title: Haploid and doubled haploid plants from developing male and female gametes of Gentiana triflora.
abstract: Protocols were developed for the generation of haploid or doubled haploid plants from developing microspores and ovules of Gentiana triflora. Plant regeneration was achieved using flower buds harvested at the mid to late uninucleate stages of microspore development and then treated at 4°C for 48 h prior to culture. Anthers and ovaries were cultured on modified Nitsch and Nitsch medium supplemented with a combination of naphthoxyacetic acid and benzylaminopurine. The explants either regenerated new plantlets directly or produced callus that regenerated into plantlets upon transfer to basal media supplemented with benzylaminopurine. Among seven genotypes of different ploidy levels used, 0-32.6% of cultured ovary pieces and 0-18.4% of cultured anthers regenerated plants, with all the genotypes responding either through ovary or anther culture. Flow cytometry confirmed that 98% of regenerated plants were either diploid or haploid. Diploid regenerants were shown to be gamete-derived by observing parental band loss using RAPD markers. Haploid plants were propagated on a proliferation medium and then treated with oryzalin for 4 weeks before transfer back to proliferation medium. Most of the resulting plants were diploids. Over 150 independently derived diploidised haploid plants have been deflasked. The protocol has been successfully used to regenerate plants from developing gametes of seven different diploid, triploid and tetraploid G. triflora genotypes.
authors.affiliation: The New Zealand Institute for Plant and Food Research Limited, Private Bag 11600, Palmerston North 4442, New Zealand.
authors.forename: Ranjith
authors.initials: R
authors.lastname: Pathirana
authors.forename: Tonya
authors.initials: T
authors.lastname: Frew
authors.forename: Duncan
authors.initials: D
authors.lastname: Hedderley
authors.forename: Gail
authors.initials: G
authors.lastname: Timmerman-Vaughan
authors.forename: Ed
authors.initials: E
authors.lastname: Morgan
mesh_terms: D001593:Benzyl Compounds / Q000494:pharmacology; D018744:DNA, Plant / Q000235:genetics; D004171:Diploidy; D005434:Flow Cytometry; D035264:Flowers / Q000187:drug effects / Q000254:growth & development; D005838:Genotype; D030012:Gentiana / Q000187:drug effects / Q000235:genetics / Q000254:growth & development* / Q000502:physiology; D055993:Germ Cells, Plant / Q000254:growth & development*; D006016:Glycolates / Q000494:pharmacology; D006238:Haploidy; D006824:Hybridization, Genetic / Q000187:drug effects; D010937:Plant Growth Regulators / Q000494:pharmacology; D011110:Polymorphism, Genetic / Q000187:drug effects; D011687:Purines / Q000494:pharmacology; D019105:Random Amplified Polymorphic DNA Technique; D012038:Regeneration / Q000187:drug effects / Q000502:physiology; D013045:Species Specificity; D046509:Tissue Culture Techniques
chemical_list: D001593:Benzyl Compounds; D018744:DNA, Plant; D006016:Glycolates; D010937:Plant Growth Regulators; D011687:Purines; C018610:1-naphthoxyacetic acid; C480551:benzylaminopurine