| Cotyledon and hypocotyl of Trigonella ruthenica L. and Medicago sativa L. . Qingshui were used as test material to study the possible factors that influence the isolation and culture of protoplast on the basis of established high quality calli. Orthogonal design was used to study the effect of different exogenous hormones: 2,4-D, 6-BA, KT and NAA on the callus formation of cotyledon and hypocotyls of Trigonella ruthenica L. and Medicago sativa L. Qingshui. Because of the high rigidity rate, it was hard for Trigonella ruthenica L. to germinate, therefore the way to increase the germination rate was studied. The floppy calli with obvious granulars were conducted with enzymolysis, and were studied the effects of different enzyme combinations, enzymolysis time, osmotic pressure, calli subculture time as well as the pretreatment measures on isolation of protoplast. The obtained protoplasts with high yield and vigour derived from cotyledon calli of Trigonella ruthenica L. were used to study the effects of protoplast density, culture methods and exogenous on protoplast differentiation. The results indicate that:1. The highest germination rate was found when Trigonella ruthenica L. seeds being soaked in 98% H2SO4 for 15min, which was increased by 59% compared with CK at 84%.2. The functional effect of hormone on calli of the two forage grass varieties: the optimum induction effect was found on condition of 0.5 mg·L-1 2,4-D + 1.0 mg·L-1 6-BA+ 1.0 mg·L-1 NAA+ 0.25 mg·L-1 KT treatment; conspicuous induction effect was found on hypocotyl of Trigonella ruthenica L.cv on condition of 0.5 mg·L-1 2,4-D + 1.0 mg·L-1 6-BA + 0.5 mg·L-1 NAA + 0.25 mg·L-1 KT treatment; 0.5 mg·L-1 2,4-D + 0.5 mg·L-1 NAA + 0.5 mg·L-1 KT treatment was found appropriate for the calli formation of the hypocotyl of Medicago sativa L. Qingshui.3. After 12-14d of subculture for cotyledon calli of Trigonella ruthenica L., the materials were soaked in CPW-10 for 1h, and then were treated on condition of 0.5 mol·L-1 mannitol osmotic pressure, and finally being treated for enzymolysis in the compound solution of 2% cellulose + 0.5% pectinase + 0.3% driselase, all together 10h, the isolated protoplasts reached 13.67×105·g -1, and the survival rate was found at 93.3%.4. After 10-12d of subculture for hypocotyl calli of Trigonella ruthenica L., the materials were placed at 25±1℃in the dark for 24h, and then were treated on condition of 0.55 mol·L-1 mannitol osmotic pressure, and finally being treated for enzymolysis in the compound solution of 2% cellulose +0.5% pectinase + 0.3% hemicellulose, all together 12h, the isolated protoplasts reached 3.57×105·g -1, and the survival rate was found at 74.37%.5. After 12-14d of subculture for cotyledon calli of Medicago sativa L. Qingshui, the materials were soaked in CPW-10 for 1h, and then were treated on condition of 0.6 mol·L-1 mannitol osmotic pressure, and finally being treated for enzymolysis in the compound solution of 2% cellulose + 0.5% pectinase + 0.3% macerozyme + 0.3% hemicellulose + 0.3% driselase, all together 10h, the isolated protoplasts reached 14.45×105·g -1, and the survival rate was found at 87.33%.6. After 12d of subculture for hypocotyl calli of Medicago sativa L. Qingshui, the materials were placed at 4℃in the dark for 24h, and then were treated on condition of 0.55 mol·L-1 mannitol osmotic pressure, and finally being treated for enzymolysis in the compound solution of 2% cellulose +0.5% pectinase + 0.3% driselase, all together 10h, the isolated protoplasts reached 7.07×105·g -1, and the survival rate was found at 82.05 %.7. The regenerated protoplast calli was finally obtained with the differentiation rate at 61.4% under the most favorable differentiation conditions when the density was at 2.5-5×105·mL-1, in the MS+KM8P medium, and being added with 0.5 mg·L-1 2,4-D, 1 mg·L-1 6-BA and 1 mg·L-1NAA, with solid-liquid culture method.All the above indicate that high capability for calli formation was found for different materials when being cultured under the optimum conditions, and under the optimum enzymolysis condition, protoplasts with high yield and vigor could also been isolated. Within which, the protoplast cells isolated from cotyledon calli of Trigonella ruthenica L. were found differentiated for many times, and was finally obtained the regenerated cali. |
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