Following preliminary experiments, studies on isolation and culture of Ranunculus sceleratus L. leaf protoplasts were performed to specify conditions inducing embryogenesis. The best developmental stage for obtaining protoplasts was the second unfolded leaf; protoplasts were incubated in cellulase Onozuka R 10 (0.1%), Driselase (0.05%), Macerozyme R 10 (0.02%), glucose (8.9%), and Murashige and Skoog major salts (×0.5) or calcium chloride (220 mg/L), yielding 8 × 105 viable protoplasts per leaf; the best inoculum has 35 × 103 protoplasts/mL, protoplasts were cultured in 1-naphthaleneacetic acid (NAA) (3 mg/L), 6-benzylaminopurine (1 mg/L), modified Heller's micronutrients, Morel's vitamins, calcium nitrate (400 mg/L), and glutamine (100 mg/L). Although survival of protoplasts was as high as 80%, only 3% of the cells divide after 3 weeks. After subculturing the suspensions embryogenesis proceeded rapidly. Use of NAA (3 mg/L) or spreading cell cultures on solid medium delays the process. Following induction, cultures become habituated; continuous embryogenesis results from the differentiation of secondary embryos arising directly from primary embryo epidermis. The capacity for embryogenesis, however, diminishes after several subcultures, particularly if media are supplemented with growth regulators. Although growth of many embryos is inhibited, most plantlets subsequently develop normally. Thus, while we have demonstrated embryogenic capacity in suspension cultures derived from mesophyll protoplasts, direct development of somatic embryos from protoplasts remains to be shown.
FLOWER PETAL PIGMENTATION AFFECTED BY THE EXPERIMENTAL PLANT GROWTH REGULATOR A-1699 DF
