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Haploid and doubled haploid plants from developing male and female gametes of Gentiana triflora

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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.

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2,4-Dichlorophenoxyacetic acid






Doubled haploid


Least significant difference


Naphthoxyacetic acid


Plant growth regulator


Randomly amplified polymorphic DNA




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We wish to thank Dr. Ross Bicknell, Sylvia Erasmuson and Dianne Hall for assistance with flow cytometry; Andrew Mullen and Sriya Pathirana for tissue culture media preparation; Bruce Dobson for maintaining the plants in the greenhouse; and Dr. Mary Christey, Dr. Simon Deroles, Dr. Sue Gardiner and Ms. Catherine Ford for critical reading of the manuscript. Partial funding from the New Zealand Foundation for Research Science and Technology (contract C02X0705) is gratefully acknowledged.

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Correspondence to Ed Morgan.

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Communicated by F. Sato.

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Pathirana, R., Frew, T., Hedderley, D. et al. Haploid and doubled haploid plants from developing male and female gametes of Gentiana triflora . Plant Cell Rep 30, 1055–1065 (2011).

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