Protoplast Culture In Three Legume Forages And Somatic Hybridization Between Alhagi Pseudal Hagi. And Astragalus Cicer L.

Mesophyll protoplasts of Astragalus melilotoides Pall, transfromed by Agrobact-erium rhizogene were obtained enzymatically. Protoplasts of sustainable divisions developed into calli, and high frequency of shoot differentiation was obtained on differentiated medium. We have compared the effect of cotyledon age, pretreatment time and enzyme digestion time on protoplast division frequency. Liquid culture method was appropriate for the protoplast division. The highest division frequency was 38% in DPD medium supplemented with 2.0 mg/L 2,4-D, 0.2mg/L 6-BA, 0.3M mannitol, 2%(w/v) sucrose and 500mg/L casein hydroIysate(CH) at a plating density of 3×105/ml. Protocalli could regenerate hairy roots and roots.Calli were induced from hairy root segments of Agrobacterium rhizogene A4-transformed Alhagi pseudal hagi. The highest yield of protoplasts (80%) was obtained from 8-day-old friable calli after subcultured on fresh medium. Protoplasts were induced to undergo sustained divisions in DPD medium aupplemented with 1.5mg/L 2,4-D, 0.2mg/L 6BA, 0.3 M mannitol, 2%(w/v) sucrose and 500mg/L casein hydrolysate(CH) at a plating density of 4×105/ml on the condition that osmotic pressure was 540±3mmol/L. The division frequency was about 42 %. Numerous buds were induced from protocalli on MS medium supplemented with Phytohormone 6-BA.Protoplasts were isolated from the methionine resistant cell line of Astragalus cicer L. .The highest yield of protoplasts was obtained from 12-day-old friable calli after subcultured on fresh medium. The viability of protoplasts reached over 80%. Protolasts underwent sustained divisions when cultured in DPD medium supplemented with 2.0mg/L 2,4-D, 0.2mg/L 6BA, 0.3 M mannitol, 2%(w/v) sucrose and 500mg/L casein hydrolysate(CH) at a plating density of 2×105/ml.