The effect of several factors on somatic embryogenesis and plant regeneration in protoplast cultures of Gentiana kurroo (Royle)

Abstract

Protoplasts were isolated from cell suspensions derived from cotyledon and hypocotyl Gentiana kurroo (Royle). Cell walls were digested with an enzyme cocktail containing cellulase, macerozyme, driselase, hemicellulase and pectolyase in CPW solution. Protoplast viability ranged from 88 to 96%. Three techniques of culture and six media were evaluated in terms of their efficiency in producing viable cultures and regenerating whole plants. With liquid culture, cell division occurred in only a low number of the protoplasts isolated, and no plant regeneration was successful. Cell division occurred within 2 or 3 days in case of agarose solidified media. After 10 days of culture, the number of dividing cells was the highest with modified MS medium in which NH 4 NO 3 was replaced with 3.0 g l -1 glutamine. The best results were obtained with agarose bead cultures: plating efficiency was 68.7% and 58.1% for protoplasts isolated from cotyledon and hypocotyl derived suspensions, respectively. The results were achieved with using medium containing 0.5 mg l -1 2,4-D + 1.0 mg l -1 kinetin or 2.0 mg l -1 BAP + 1.0 mg l -1 dicamba + 0.1 mg l -1 NAA + 80 mg l -1 adenine sulfate. Protocalluses transferred on the following composition of plant growth regulators: 0.5 mg l -1 2,4-D + 1.0 mg l -1 kinetin or 1.0 mg l -1 kinetin + 0.5 mg l -1 GA 3 + 80.0 mg l -1 adenine sulfate developed in embryogenic cultures. However, the best embryo production occurred with the first one. Later embryos were transferred to halfstrength MS mineral salts to promote plants formation. Flow cytometry studies revealed increased amounts of DNA in about one third of the regenerants.

Plant regeneration from protoplasts of Gentiana straminea Maxim

“…The calli derived from sLD and liquid culture systems were non-embryogenic, while those derived from aPL culture system showed both embryogenic and non-embryogenic characteristics. The effects of various culture systems on callus formation from protoplasts have also been reported in other plants [7,8,15,24]. In Gentiana kurroo, globular somatic embryos were detected one week after the microcolonies obtained in the thin layer culture and bead culture had been transferred to induction medium, but the cell aggregates obtained in the liquid culture died when transferred to the same medium [15].…”

“…In Oenothera hookeri, embedding protoplasts in thin alginate layers was considered one of the most crucial parameters for cell division and colony formation [23]. In Gentiana kurroo, the plating efficiency of protoplasts reached 68.7% using agarose bead culture [15]. We also tested the effect of culture methods on protoplast division and colony formation.…”

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

“…The presence of aneuploid cells (5-6C) in some calli of G. kurroo in the present study resulted in the regeneration of a single plant, which, however, showed abnormal morphology and did not survive. In our previous studies on G. kurroo, about 30-37 % polyploids (tetraploids and hexaploids) was regenerated from cell suspensionderived protoplasts (Fiuk and Rybczyński 2007), and 14 % tetraploids from hypocotyl-derived cell suspension (Fiuk and Rybczyński 2008a).…”

“…Recently, we succeeded in regenerating plants from leaf mesophyll protoplasts of G. decumbens by indirect somatic embryogenesis (Tomiczak et al 2015). PRM3 medium containing 1.0 mg l -1 kinetin, 0.5 mg l -1 GA 3 , 80 mg l -1 adenine sulfate, which led to the formation of somatic embryos from protoplast-derived tissues of G. decumbens, was the same medium as was widely used for the induction of somatic embryogenesis from callus and cell suspensions of G. pannonica, G. cruciata, G. tibetica (Mikuła and Rybczyński 2001;Mikuła et al 2002), and G. kurroo (Fiuk and Rybczyński 2008a), as well as from protoplasts of G. kurroo cell suspensions (Fiuk and Rybczyński 2007). The greatest number of somatic embryos regenerated from mesophyll protoplast-derived callus of G. tibetica and G. kurroo was also achieved by using this medium.…”

“…So far, plants of the genus Gentiana have been regenerated only from green leaf mesophyll protoplasts of G. scabra (Takahata and Jomori 1989), G. triflora and G. scabra x G. triflora (Nakano et al 1995), from callus protoplasts of G. crassicaulis (Meng et al 1996) and from cell suspension protoplasts of G. kurroo (Fiuk and Rybczyński 2007;Wójcik and Rybczyński 2015) and G. macrophylla (Hu et al 2015). Although leaves are the most widely accessible plant material for protoplast isolation, cultures derived from leaf mesophyll protoplasts of gentians are usually associated with low frequency of morphogenetic processes.…”