摘要
用草木樨状黄芪甲硫氨酸抗性变异系植株茎切段诱导的松软愈伤组织为材料，通过酶法分离出大量有活力的原生质体。原生质体经持续分裂形成了愈伤组织，并高频率地分化出再生苗。比较了不同培养基、培养方法和培养密度对原生质体分裂和再生的影响。结果表明，原生质体以3×10~5／ml的植板密度，采用琼脂糖岛法培养在附加2,4-D1．0mg／L、6BA0．5mg／L、水解酪蛋白500mg／L、蔗糖3％、甘露醇0．3mol／L的~KM_8D培养基中，可获得最佳效果，其细胞分裂频率达38％左右。原生质体再生的植株仍然保持对甲硫氨酸的抗性，同时对乙硫氨酸表现交叉抗性。染色体检查，同工酶(过氧化物酶、细胞色素氧化酶、酯酶)和RAPD分析表明，来源于抗性系原生质体的再生植株具一定的遗传稳定性，但与野生型相比亦发生了一些变异。
本研究对紫花苜蓿发根农杆菌转化系愈伤组织分离的原生质体进行了培养，并获得了愈伤组织和分化的发状根。结果表明，用转代后生长12d的愈伤组织，分离的原生质体产率为4．2×10~6个／g，原生质体活力在80％以上。原生质体以1．0×10~5／ml的植板密度培养于附加2mg／L2,4-D，0．2mg／L KT，0．3mol／L甘露醇，2％蔗糖，500mg／L水解酪蛋白的DPD培养基上，分裂频率可达24％。原生质体分裂形成的愈伤组织在无激素MS培养基上再分化出的发状根仍具冠瘿碱合成酶活性。
通过PEG诱导原生质体融合和杂种细胞的筛选培养，首次得到沙打旺(+)紫花苜蓿的属间体细胞杂种。尽管双亲均已丧失分化植株的能力，但由于再生互补效应，杂种细胞系R_1仍出现分化，并得到小苗。杂种R_1细胞的染色体数检查、冠瘿碱检测、同工酶分析，可溶性蛋白SDS-PAGE电泳和RAPD分析结果，都证明了其杂种特性。
Calli were induced from internode segments of the methionine resistant plantlets of Astragalus melilotoides. The highest yield of protoplasts (2.1 X 106/g F.Wt.) was obtained from 8-day-old friable calli after subcultured on fresh medium. The enzyme combination of 2% cellulase Onozuka R-10, 0.5% Hemicellulase and 0.5% Pectinase was effective for protoplast isolation. Protoplasts were induced to undergo sustained divisions in KMgp medium supplemented with l.Omg/L 2,4-D, 0.5mg/L 6BA, 0.3M marmitol, 2% (w/v) sucrose and 500mg/L casein hydrolysate at a plating density of 3 X 105/ml. Agarose-beads culture method was appropriate for the protoplast division of the methionine resistant cell line. The division frequency was over 38%. High frequency of shoot differentiation was obtained from the protocalli on differentiated medium. The regenerated plants still preserved resistance to methionine and ethionine. Chromosome numbers were no remarkable difference with the control. Analysis of isoenzyme ( Peroxidase, Cytochrome oxidase, Esterase) and RAPD indicated that the regenerated plants from protocalli were different with the control.
Protoplasts were isolated from Agrobacterium rhizogenes A^ transformed cell line of Medicago Sativa L.. The highest yield of protoplasts (4.2 X 106/g F.Wt.) was obtained from 12-day-old calli after subculturing on fresh medium. The viability of protoplasts reached to over 80%. Protoplasts underwent sustained divisions when cultured in DPD medium supplemented with 2mg/L 2,4-D, 0.2mg/L KT, 0.3M mannitol, 2% (w/v) sucrose and 500mg/L casein hydrolysate (CH) at a plating density of 1.0 X 105/ml. The division frequency was about 24%. Numerous hairy roots were induced from protocalli on MS medium without any growth regulator. The paper electrophoresis revealed that all of the regenerated hairy roots tested synthesized the corresponding opines.
Somatic hybrid cells were obtained between ethionine resistant cell line of Astragalus adsurgens Pall, and Agrobacterium /"/z/zogerae.s'-transformed cell line of
Medicago Saliva L. using protoplast fusion by PEG method. The hybrid cells were selected efficiently using IOA pretreatment of protoplasts and the genetic marker of transformed Medicago Sativa. Although both of the parents were lost the regeneration capacity, the hydrid cell line RI was capable of differentiating small shoots due to complementation of regeneration capacity. Examination of chromsome number of the hybrids RI showed the sum of both parent chromsome besides aneuploid. The hydrid cell line RI was characterized by isoenzyme analyses, SDS-PAGE, RAPD and opine synthetase assay.
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