Comparison of the morphogenic potential of five Gentiana species in leaf mesophyll protoplast culture and ploidy stability of regenerated calli and plants

Abstract

The morphogenic potential of five Gentiana species of medicinal and ornamental value, i.e.G. cruciata, G. kurroo, G. lutea, G. septemfida, and G. tibetica, was compared using agarose-bead leaf mesophyll protoplast culture. Modified Murashige and Skoog medium containing 2.0 mg l -1 1-naphthaleneacetic acid and 0.1 mg l -1 thidiazuron ensured the highest cell division frequency during both protoplast and protoplast-derived cell culture or callus formation. Indirect plant regeneration mainly via the somatic embryogenesis pathway was achieved for G. kurroo and G. tibetica with the greatest efficiency occurring with MS medium supplemented with 1.0 mg l -1 kinetin, 0.5 mg l -1 gibberellic acid, and 80 mg l -1 adenine sulfate. A considerable percentage of autopolyploid and aneuploid cells was detected in callus lines obtained from protoplasts using flow cytometry. The presence of polyploid cells in calli resulted in the regeneration of 85 % polyploid plants of G. kurroo and 14 % polyploids of G. tibetica. Chromosome counting and stomatal characteristic confirmed the ploidy variation among regenerants.

Protoplast fusion in the genus Gentiana: genomic composition and genetic stability of somatic hybrids between Gentiana kurroo Royle and G. cruciata L.

“…For over 20 years, we have studied the morphogenic abilities of various gentian species in vitro, aimed at conserving and broadening their genetic diversity (Mikuła et al 1996(Mikuła et al , 2011Mikuła and Rybczyński 2001;Fiuk and Rybczyński 2008;Tomiczak et al 2016). The results of our experiments revealed the tremendous embryogenic potential possessed by Gentiana kurroo Royle.…”

“…1). All calli and plants were obtained following electrofusion of protoplasts isolated from 4-year-old highly embryogenic and cytogenetically stable cotyledon-derived cell suspension culture (K/C) of diploid G. kurroo (2n = 2x = 26; Fiuk and Rybczyński 2007) and protoplasts released from leaf mesophyll of in vitro-grown plants of tetraploid G. cruciata (2n = 4x = 52; Tomiczak et al 2016). Their hybrid nature was previously confirmed (Tomiczak et al 2015b).…”

“…Average nuclear DNA content for G. kurroo and G. cruciata was estimated based on flow cytometric measurements of 24 and 19 seed-derived plants, respectively. Analyses were carried out using Partec CCA flow cytometer according to Tomiczak et al (2016). Pisum sativum L. 'Set' (9.11 pg/2C; Sliwinska et al 2005) served as an internal standard for the estimation of the nuclear DNA content of G. kurroo and all somatic hybrids.…”

“…This could be due to either genome duplication at the early stage of post-fusion cell culture or fusion of four protoplasts. However, taking into consideration the tendency of gentian protoplast-derived cells to undergo spontaneous polyploidization (Fiuk and Rybczyński 2007;Tomiczak et al 2015aTomiczak et al , 2016, the first hypothesis seems more likely. Those highly polyploid forms appeared to be very unstable during the next years of in vitro culture, leading to downsizing of the genome.…”

“…The last two are tetraploid species of medicinal and ornamental importance (Köhlein 1991) that are easy to cultivate. Although the results of studies conducted so far have indicated, that G. cruciata is recalcitrant in cultures of leaf mesophyllderived protoplasts (Tomiczak et al 2016), we managed to obtain three somatic hybrid callus lines following electrofusion of their protoplasts with those isolated from cotyledon-derived cell suspension of G. kurroo and thereby, regenerated a total of 87 plants via somatic embryogenesis from two of these (Tomiczak et al 2015b). The aim of the work presented here was to characterize the genomic composition and genetic stability of selected somatic hybrids between G. kurroo and G. cruciata.…”