Study On Tissue Culture And In Vitro Polyploid Induction Of Gentiana Macrophylla Pall.

Using the leaves of Gentiana macrophylla Pall, as explants, the embryogenic calli were induced and regenerated plants were produced. The proper way for leaf sterilization was: 70% ethanol 25S + 0.1% HgCl₂ 10～12min or 70% ethanol 40S + 0.1% HgCl₂ 8min.The leaf base was proved to be the best explants for the initiation of calli. The optimal medium for callus induction was the MS medium added with 2.0mg/L 2,4-D, 0.5 mg/L KT and 500mg/L LH. The rate of callus induction was up to 95%.When the induced calli were transferred and cultured on the MS medium supplemented with 1.5mg/L 2,4-D,0.2mg/L 6-BA and 500mg/L LH , the embryogenic calli were induced and subcultured well. After transferred on 1/2MS medium with 0.1 mg/L 2, 4-D , 2.0 mg/L 6-BA and 500 mg/L LH , embryogenic calli gave rise to embryoids. The differentiation rate could reach 93.9%.When shoots differentiated from the mature embryoids were transplanted and cultured for two weeks on MS medium without any hormones, they developed into plantlets.The embryogenic calli were used for protoplast isolation. The highest yield of protoplasts(80%) was obtained from 7-day-old friable calli after subculturing on fresh medium. Protoplasts were induced to undergo sustained division in the DPD medium supplemented with 1.5mg/L 2,4-D, 0.2mg/L 6-BA, 0.4M mannitol, 2%(w/v) sucrose and 500mg/L lactolbumin hydrolysate(LH) at a plating density of 3.5×105/ml.The division frequency was about 38%.Buds could be induced from protocalli on MS medium supplemented with 0.1mg/L 2,4-D,2mg/L 6-BA and 500mg/L LH.The embryogenic calli maintained on the MS medium supplemented with 1.5 mg/L 2, 4-D ,0.2 mg/L 6-BA and 500 mg/L LH were also adopted for polypoid induction. The diploid embryogenic calli were treated with two methods:1.adding colchicine in the medium; 2.immersing the materials into colchicine solution with different concentrations and period. The results showed that both methods could induce polypoid cells, however the latter was far better than the former in respect of the induction rate. The appropriate explants for polyploid inducing were those embryogenic calli which have initiated differentiation. When the these calli which have initiated differentiation treated in 0.1% colchicine solution for 6h,the morphorlogical variation plants were observed. Microscopic examination of shoot tips showed that the chromosome number was 4n=4x=52, while that of normal plants was 2n=2x=26.