Plant Cell Reports

, Volume 30, Issue 6, pp 1055–1065 | Cite as

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

  • Ranjith Pathirana
  • Tonya Frew
  • Duncan Hedderley
  • Gail Timmerman-Vaughan
  • Ed MorganEmail author
Original Paper


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.


Anther culture Breeding Callus Flow cytometry Ovary culture RAPD markers 



2,4-Dichlorophenoxyacetic acid






Doubled haploid


Least significant difference


Naphthoxyacetic acid


Plant growth regulator


Randomly amplified polymorphic DNA





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.


  1. Cai X, Xu SS (2007) Meiosis-driven genome variation in plants. Curr Genomics 8:151–161PubMedCrossRefGoogle Scholar
  2. Cai YF, Liu YL, Liu ZH, Zhang F, Xiang FN, Xia GM (2009) High-frequency embryogenesis and regeneration of plants with high content of gentiopicroside from the Chinese medicinal plant Gentiana straminea Maxim. In Vitro Cell Dev Biol Plant 45:730–739CrossRefGoogle Scholar
  3. Chen LY, Chen QL, Xu D, Hao JG, Schlappi M, Xu ZQ (2009) Changes of gentiopicroside synthesis during somatic embryogenesis in Gentiana macrophylla. Planta Med 75:1618–1624PubMedCrossRefGoogle Scholar
  4. Coleman A, Goff L (1985) Applications of fluorochromes to pollen biology. 1. Mithramycin and 4′,6-diamidino-2-phenylindole (DAPI) as vital stains and for quantitation of nuclear DNA. Stain Technol 60:145–154PubMedGoogle Scholar
  5. Datta SK (2005) Androgenic haploids: factors controlling development and its application in crop improvement. Curr Sci 89:1870–1878Google Scholar
  6. Doi H, Takahashi R, Hikage T, Takahata Y (2010) Embryogenesis and doubled haploid production from anther culture in gentian (Gentiana triflora). Plant Cell Tissue Organ Cult 102:27–33CrossRefGoogle Scholar
  7. Duan YW, He YP, Liu JQ (2005) Reproductive ecology of the Qinghai-Tibet Plateau endemic Gentiana straminea (Gentianaceae), a hermaphrodite perennial characterized by herkogamy and dichogamy. Acta Oecol 27:225–232CrossRefGoogle Scholar
  8. Eason JR, Debenham M, McLachlan A, Morgan E (2007) Novel red-flowered Gentiana: an emerging export cut flower crop from New Zealand. In: Kanlayanarat S, Nell T, Eason J (eds) Proceedings of the international conference on quality management in supply chains of ornamentals. King Mongkuts University of Technology, Thonburi, pp 259–266Google Scholar
  9. Fiuk A, Rybczynski JJ (2008a) Genotype and plant growth regulator-dependent response of somatic embryogenesis from Gentiana spp. leaf explants. In Vitro Cell Dev Biol Plant 44:90–99CrossRefGoogle Scholar
  10. Fiuk A, Rybczynski JJ (2008b) Morphogenic capability of Gentiana kurroo Royle seedling and leaf explants. Acta Physiol Plant 30:157–166CrossRefGoogle Scholar
  11. Forster BP, Heberle-Bors E, Kasha KJ, Touraev A (2007) The resurgence of haploids in higher plants. Trends Plant Sci 12:368–375PubMedCrossRefGoogle Scholar
  12. Gaspar T (1991) Vitrification in micropropagation. In: Bajaj YPS (ed) Biotechnology in agriculture and forestry, vol 17. High-tech and micropropagation I. Springer, Berlin, pp 116–126Google Scholar
  13. Hosokawa K, Oikawa Y, Yamamura S (1998) Mass propagation of ornamental gentian in liquid medium. Plant Cell Rep 17:747–751CrossRefGoogle Scholar
  14. Hou QZ, Meng LH, Yang HL (2008) Pollination ecology of Gentiana siphonantha (Gentianaceae) and a further comparison with its sympatric congener species. J Syst Evol 46:554–562Google Scholar
  15. Ivanova M, Van Staden J (2010) Natural ventilation effectively reduces hyperhydricity in shoot cultures of Aloe polyphylla Schönland ex Pillans. Plant Growth Regul 60:143–150CrossRefGoogle Scholar
  16. Kim M, Jang I (2000) Rapid assessment of microspore development stage in pepper using DAPI and ferric chloride. J Plant Biotech 2:129–134Google Scholar
  17. Kim M, Kim J, Yoon M, Choi DI, Lee KM (2004) Origin of multicellular pollen and pollen embryos in cultured anthers of pepper (Capsicum annuum). Plant Cell Tissue Organ Cult 77:63–72CrossRefGoogle Scholar
  18. Lassner MW, Peterson P, Yoder JI (1989) Simultaneous amplification of multiple DNA fragments by polymerase chain reaction in the analysis of transgenic plants and their progeny. Plant Mol Biol Rep 7:116–128CrossRefGoogle Scholar
  19. Lincoln S, Daly M, Lander E (1992) Constructing linkage maps with MAPMAKER/EXP 3.0. Whitehead Institute Technical Report, 3rd edn. Whitehead Institute, Cambridge, MAGoogle Scholar
  20. Linsmaier EM, Skoog F (1965) Organic growth factor requirements of tobacco tissue cultures. Physiol Plant 18:100–106CrossRefGoogle Scholar
  21. Morgan ER (2004) Use of in ovulo embryo culture to produce interspecific hybrids between Gentiana triflora and Gentiana lutea. N Z J Crop Hortic Sci 32:343–347CrossRefGoogle Scholar
  22. Morgan ER, Hofmann BL, Grant JE (2003) Production of tetraploid Gentiana triflora var. japonica ‘Royal Blue’ plants. N Z J Crop Hortic Sci 31:65–68CrossRefGoogle Scholar
  23. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  24. Nitsch JP, Nitsch C (1969) Haploid plants from pollen grains. Science 163:85–87PubMedCrossRefGoogle Scholar
  25. Nixon J (2010) Testing for segregation distortion in genetic scoring data from backcross or doubled haploid populations. Heredity 96:290–297Google Scholar
  26. Pintos B, Manzanera JA, Bueno MA (2007) Antimitotic agents increase the production of doubled-haploid embryos from cork oak anther culture. J Plant Physiol 164:1595–1604PubMedCrossRefGoogle Scholar
  27. Raina R, Behera MC, Chand R, Sharma Y (2003) Reproductive biology of Gentiana kurroo Royle. Curr Sci 85:667–670Google Scholar
  28. Rodrigues LR, Oliveira JMS, Mariath JEA, Iranco LB, Bodanese-Zanettini MH (2005) Anther culture and cold treatment of floral buds increased symmetrical and extra nuclei frequencies in soybean pollen grains. Plant Cell Tissue Organ Cult 81:101–104CrossRefGoogle Scholar
  29. Segui-Simarro JM, Nuez F (2008) How microspores transform into haploid embryos: changes associated with embryogenesis induction and microspore-derived embryogenesis. Physiol Plant 134:1–12PubMedCrossRefGoogle Scholar
  30. Shalaby TA (2007) Factors affecting haploid induction through in vitro gynogenesis in summer squash (Cucurbita pepo L.). Sci Hortic 115:1–6CrossRefGoogle Scholar
  31. Takahata Y, Jomori H, Miyono S, Kunitake H, Mii M (1995) Regeneration of plants from protoplasts of Gentiana species (Gentian). In: Bajaj YPS (ed) Biotechnology in agriculture and forestry, vol 34. Plant protoplasts and genetic engineering. Springer, Berlin, pp 55–62Google Scholar
  32. Timmerman GM, Frew TJ, Miller AL, Weeden NF, Jermyn WA (1993) Linkage mapping of sbm-1, a gene conferring resistance to pea seed-borne mosaic virus, using molecular markers in Pisum sativum. Theor Appl Genet 85:609–615CrossRefGoogle Scholar
  33. Tomiczak K, Rybczynski JJ, Wojcik A, Mikula A (2009) Morphogenic potential of Gentiana kurroo (Royle) cell suspension after cryotreatment. Cryo Lett 30:396–396Google Scholar
  34. Winkelmann T, Geier T, Preil W (2006) Commercial in vitro plant production in Germany in 1985–2004. Plant Cell Tissue Organ Cult 86:319–327CrossRefGoogle Scholar
  35. Zeleznik A, Baricevic D, Vodnik D (2002) Micropropagation and acclimatization of yellow gentian (Gentiana lutea L.). Zb Bioteh Fak Univ Ljubl 79:253–259Google Scholar
  36. Zhang ZM, Leung DWM (2002) Factors influencing the growth of micropropagated shoots and in vitro flowering of gentian. Plant Growth Regul 36:245–251CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Ranjith Pathirana
    • 1
  • Tonya Frew
    • 2
  • Duncan Hedderley
    • 1
  • Gail Timmerman-Vaughan
    • 2
  • Ed Morgan
    • 1
    Email author
  1. 1.The New Zealand Institute for Plant and Food Research LimitedPalmerston NorthNew Zealand
  2. 2.The New Zealand Institute for Plant and Food Research LimitedChristchurchNew Zealand

Personalised recommendations