泡桐丛枝病病原MLO在寄主离体培养组织中的保存与增殖

 感染丛枝病病原MLO的泡桐幼苗茎梢,经表面灭菌后,置于附加0~3mg/1 6-BA的MS培养基中进行离体培养,由腋芽或顶芽直接伸长生长所形成的试管苗或经不定芽发生途径所形成的试管苗在1个月内均表现出典型的丛枝症状。在高浓度6-BA(2~3mg/1)处理培养基中,丛枝试管苗1月左右病死。当外植体为0.5mm长的微茎尖时,试管苗的丛枝症状有所减轻。DAPI荧光染色镜检结果表明,这种已表现出丛枝的试管苗的根、茎、叶柄的韧皮部筛管,以及已具维管束分化的愈伤组织的筛管部位发出很强的MLO特异荧光。超薄切片电镜观察发现,离体培养组织中的MLO形态具有较多处于活跃增殖期的哑铃形和芽殖形。以30天为一个继代周期,在无激素培养基或低浓度6-BA(lmg/1)与无激素培养基交替继代培养1年之后,来源于染病愈伤组织和试管苗中的MLO深度一直维持在相当高的水平。试验表明,利用寄王组织进行离体培养是保存、繁殖并进一步深入研究泡桐丛枝病病原MLO的一条良好途径。

PRESERVATION AND MULTIPLICATION OF PAULOWNIA WITCHES' BROOM PATHOGEN MLOs BY HOST PLANT TISSUE CULTURE

Morphologically normal buds were collected as explants from the root-sprouting seedlings infected by MLOs. After being sterilized with 0.1% HgCl₂ for 10 minutes, the explants were cultured into MS basal medium supplemented with 0~3 mg/1 6 -BA. 30 days later, all in vitro shoots, from the elongation of the original buds or adventitious organogenesis, exhibited typical symptoms of witches' broom. With the treatment of a high concentration of cytokinin (2~3 mg/16-BA), the witches' broom shoots wilted within 30 days. However, the degree of witches' broom decreased when 0.5mm meris-tems were used as explants. After the tissues from the root, shoot, petiole and callus of witches' broom plants in vitro were dyed with DAPI, fluorescence microscopy showed that strong MLO specific fluorescence emitted from the sieve tubes of these tissues. Compared with the morphology of MLOs in the field plants, more MLOs in the in vitro cultures were found with electron microscopy to be at the stage of active reproduction (budding and dumb-bell stage). The intensity of MLOs in the in vitro cultures was kept at a high level, throughout the whole experimental period (one year) with one subculture every month. This study indicates that tissue culture of host plants is a good solution for the preservation and multiplication of pathogen MLOs.