Winged-bean protoplasts: Isolation and culture to callus

Abstract

Abstract. A system was developed for protoplast isolation and culture from suspension cultured cells of winged bean, Psophocarpus tetragonolobus. Cells from a three-day-old suspension were incubated in an enzyme mixture containing 6% cellulysin, 1% Macerase, 1% desalted Rhozyme, 0.4M sorbitol, and 0.1M CaCl: at pH 5.5. Average yields of protoplasts were 6.5 × 106 per gram fresh weight of cells. Protoplasts were cultured in modified B5 medium containing 68.4g/1 glucose, 250mg/l xylose, 0.1mg/l 2,4-D, 0.5 mg/1 BAP, 250 mg/l N-Z amine type AS, and 20 ml/1 coconut water.After 24 h of culture, the protoplasts had synthesized a new wall, and in three days had begun division. The optimum plating density was 1-2 × i0 ~ protoplasts/ml. The division frequency ranged between 40%-60% for most experiments with a high of 72% in one experiment. After three weeks, cell colonies could be transferred to solid MS medium containing N-Z anfine and coconut water where callus developed. This protoplast system is technically comparable to soybean for experiments concerned with genetic manipulation involving legumes.

A Comparative Mechanical Analysis of Plant and Animal Cells Reveals Convergence across Kingdoms

“…After 10 min of gentle shacking (30 rpm), protoplasts were sorted from aggregates by filtration on a 300 mm mesh. Rheological measurements on protoplasts were performed around 5 min after cell wall removal and for no more than 4 h to keep the protoplast wallless (synthesis of a new cell wall generally takes 24 h after cell wall removal (25,26)). …”

Morphogenetic responses from in vitro cultured seedling explants of mung bean (Vigna radiata L. Wilczek)

“…The recalcitrant callus of grain legumes has always been a subject of discussion in literature [4,14,15]. Except for the limited success in pea callus [11] and the recent report in soybean [2] the differentiation in grain legumes is often restricted to primary callus and not in established callus cultures [6,9,19]. However judicial selection of explants and medium components would help to raise in vitro plants in large numbers.…”

“…2C). On MS + BA (3.4#M), the number of shoots (6)(7)(8) were more than that in MS + IBA (2-4 shoots) -but the shoots in the latter were bigger and well developed compared to small and underdeveloped shoots in the former. In subsequent experiments, the cotyledonary segment was cultured initially on MS + BA 3.4#M for a period of 20 days and then transferred to MS + IBA (6.1#M) where most of the shoots induced in cytokinin medium, grew into well developed shoots (Fig.…”

Intra-varietal variation for in vitro plant regeneration in the genus Trifolium

“…In primary callus cultures, shoots may developed adventitiously from organised tissues of the original explant embedded in the callus (CURE & MOTT, 1978 ;HERMAN & HAAS, 1978), or they may arise from preformed buds (see WIDHOLM & RICK, 1983) . There are at least two leguminous species where, from the same laboratories, primary callus cultures have been reported to differentiate shoots (GAMBORG et al ., 1974 ;GUDDIHY & BOTTINO, 1982) but their protplasts did not (CUDDIHY & BOTTINO, 1982 ;GAMBORG et al ., 1975) . The Tr-20 line selected in the present work on the basis of shoot differentiation from leaf calli has now shown good regeneration from protoplast calli .…”