【作者】 艾鹏飞；

【导师】 罗正荣；

【作者基本信息】 华中农业大学 ， 果树学， 2004， 博士

【摘要】 植物遗传资源是人类赖以生存和发展的重要物质基础，保持其遗传多样性是人类实现可持续发展的重要保证。柿，资源丰富，为创制其新种质提供了广泛的背景材料。可是，传统的田间种植保存，易受病虫害、自然灾害及经费短缺等弊端的困扰，使得大量的柿资源面临或已经流失。离体保存经济、安全和实用，正在成为田间种植保存的重要补充技术。鉴于此，我们进行了柿种质资源离体保存及其遗传稳定性研究。主要结果如下： 1．建立了柿35份试材的离体培养体系：茎尖在含ZT 2.0mg／L或CPPU 0.2mg／L和0～0.05mg／L的IAA或IBA的MS（1／2N）+PVP 600mg／L+蔗糖30g／L+琼脂7mg／L培养基上培养，繁殖系数高。本体系用CPPU代替ZT，大大降低成本。 2．优化了柿试管苗生根条件：试管苗嫩梢经IBA 250mg／L浸基部15s后转接到1／2MS（1／2N）+活性炭1000mg／L的生根培养基中或直接接种到添加有IBA 1mg／L的上述生根培养基中诱导生根，生根率高，根条数多。 3．建立了柿7个基因型试管苗单芽姊妹系，为保存后再生植株的遗传稳定性检测提供了材料。 4．采用常温继代培养对柿试管苗保存了4～5年，并对保存后的5种基因型进行了遗传稳定性检测。结果核DNA含量、染色体数没有改变；RAPD分析，次郎3648条扩增带和罗田甜柿4752条扩增带中均没有出现变异带；前川次郎变异率为0.028％；松本早生变异率为0.031％；君迁子变异率为0.064％；磨盘柿变异率为0.016％。这说明常温继代保存柿试管苗易引起DNA水平上的变异。 5．建立起柿试管苗缓慢生长法保存体系：试管苗在添加有甘露醇20g／L或PP₃₃₃ 1.0mg／L的MS（1／2N）+蔗糖 20g／L+琼脂7g／L+PVP 500mg／L培养基上或在含有CPPU 0.2mg／L的1／2MS（1／2N）+蔗糖 15g／L+琼脂7g／L+PVP 500mg／L培养基上于6±1℃和800lx下保存18个月时，90％以上仍存活；分别对上述3种方法保存的再生植株遗传稳定性检测，核DNA含量和染色体数没有发现改变；RAPD分析，PP₃₃₃保存的次郎变异率为0.16％，君迁子变异率为0.86％；另2种方法保存的再生植株中没有发现变异带。 6．对柿休眠芽茎尖进行了两步冷冻法超低温保存：腋芽枝段依次经过0，-5，-10和-20℃各12h后投入液氮保存，40℃水浴化冻后切取腋芽，灭菌后剥取茎尖接种于MS（1／2N）培养基上培养，再生率超过60％。对柿休眠芽茎尖进行了玻璃化法超低温保存研究:茎尖在含有蔗糖0.3、0.5和0.7 moFL的培养基上预培养各ld，20℃下装载液过渡20面n，0℃下玻璃化液少V凡或Pv凡)处理30⁴0^后液氮保存，化冻、洗涤后在M以l瓜N)培养基上培养，再生率超过70%。分别对柿休眠芽茎尖两步冷冻法和玻璃化法超低温保存的5种和6种基因型的再生植株进行了遗传稳定性检测:细胞染色体数没有发生改变:月几P分析，不同基因型的756¹026条带中，也没有发现变异带。这说明柿休眠芽茎尖超低温保存可能没有发生遗传变异。9.建立起柿试管苗茎尖玻璃化法超低温保存体系:试管苗在加有蔗糖0.3 mol几培养基上于 6土1℃下锻炼6周后，切取茎尖在含蔗糖0.7 mol几的培养基上预培养2d，20℃下装载液 过渡2伪正n，o℃下PVS:处理80面n后液氮保存，化冻、洗涤后恢复培养，再生率超过 3识/o。君迁子、次郎、罗田甜柿和罗田甜柿加倍体的再生单芽姊妹系的细胞学检测，核DNA 含里和染色体数没有发生改变;次郎和罗田甜柿各10个单芽姊妹系川几P分析，在分别 扩增出的11 020条和10670条谱带中，均没有发现变异带。这为柿试管苗茎尖玻璃化法超 低温保存提供了可行性依据。10.对柿品种禅寺丸花粉进行了干燥脱水法和玻璃化法超低温保存研究，结果表明:0℃下硅 胶干燥肠或20士l℃下PVSZ处理40一60min后直接投入液氮保存，高温40℃解冻后，相 对存活率（TTC检测）和萌发率均在90%以上，且花粉活力与保存时间长短无关。这为 柿花粉长期保存提供了有效途径。更多还原

【Abstract】 Plant genetic resources are one of the most important substance bases for human to survive and develop, and to keep their diversity is the important guarantee for sustainable development. China is one of the primary origins of persimmon, and has variant genotypes of persimmon, which provide selectable breeding materials for creating better cultivars. However, planting in open field, as a traditional method for conservation of persimmon germplasm, has emerged so many faults such as diseases and insect pests, natural disasters, shortage of fund and so on, which even leads to facing loss or loss of a mass of germplasm. In vitro conservation has been becoming the key complementary conservation technique for its advantages of economy, safety and practicality’. Therefore, We have been beginning to study persimmon germplasm conservation in vitro and genetic stability of regenerated plantlets And, the major results listed as follows:1. Studies were carried out on in vitro culture of 35 genotypes of persimmon including species, cultivars and clones. The results showed that shoot tips, which were cultivated on the modified MS medium with nitrates reduced to half strength [MS(1/2N)] added with PVP 600 mg/L, sucrose 30 g/L, agar 7.0 mg/L, ZT 2.0 mg/L or CPPU 0.2 mg/L, and IAA or IBA 0~0.05 mg/L, could difference and grow better. And in vitro shoots increased by several times. The cost had been reduced greatly when CPPU was adopted instead of ZT above in vitro culture system of persimmon.2. The factors influencing rooting of persimmon shoots in vitro were studied: The results showed mat shoots subcultured constantly for certain times were dipped in IBA solution 250mg/L for 15s, and were incubated on the 1/2MS(1/2N) medium containing activated charcoal 1000mg/L, or were directly inoculated on the rooting medium above added IBA Img/L, which was better for rooting.3. Single-bud sibling lines of 7 persimmon genotypes were established, which provided materials for the studies on genetic stability of conserved regenerated plantlets.4. In vitro shoots were conserved for 4~5 years by subculture at normal temperature and genetic stability of 5 in vitro conserved genotypes was detected The results showed no changes of DNA content and chromosome number were found. And when variation at DNA level was tested by RAPD, there were no aberrant bands presenting among amplified 3648 bands of 6 clones of Jirou and 4752 bands of 8 clones of Luotiantiansbi, the aberrant frequency being 0.028% among 3624amplified bands of 6 clones of Maekawa-Jirou, the aberrant frequency being 0.031% among 3670 amplified bands of 10 clones of Matsumoto-Wase, the aberrant frequency being 0.064% among 4696 amplified bands of 8 clones of Date plum, and the aberrant frequency being 0.016% among 6380 amplified bands of 10 clones of Mopanshi. It revealed that conservation in vitro shoots of persimmon by subculture for long term resulted in variation at DNA level potentially.5. Approaches for conservation of in vitro shoots by slow growth and detecting the genetic stability of regenerated plantlets were established in persimmon. On an average, 90% among in vitro shoots conserved for 18 months at 6 and 800 be (12h photoperiod) with 3 methods by which shoots were cultured on MS(1/2N) medium supplemented with 20g/L sucrose, 7g/L agar and 500mg/L PVP added with 20.0 g/L mannitol or 1.0 mg/L PP333 or on 1/2MS(1/2N) supplemented with 20g/L sucrose, 7g/L agar and 500 mg/L PVP added with 0.2 mg/L CPPU maintained viability. And those regenerated plantlets conserved for 18 months by slow growth were analyzed on genetic stability. As a result, among 3 single-bud sibling lines of Jirou and Date plum conserved with PP333, there were no changes of DNA content and chromosome number, and 0.16% aberrant frequency existing among 1827 bands and 0.86% aberrant frequency existing among 1736 bands amplified by RAPD respectively. And no variations of regenerated plantlets conserved by another two methods were found by cytological and molecular measures.6. A p更多还原