Salicylic acid (SA), when added to the differentiation medium below 200mol/l, significantly enhanced somatic embryogenesis in callus culture of Astragalus adsurgens Pall. The highest frequency of somatic embryogenesis occurred at 150mol/l SA. Enhanced somatic embryogenesis by SA was accompanied by an increase in the endogenous H2O2 level as compared with controls. This increased endogenous H2O2 level was related to the inhibition of the activities of ascorbate peroxidase (APX) and catalase (CAT). Although the promoting effect of exogenous H2O2 was significantly lower than that of exogenous SA on the development of somatic embryos, the pre-treatment of callus with dimethylthiourea (a trap for H2O2) significantly inhibited somatic embryogenesis, even if callus was cultured on the differentiation medium supplemented with 150

The inter-relationship between exogenous calcium (Ca2+) during cold pretreatment and cold-enhanced somatic embryogenesis was investigated using cell suspension cultures of Astragalus adsurgens Pall. Cell suspension was obtained from embryogenic callus and could be induced to form somatic embryos in the differentiation medium. Suspension cells, after cold-treatment at 8℃ for 2 to 3 wk, displayed an enhanced capacity for somatic embryogenesis as compared to those without cold pretreatment. Longer cold pretreatment (>4wk) resulted in the inhibition of somatic embryogenesis. The enhanced embryogenic response of cells to cold pretreatment was dependent on the Ca2+ level in the pretreatment medium. Ca2+ levels below 1 mM suppressed the cold-enhanced response. Addition of lanthanum into the pretreatment medium completely abolished the cold induced enhancement of somatic embryogenesis. These results suggest that embryogenic cells require a minimal concentration of Ca2+ during pretreatment for the expression of this cold-enhanced capacity for somatic embryogenesis in A.adsurgens and the influx of exogenous Ca2+ during pretreatment might also be involved.

The inter-relationship between exogenous calcium (Ca2+) during cold pretreatment and cold-enhanced somatic embryogenesis was investigated using cell suspension cultures of Astragalus adsurgens Pall. Cell suspension was obtained from embryogenic callus and could be induced to form somatic embryos in the differentiation medium. Suspension cells, after cold-treatment at 8℃ for 2 to 3wk, displayed an enhanced capacity for somatic embryogenesis as compared to those without cold pretreatment. Longer cold pretreatment (>4wk) resulted in the inhibition of somatic embryogenesis. The enhanced embryogenic response of cells to cold pretreatment was dependent on the Ca2+ level in the pretreatment medium. Ca2+ levels below 1 mM suppressed the cold-enhanced response. Addition of lanthanum into the pretreatment medium completely abolished the cold induced enhancement of somatic embryogenesis. These results suggest that embryogenic cells require a minimal concentration of Ca2+ during pretreatment for the expression of this cold-enhanced capacity for somatic embryogenesis in A. adsurgens and the influx of exogenous Ca2+ during pretreatment might also be involved.

An efficient procedure was developed for inducing callus and plant regeneration using hypocotyls segments of Astragalus adsurgens. The combinations and concentrations of different growth regulators were shown to be critical factors for both the frequency and the type of callus formation as well as for the potential of callus differentiation. Of the four morphologically distinct types of calli that were induced, a friable, yellow callus, i.e. type I, induced on MS medium supplemented with 9.0 mM 2,4-dichlorophenoxyacetic acid and 2.2 mM N6-benzylaminopurine (BA), and then transferred to MS medium containing 0.5 mM a-naphthaleneacetic acid and 8.9 mM BA, exhibited the maximum frequency of shoot regeneration (75%). After regenerated shoots were transferred onto halfstrength MS medium without growth regulators, they rooted and complete plants were obtained. Plantlet regeneration from callus cultures required 7-8 weeks.