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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。
The stem shoots of P. paulownis seedlings infected with the pathogen MLO of cucumbers were sterilized on the surface and placed in MS medium supplemented with 0 ~ 3mg / 1 6-BA for in vitro culture and directly grown by axillary buds or terminal buds Of test-tube seedlings or viable buds formed by the way of tube seedlings within 1 month showed typical symptoms of Congzhi. In high concentrations of 6-BA (2 ~ 3mg / 1) treatment medium, Congzhi testicular disease died about January. When explants were 0.5 mm long, the symptom of the twigs in the test tube was relieved. The results of DAPI fluorescence microscopy showed that the phloem sieve tube of roots, stems and petioles showing the branches of cucumbers and the screen tube part of the vascular bundles with vascular bundle differentiation gave strong MLO specific fluorescence . The results of ultrathin section electron microscopy showed that the morphology of MLO in vitro was more dumbbell-shaped and budding at the stage of active proliferation. With 30 days as a successor cycle, MLOs derived from infected calli and plantlets after subculture in hormone-free or low-concentration 6-BA (1 mg / l) and hormone-free medium for 1 year Depth has been maintained at a very high level. Experiments show that the use of tissue-in-vitro culture in vitro is a good way to preserve, reproduce and further study the pathogens of paulownia witches broom pathogen MLO.