Embryogenesis and doubled haploid production from anther culture in gentian (Gentiana triflora)

Abstract

The overall goal of this study is to develop an anther culture system to produce doubled haploid (DH) lines of gentian (Gentiana triflora), an ornamental flowering plant, for use in an F1 hybrid breeding program. Embryogenesis was induced from anther cultures incubated on half-strength modified Lichter (NLN) medium containing a high concentration of sucrose (130 g/l) and subjected to heat shock treatment. Among the various parameters investigated, anthers collected from buds 9-12 mm in length induced the highest frequency of androgenesis. Moreover, among three genotypes tested, cvs. Ashiro-no-Aki and Ashiro-no-Natsu produced 21.3 and 3.7 embryos per 100 anthers, respectively, whereas, cv. Lovely-Ashiro failed to produce embryos. Among a total of 427 embryos transferred to a regeneration medium consisting of Murashige and Skoog (MS) medium, 138 plants were regenerated. The ploidy levels of regenerants were determined by flow cytometry and chromosome counts, revealing the presence of 5% haploids, 25% diploids, and 70% triploids. Inter simple sequence repeat (ISSR) analysis using the 6PS line obtained following self-pollination of the diploid plant obtained from anther culture confirmed that the diploid plant was indeed a DH.

An Efficient Method for Plant Regeneration from Calli of <i>Swertia mussotii</i>, an Endangered Medicinal Herb

“…Most importantly, micropropagation provides an efficient protocols for conservation of plant biodiversity and multiplication of the endangered species. Recently, in vitro propagation procedures have been adopted for large-scale propagation of a number of medicinal plants in the family Gentianaceae [5][6][7][8][9][10]. Despite being a valuable medicinal plant, very little work has been done on tissue culture of S. mussotii.…”

Haploid and Doubled Haploid Plant Production in Gentian (Gentiana spp.)

“…After surface sterilization of buds, five anthers dissected from buds are cultured in a 60-mm plastic Petri dish containing 3 ml of 1/2 NLN medium (Takahata and Keller 1991) supplemented with 13 % (w/v) sucrose. The cultures are incubated at 32.5°C This unknown species is considered to be G. scabra from SNP (single nucleotide polymorphism) analysis (Doi et al 2010) for 1 day in darkness, prior to maintenance at 25°C with a 16/8 h photoperiod. After 2-4 months of culture, embryos emerge from the yellowish and/or brownish anthers.…”

In Vitro Studies and Biotechnology of Taiwan Native Species of the Gentianaceae

“…They established plant regeneration system in G. cruciata and G. purpurea, and a callus induction in G. punctata and G. pannonica. Since then, several reports have been published on different members of the Gentianaceae, for example, G. lutea (Lamproye et al 1987;Viola and Franz 1989), G. pneumonnthe (Lamproy et al 1987), G. tibetica (Skrzypczak-Pietraszek et al 1993), G. kurroo (Sharma et al 1993), G. triflora (Tariba 1994), G. lutea, G. cruciata, G. purpurea, G. acaulis (Momčilović et al 1997), G. davidii (Chueh et al 2000), G. lutea (Železnik et al 2002), interspecific hybrids between G. triflora and G. lutea (Morgan 2004), G. punctata (Butiuc-Keul et al 2005), G. asclepiadea (Dević et al 2006), G. straminea (Cai et al 2009), G. pannonica (Fiuk et al 2010), and somatic embryogenesis and the generation of double haploid (dihaploid) plant in G. triflora (Doi et al 2010).…”