墨兰组织培养结合化学处理脱除建兰花叶病毒(CymMv)的研究

Eradication of CymMv from Tissue Cultures of Cymbidium sinense with Chemotherapy

对墨兰原球茎顶端分生组织培养结合化学处理脱除建兰花叶病毒(CymMv)病原的效果进行了研究.取1～2 mm大小的墨兰原球茎顶端分生组织,经0,20和40 mg·L-1的三氮唑核苷浸泡15 min处理和继代培养,诱导再生植株.RT-PCR检测表明:从带病叶样提取的RNA经反向转录和PCR扩增反应,在琼脂糖凝胶电泳中检测到长为767 bp的CymMv病原特异扩增产物,而健康墨兰叶样中未检测到该扩增产物.单纯利用原球茎顶端分生组织培养获得的试管苗,脱毒率为72.9%;原球茎顶端分生组织经过20 mg·L-1的三氮唑核苷处理,可以获得100%的无病毒苗.虽然三氮唑核苷处理造成原球茎顶端分生组织细胞一定程度的伤害,但经过5～6个月的培养,分生组织能恢复生长,不定芽能有效地增殖,并获得了再生植株.当三氮唑核苷处理浓度为40 mg·L-1时,原球茎的顶端分生组织出现透明和褐变现象,细胞活力难以恢复.

Meristem-tip of protocorms of Cymbidium sinense was employed to study eradication of CymMv by combining tissue culture and chemotherapy. Meristem-tip of protocorms about 1 -2 mm in length were ex- cised and immersed separately with 0, 20 and 40 mg · L^-1 of antiviral agent, ribavirin for 15 mins, followed by the sub-culture, the regenerated plantlets were achieved. Above materials were determined for virus infection by RT-PCR assay. Experiment indicated that RNA of virus-infected samples were determined and the amplified 767 bp specific fragments of CymMv could be visibled clearly in agarose gel electrophoresis, and no product appeared in the health control ; only 72.9% of the plantlets obtained merely by meristem of protocorms culture were virus-free, while the percentage was 100% for plantlets regenerated from meristem of protocorms, ever treated by 20 mg · L^-1 of ribavirin, though the chemical injured tissue to certain extents, which were still recovered, efficient proliferation of adventitious buds were achieved, and plantlets were regenerated subsequently after 5 - 6 months. When treated with 40 mg · L^-1 of ribavirin following above-mentioned method, meristem of protocorms became transparent and browned, and the cell vigor could not be recovered.