Fertile wheat (Triticum aestivum L.) regenerants from protoplasts of embryogenic suspension culture

Abstract

We report regeneration of fertile, green plants from wheat (Triticum aestivum L. cv. Aura) protoplasts isolated from an embryogenic suspension initiated from somatic early-embryogenic callus. The present approach combines the optimization of protoplast culture conditions with screening for responsive genotypes. In addition to the dominant effect of the culture media, the increase in fresh mass and the embryogenic potential of somatic callus cultures varied considerably between the various genotypes tested. Establishment of suspension cultures with the required characters for protoplast isolation was improved by reduction of the ratio between cells and medium and by less frequent (monthly) transfer into fresh medium. A new washing solution was introduced to avoid the aggregation of protoplasts. However, the influence of the culture medium on cell division was variable in the different genotypes. We could identify cultures from cultivar Aura that showed approximately a 9% cell division frequency and morphogenic response. The protoplast-derived microcolonies formed both early and late-embryogenic callus on regeneration medium and green fertile plants were obtained through somatic embryogenesis. The reproducibility of plant regeneration from protoplast culture based on the cultivar Aura was demonstrated by several independent experiments. The maintenance of regeneration potential in Aura suspension cultures required establishment of new cultures within a 9-month period.

Somatic embryogenesis and fertile green plant regeneration from suspension cell-derived protoplasts of rye ( Secale cereale L.)

“…A 4-day-old suspension, which was in exponential growth, yielded the maximum number of protoplasts in our experiments. This finding is in agreement with the results of most studies in other cereals employing a 3-to 5-day-old suspension for protoplast isolation (Jähne et al 1991;Funatsuki et al 1992;Pauk et al 1994;Thompson et al 1986;Utomo et al 1995).…”

“…Approximately 1-g (fresh weight) aliquots of suspension cell aggregates, obtained 3-5 days after subculture, were incubated with 7 ml filter-sterilised enzyme solution in a petri dish (5 cm in diameter). Enzymatic digestion was carried out on a gyratory shaker (50 rpm) at 28C in the dark for 3-4 h. The mixture was filtered through a 60-mm nylon mesh sieve with Wms washing solution (Pauk et al 1994), which consists of MS 5519 powder medium with mannitol at 660-700 mOsm kg 1 H 2 O, to remove undigested cell clumps. The filtrate was then centrifuged at 80 g (Sorval TC 6) for 5 min, and the protoplast pellets were collected and washed twice with Wms washing solution by centrifugation at 80 g for 5 min.…”

Time saving method for protoplast isolation, transformation and transient gene expression assay in barley

“…Considerable progress has been made in protoplast culture of important monocot species and the first protoplast derived regeneration in cereals was reported in rice (Oryza saliva L.) by Fujimura and his group (1985). Succesful protoplast-plant regeneration systems have also been reported in wheat (Vasil et al 1990, Chang et al 1991, Yang et al 1993, Ahmed and SAgi 1993, Pauk et al 1994, maize (Mörocz et al 1990) and barley (Jähne et al 1991). Improvements in protoplast culture and regeneration have significantly given a contribution to the genetic transformation of monocots.…”