Double haploids
- 2. What is Double Haploid? • A doubled haploid (DH) is a genotype formed when haploid cells undergo chromosome doubling.
- 3. What is Haploid? Plant or cell with gametophytic chromosome number {n}. meiosis results in haploidy : Haploid organism/cell is the result of normal meiosis, in which chromosome number gets halved. So, be it diploid, tetraploid, hexaploid, etc E.g.: A hexaploid organism has 42 chromosomes; When its meiocytes undergo meiosis, each of the four resultant cells will have 21 chromosomes each and will be called haploid cells. If it's tetraploid with 42 chromosomes, result of meiosis will still remain 21. So, basically, haploid is term used for half the number of chromosomes an organism has, as a result of meiosis.
- 4. Monoploids • Monoploid : is basic set of chromosomes in an organism{x} • If the original plant was diploid {2n}, the haploid cells are monoploid {x} • Therefore n=x • If a hexaploid organism has 42 chromosomes, that means its diploid cell had 14 chromosomes and thus, its monoploid will be 7. Remember, this is different from haploid.
- 5. Difference b/w haploid and monoploid haploid monoploid haploid is (cytology) of a cell having a single set of unpaired chromosomes, such as a gamete monoploid is (genetics) having a single set of chromosomes Denoted by “n” Denoted by “x” If an organism is diploid, it's monoploidy and haploidy will be same.
- 6. Difference b/w double haploids and dihaploids? double haploids dihaploids Double haploid organism is produced by doubling chromosomes in a haploid cell. E.g: If a diploid organism has 14 chromosomes, it's haploid cells would naturally contain 7 chromosomes. If that haploid cell is subjected to chromosome duplication, resultant organism would be double haploid. Dihaploid organism is produced when haploid cells of tetraploid, hexaploid, etc. organisms are subjected to chromosome duplication. E.g. If a hexaploid organism has 42 chromosomes, it's haploid cells will have 21 chromosomes and it's dihaploid cells will have 42 chromosomes, where 21 chromosomes will be same as other 21. Note : resultant organism would be homozygous for every gene Note : this organism might be heterozygous, as genes may have different alleles in it's haploid cells itself. Of course, it's a hexaploid. Double haploid is strictly used for diploid organisms while dihaploid can be used for tetraploid, hexaploid, etc. organisms
- 7. History • 1922- Blackslee et al., reported first spontaneous haploid in Datura stramonium. • 1964: Guha and Maheshwari, first haploid via anthers of Datura inoxia
- 8. Traditional breeding / DH Breeding Traditional breeding DH Breeding After 7 yrs homozygosity attained is 98.4% After 2yrs homozygosity attained is 100% Hetrosis cannot be fixed Hetrosis can be fixed New cultivar is formed in 8-10 yrs New cultivar is formed in 3-5 yrs Recessive mutant identification is difficult Recessive mutant identification is easy Is not cost effective expensive Temporary mapping population Permanent mapping population achieved
- 9. Application of DH • Homozygosity Achieved Is 100% – In SP crops used directly as cultivar – In CP crops used as inbreds • Used to identify recessive mutants • Used as permanent mapping population • Used to identify molecular markers for trait selection • Used in QTL analysis • Constructions of genetic maps
- 10. Disadvantages of DH Frequency of haploid production is very low Highly genotype dependent Highly unpredictable Costly Chemicals used are harmful
- 11. Methods for haploid production • Chromosome elimination • Bulbosome method • Parthenogesis: pseudogamy, semigamy, apogamy • Inducer based approach In vivo method • Androgenesis: anthers • Gynogenesis: ovary, egg, ovule In vitro method
- 12. How are haploids? smaller Lower plant vigour Sterile {as unable to pair during meiotic pairing} Note : in order to use these Haploids in breeding programes and propogate them through seeds , their fertility has to be retained by either spontaneously or induced chromosome doubling
- 13. Haploid techniques Induction of maternal haploids In situ induction In vitro induction Induction of paternal haploids Androgenesis
- 14. In situ induction of maternal haploid It can initiated by pollination: pollens from Same species {maize} Irradiated pollen Wild relatives {barley, potato} Unrelated species {wheat}
- 15. Procedure pollination Fertilization of egg Hybrid developed Paternal chromosome eliminated in early embryogenesis Egg is not fertilized Haploid embryo developed Polar nuclei pollinated forming endosperm
- 16. Pollination pollen of same species Majority of regular hybrids formed Small proportion of haploid maternal embryo with normal triploid endosperm formed Irradiated pollen Irradiated pollen unable o fertilize egg cells Eg. Sunflower, nicotiana Disadvantages: need efficient emasculation Too laborius Dose of irradiation •If low: generative nucleus partly damage hence maintain the capacity to fertilize egg cells •If dose increased: decrease the no. of developed embryo but obtained regenerants are mostly haploids Wide hybridization Very effective Involves interspecific and intergeneric pollination Bulbosome method
- 17. Bulbosome method • First ever used induction method to produce large no. of haploids • In Barley: wide hybridization between barley and wild relative Hordium vulgare • Female • 2n=2x=14 H. Bulbosome • Male • 2n=2x=14 Hybrid embryo {chromosom e of both parents }
- 18. chrromosome of wild relative is preferentially eliminated from developing embryo. Due to failure of endosperm development, haploid embryo is formed. This embryo is extracted and grown invitro During embryogenesis:
- 19. Bulbosome method: wheat X maize Hybrid grown in vitro: because endosperm fails to develop in such seeds Maize chromosome eliminated after hybrid embryo formed to get wheat haploid After pollination
- 20. In vitro induction of maternal haploids Used where androgenesis and pollination failed Used in many species like cucumber, onion, sugarbeat, squash, sunflower, wheat, barley In vitro Culturing : • Ovule • Placenta attached ovule • Ovaries/whole flower bud
- 21. Induction of Paternal Haploids: Androgenesis Androgenesis : Direct: embryo Indirect: callus Used in crops like barley, wheat, maize, rice, triticale, rye, tobacco, rapeseed and other brassica Haploids are produced by using male gametophyte: Anther Microspore
- 22. Direct Androgenesis Similar to zygotic embryogenesis At globular stage: most of the embryo released from pollen call wall After 4-5 weeks: •cotyledons unfolds •Plantlet emerged from anthers Eg. Mustard and tobacco
- 23. Indirect Androgenesis At globular stage: irregular and asynchronous division occurs callus formed Organogenesis Haploid plant Eg: cereals
- 24. Major drawbacks: Androgenesis Highly genotype dependent • gametophytic to saprophytic. Relies on ability of microspore and mature pollen grain to covert their pathway : • Woody plants • Leguminous plants • A. thaliana Recalcitrance of some important agriculture species:
- 25. Factors affecting Androgenesis Development stage of male gamete Anther/ microspore stage: •uninucleated or binucleated Stress treatment: arrests the microspore in gamatophytic pathway and triggers embryogeneis by promoting cell divison and formation of embryo Temperature pretreatment Sucrose and nitrogen starvation Osmotic stress Irradiation Colchicine Auxin Water stress
- 26. Factors affecting haploid induction Genotype of donor plant • temperature • humidity Physiological conditions • Late uninucleated • Early binicleate Developmental stage of microspore • Cold • Hot Pretreatment Culture medium composition • Light • temperature Physical factors
- 27. Chromosome Doubling: Why? Haploid plants: reduced vigour Smaller One set of chromosome- no meiosis- no seed set.
- 28. Colchicine Antimicrobial drug Extracted from: “Colchicum autumnale” It binds to tubulin and inhibits the microtubule polymerization Highly toxic Used in millimolar cocentration Other options: • Oryzaline • Trifluraline • Pronamide • APM{amiprophosmethyle}
- 29. Maintenance of DH SP crops: normally selfed and maintained • Treating with co2 rich environment • Gibberlic acid • Nacl, urea, ammonium sulfate used on stigmas CP crops: for strongly self incompatible crops various techniques been used: Clonally propagated: micropropagation