黄独的茎尖培养和病毒检测

Shoot tip culture and virus detection of Dioscorea bulbifera

目的以黄独为试材,研究不同因素对黄独茎尖培养的影响,以期找到适合黄独茎尖分化成苗的培养基和脱毒检测方法。方法采用植物组织培养的方法进行茎尖培养,采用症状学法、指示植物法和RT-PCR法对茎尖脱毒植株进行病毒检测。结果黄独茎尖的最佳消毒方式是先用70%酒精消毒30s,再用0.1%HgCl2消毒12min;在37℃中热处理7d后再切离0.5mm茎尖效果较佳。茎尖再生芽增殖的最佳培养基是MS+KT2mg/L+NAA0.5mg/L;黄独茎尖再生芽的最适生根培养基为1/2MS+NAA0.5mg/L;黄独脱毒苗最好的移栽基质为珍珠岩-蛭石(2:1);RT-PCR法是检测黄独马铃薯Y病毒(PVY)感染的主要方法,通过病毒学检测,其平均脱毒率为86.5%。结论首次建立了黄独茎尖脱毒培养的体系,为黄独脱毒苗的快速繁殖及工厂化生产奠定了技术基础。

Objective Effects of different factors on shoot tip culture in vitro were studied by using Dioscorea bulbifera as test material to find the media suitable to the shoot tip differentiation of D. bulbifera and the method of virus delection. Methods Plant tissue culture method was used in shoot tip culture and the symptom observation method, instruction plant method, and RT-PCR method were used in virus detection of virus-free plantlets. Results The best disinfection method for D. bulbifera shoot tips was firstly disinfected for 30 s with 70% alcohol and then disinfected for 12 rain with 0.1% HgCl2 ; It is better for D. bulbifera to cut shoot tips in 0.5 mm length after 37 ℃ heat treatment for 7 d; The best proliferation medium of D. bulbifera shoot tips was MS＋KT 2 mg/L＋NAA 0.5 mg/L; The best rooting medium of regeneration buds from D. bulbifera shoot tips was 1/2 MS＋NAA 0.5 mg/L; The best matrix of regeneration plantlets from D. bulbifera shoot tips was perlite-vermiculite （2 ： 1） ; RT-PCR Method was primary mean to detect potato virus Y （PVY） of regeneration plantlets from D. bu[bifera shoot tips, and average rate of PVY free was 86.5% by RT-PCR. Conclusion The virus-free culture system of D. bulbifera shoot tips is established for the first time, providing a technological basis for the rapid propagation and factory production of D. bulbifera virus-free plantlets.