Protoplast Culture And Somatic Hybridization In Three Legume Forages

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 ofMedicago 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.