【作者】 李丽；

【导师】 徐昌杰； 徐建国；

【作者基本信息】 浙江大学 ， 园艺（专业学位）， 2014， 硕士

【摘要】 本研究选用本地早、温州蜜柑、红玉柑的7～8年龄当年生新梢茎段为试材,开展了离体诱导培养不定芽、应用再生不定芽开展茎尖微芽嫁接等提高茎尖微芽嫁接成活率的技术研究和茎尖嫁接成活苗的PCR检测。主要试验结果如下：1.通过不同消毒方法对比试验,筛选出较适宜的消毒方法,有效改善了柑橘茎段离体诱导培养的污染严重的问题。试验结果表明,炎热干燥的夏季是半木质化茎段最佳的外植体取材时期,此时期的茎段外植体表面携带的外生菌较少,相同条件的消毒处理后,茎段培养的污染率最低；采用5%次氯酸钠间隔48h的两次消毒方法,能有效降低茎段培养的污染,污染率为22.53%,且材料死亡少,成活率最高达72.41%。2.应用6-BA、IAA、GA33因子3水平的正交试验组合,对茎段进行离体诱导培养试验,结果表明以6-BA0.5mg/L、IAA0.05mg/L和()Aa0.5mg/L这一植物生长调节剂组合诱导再生芽效果最好。3.对不同来源的茎尖进行微芽嫁接成活对比试验,发现茎段诱导的离体再生芽茎尖的微芽嫁接成活率为73.5%,田间嫩梢接穗的茎尖微芽嫁接成活率为51.6%,前者优于后者,两者之间的影响差异较显著。田间接穗茎尖的微芽嫁接成活苗的长势优于离体再生芽茎尖嫁接的成活苗,可以考虑7-8月分以田间新生嫩梢取茎尖进行微芽嫁接,可有效保证嫁接苗的质量；其它几个月分则从离体再生嫩梢取茎尖进行微芽嫁接,可有效延长嫁接周期。4.根据柑橘黄龙病病原的核苷酸序列设计了PCR引物检测茎尖微芽嫁接苗的黄龙病病毒。结果表明,阳性对照带毒症状植株和田间疑似黄龙病症状的温州蜜柑、本地早蜜橘、秋辉、红玉柑和黄皮植株中均扩增到1条大于703bp的特异性条带,而用3-4个叶原基茎尖的微芽嫁接成活苗则无扩增到特异性条带,表明试验不同品种的植株是带毒的,通过茎尖微芽嫁接成功脱除了柑橘黄龙病病原,证明茎尖微芽嫁接脱除柑橘病毒是有效的。更多还原

【Abstract】 The study was carried out with citrus mature stem segments of Bendizao mandarin(Citrus reticulata), Satsuma mandarin (C. unshiu), Hongyugan tangor [C. reticulatax (C. sinensisxC. reticulata)] as explants. The in vitro induction system for adventitious buds was established, and then with these buds, shoot-tip micrografting was performed and plantlets were obtained. Finally, virus detection of these plantlets was carried out by PCR. The main findings are as follows.1. Different disinfection methods were compared and a best disinfector was screened out, which markedly reduced the contamination rate of mature stem segment explants. It was ob-served that for a sunny day in summer, with mature, half lignified stem segment explants ex-cised after2p.m., and with a disinfection protocol of48h interval, the contamination rate was22.53%, and survival rate was the highest.2. Plant hormone concentration combinations of6-BA, IAA and GA3were tested, and it was found that the combination0.5mg/L6-BA+0.05mg/L IAA+1.0mg/L GA3was best for in vitro induction of adventitious buds from the stem explants.3. Shoot-tips from different sources were compared as regarding to micrografting survival rate, and it was found that the in vitro cultured adventitious buds are better than the buds directly collected from field trees, for having a survival rate of73.5%, as compared with51.6%of the latter, buds from field trees of micrograftin survival seedling grew better than that of in vitro cultured adventitious buds of micrografting survival seedlings, It can be considered to7-8months in the field of newborn tender tip with stem tip micrografting, which can effectively guarantee the quality of micrografting; Others specific provisions of a few months from in vitro regeneration tender tip take stem tip for micrografting which can effectively prolong graft cycle.4. PCR primers were designed based on the sequence of citrus huanglongbing virus nucle-otide, Through PCR, virus was not found on the plantlets from shoot-tip micrografting while the filed plants, were swell to the specificity of1is greater than703bp band, with3-4leaf primor-dium shoot-tip micrografting survival seedling bud grafting, no to a specific bands, showed that test of different varieties of citrus plant is poisonous, through the shoot-tip micrografting itwas successful removal the citrus huanglong virus, Therefore, it can be concluded that virus free plantlets can be obtained through shoot-tip micrografting on citrus, and the techniques estab-lished here are valuable for industrial application.更多还原