In vitro selection was carried out to obtain ethionine-resistant plants with increased contents of free methionine in the vegetative tissues of the forage legume Astragalus adsurgens Pall. Three-week-old cell colonies were derived from protoplasts mutagenized with N-methyl-N-nitrosoguanidine from embryogenic callus and were selected with 0.6 mM ethionine. Four colony lines were isolated and their resistance to ethionine was 7-8 times that of the wild-type callus. No plant regeneration occurred on these colony lines in the differentiation medium containing ethionine. Only one colony line (R-1) regenerated plants through somatic embryogenesis in the absence of ethionine. Stem and leaf segments from the regenerated plants showed the same potential to produce callus in the presence of ethionine as in the absence of ethionine. The formed callus kept continuously growing in ethionine-containing medium. Free amino acid analysis revealed that colony line R-1, its regenerated plants and callus from the regenerated plants accumulated methionine at levels at 5-9 times higher than in wild-type. These results suggested that ethionine resistance and methionine over-accumulation were also expressed at plant level. Thus, the obtained resistant colony line that could regenerate plants with over-accumulation of methionine might provide an alternative approach to improve the nutritional quality of this forage.

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.

In vitro selection was carried out to obtain ethionine-resistant plants with increased contents of free methionine in the vegetative tissues of the forage legume Astragalus adsurgens Pall. Three-week-old cell colonies were derived from protoplasts mutagenized with N-methyl-N-nitrosoguanidine from embryogenic callus and were selected with 0.6mM ethionine. Four colony lines were isolated and their resistance to ethionine was 7-8 times that of the wild-type callus. No plant regeneration occurred on these colony lines in the differentiation medium containing ethionine. Only one colony line (R-1) regenerated plants through somatic embryogenesis in the absence of ethionine. Stem and leaf segments from the regenerated plants showed the same potential to produce callus in the presence of ethionine as in the absence of ethionine. The formed callus kept continuously growing in ethionine-containing medium. Free amino acid analysis revealed that colony line R-1, its regenerated plants and callus from the regenerated plants accumulated methionine at levels at 5-9 times higher than in wild-type. These results suggested that ethionine resistance and methionine over-accumulation were also expressed at plant level. Thus, the obtained resistant colony line that could regenerate plants with over-accumulation of methionine might provide an alternative approach to improve the nutritional quality of this forage.

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.

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