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

Efficient plant regeneration through somatic embryogenesis was achieved in Astragalus adsurgens. Embryogenic callus was induced from hypocotyl, not root or cotyledon explants, and the maximum induction frequency (62%) was obtained on Murashige and Skoog (1962) basal medium (MS) containing 2.0 mg:l 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5mg/l N6-benzylaminopurine (BA). Transfer of embryogenic calli to MS medium with 1-2mg/l BA and 0.1mg/l a-naphthaleneacetic acid (NAA) resulted in somatic embryogenesis at high frequencies (63-74%) with an average of 280 somatic embryos per gram of embryogenic callus. After transfer onto half-strength MS medium without growth regulators, approximately 80% of somatic embryos developed into complete plantlets. Chromosome count of root tips from regenerants revealed a normal diploid. The rooted plantlets were successfuly transferred to soil with 60-80% survival, and the plants showed normal morphological characteristics. On MS medium containing 1.0mg/l 2,4-D and 0.5mg/l BA, embryogenic calli have retained morphogenetic potential for 12 months.

Viable protoplasts of Taxus yunnanensis were isolated from friable, light yellow callus. Protoplast yield was dependent on callus age, with a maximum from 20-day-old callus. Protoplasts were induced to undergo sustained divisions and to form cell colonies when cultured in medium consisting of B5 salts, KM vitamin and organic components, 0.45 M fructose, 3.0mg l-1 2,4-dichlorophenoxyacetic acid and 0.1 mg l-1 kinetin. The planting density was 2.5-3.0

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.

A reproducible release of viable protoplasts was obtained from friable calli of Astragalus adsurgens. Protoplasts underwent sustained divisions and formed cell colonies when cultured in either liquid or agarose-solidified Kao and Michayluk (1975) protoplast medium (KM8P) supplemented with 1.5mg/l 2,4-D, 0.5mg/l BA and 0.5 Mglucose. Compared to liquid culture, agarose bead culture improved division frequency effectively, the two culture systems showing a plating efficiency of 0.8