 5 o C for 2 to 4 days), symmetric rather than asymmetric division of the microspore nuclei or division of the vegetative nucleus, examples

18. 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 VegetativeNuclei

19. 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 Tobacco Days in Culture 5°C for 72 h Control 0 0 3712 5 10 % Pollen w/identical nuclei

20. 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 Hormone dependency as follows: Hormone independent group - embryos directly from the microspores w/o callus, 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. Microspore/pollen culture – bi-cellular pollen types only - basal components + glutamine, serine and elevated levels of i-inositol, example

21. 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.