半夏试管块茎直接发生途径的离体快繁研究

Studies on Rapid Propagation of Pinellia ternata(Thunb.) Breit.in Vitro Tuber via Direct Organgenesis

目的建立半夏试管块茎直接发生途径的离体快繁技术体系。方法以块茎、总叶柄、小叶柄、佛焰苞中部、佛焰苞下部、珠芽为外植体,研究不同消毒方法、激素组合和外植体长度对半夏试管块茎诱导的影响。结果叶柄以75%乙醇10s→0.1%升汞5min(1滴吐温80)→2%NaClO 8min→无菌水冲洗3遍为适宜的消毒方法;块茎以75%乙醇10s→0.1%升汞5min(1滴吐温80)→无菌水冲洗3遍为适宜的消毒方法;总叶柄和块茎均为最佳的外植体,适宜的初代培养基为1/2MS+6-BA 0.1mg/L+NAA 0.01mg/L+3%蔗糖+0.8%琼脂,试管块茎诱导率为100%,块茎的数量为8.5个;1/2MS+6-BA0.01mg/L+NAA0.01mg/L+3%蔗糖+0.8%琼脂和1/2MS+6-BA0.001mg/L+NAA0.001mg/L+3%蔗糖+0.8%琼脂培养基是适宜的继代增殖培养基,增殖倍数分别为6.6和6.3;二次成苗时间只需要20天左右,增殖倍数分别为5.8和5.7。1cm总叶柄平放,叶柄下端产生的饱满的试管块茎可用作人工种茎。结论该研究建立了增殖倍数高,遗传稳定性高,生产成本低的半夏快速繁殖技术体系,可为半夏工厂化育苗提供技术支撑。

Objective To establish a protocol of in vitro tuber rapid propagation of Pinellia ternate(Thunb.)Breit.via direct organgenesis.Methods Explants of tuber,primarypetiole,petiolule,middle and underpart of capullo were used to study the effects of different disinfection methods,combination of hormones and explants size on the induction of Pinellia ternata(Thunb.)Breit in vitro tuber.Results The optimal disinfection for petioles were firstly soaked in 75%ethanol for 10s followed by 0.1%HgCl_(2) for 5min(1 drop of Tween-80 added)and 2%NaClO for 8min,and finally they were rinsed in sterilized water for three times.For tuber,they were surface-sterilized with 75%ethanol for 10s followed by 0.1%HgCl_(2) for 5min(1 drop of Tween-80 added)and finally rinsed in sterilized water for three times.1/2MS medium supplemented with 0.1mg/L 6-BA,0.01mg/L NAA,3%sucrose and 0.8%agar,was the optimal medium for primary culture.The induction rate of little tuber was 100%,and the average number of tuber was 8.5 per explant.1/2MS medium with 0.01 mg/L 6-BA and 0.01mg/L NAA or 0.001mg/L 6-BA and 0.001mg/L NAA,3%sucrose,0.8%agar were suitable for subculture.The proliferation rate was 6.6 and 6.3 individually.The shoots originated from tuber again only need 20 days,which proliferation rate reached up to 5.8 and 5.7.The 1cm-length of total petiole horizontally placed on medium generated a full little tuber at the end of the petiole,which can be used for artificial tuber.Conclusion This study established a protocol for Pinellia ternata(Thunb.)Breit.rapid propagation which owns high proliferation rate,genetic stability,low production cost,and can provide technical support for industrializing propagation.