野葛试管苗茎尖玻璃化法超低温保存及植株再生

对野葛茎尖玻璃化法超低温保存技术的研究结果表明,H号野葛茎尖较佳的保存技术体系是:继代生长30 d的野葛无菌苗置于4℃冰箱炼苗5 d;在无菌条件下切取含1~2个叶原基(1.5~2.5 mm)的茎尖,转至含5% DMSO+5%蔗糖的MS培养基内,置于4℃冰箱预培养1 d;用60%、100% PVS₂(30%甘油+15%乙二醇+15% DMSO+13.7%蔗糖)分别在0℃下过渡和脱水各30 min,随即迅速投入液氮;保存24 h后,在40℃水浴中快速化冻90 s,用含41.1%蔗糖的MS培养液洗涤2次,每次停留10 min;转至再生培养基(MS+6-BA 2 mg/L+NAA 1 mg/L)暗培养7 d,然后光下培养,再生率可达60%以上。再生苗与常温苗形态指标差异不显著。

Cryopreservation of Pueraria lobata Shoot-tip in vitro by Vitrification and Plant Regeneration

The cryopreservation technique of Pueraria lobata shoot-tip was studied in this paper.The results showed that the better procedure of cryopreservation by vitrification was as follows.The aseptic plantlets of H^# Pueraria lobata subcultured in vitro for 30 days were treated at 4℃ for 5 days.The shoot-tips 1.5~2.5 mm in length with one or two leaf primordium of H^# Pueraria lobata were cut and precultured at 4℃ for 1 day in MS supplemented with 5% DMSO+5% sucrose.They were dehydrated with 60% and 100% PVS₂(PVS₂:30% glycerol+15% glycol+15% dimethyl sulfoxide+13.7% sucrose) at 0℃ for 30 minutes, respectively.The shoot-tips were immersed immediately into liquid nitrogen directly and conserved for 24 hours.After rapidly thawing in a water bath at 40℃ for 90 seconds, the shoot-tips were washed two times with MS supplemented with 41.1% sucrose (10 minutes each time), then transferred and re-cultured on the MS medium supplemented with BA 2 mg/L and NAA 1mg/L in dark for 7 days and then in light.The survival rate was up to 60%.The regenerated plantlets of Pueraria lobat were the same as the normal plantlets in morphology.