以下胚轴为外植体的棉花组织培养和转基因体系已经广泛应用于棉花转基因研究，但是该体系试验过程需要反复转移培，易污染、养耗时长。为了探索一种耗时短、实验过程简化的转基因体系，本研究探索以子叶节为外植体诱导不定胚产生多芽，同时，结合农杆菌介导法探索建立遗传转化体系。分别选用新陆早15号、泗棉3号、W0、coker 315四个陆地棉品（种）系作为实验材料，取子叶节为外植体，分别分析了不同激素或激素配比、AgNO3等对通过器官发生途径产生多芽的诱导效应，探索通过农杆菌侵染子叶节将ABD2-mCherry荧光标记基因导入棉花植株的效果。结果表明培养基中单独添加添加6-BA或KT不能诱导多芽产生，而激素联合使用1.5 mg/L 6-BA和1.5 mg/L KT或2 mg/L 6-BA和2 mg/L KT诱导多芽的效率比单独使用时更高。而单独使用AgNO3较激素诱导多芽效率更高，最高可达88.9%。培养基中添加GA3或玉米素对多芽的伸长诱导作用不明显，而在不加激素的MSB培养基中芽伸长率最高，平均芽长可达2.3 cm。不同品种多芽的诱导率不同，多芽诱导率受基因型影响。但是多芽生根率较低，受基因型的限制较大。使用AgNO3，在新陆早42号中生根率最高，可达到42.9%。用农杆菌介导法，将ABD2-mCherry导入到新陆早15号、泗棉3号以及W0，25%多芽抗500mg/L卡那霉素，PCR检测目的基因为阳性，回收PCR扩增片段并进行测序，证明与目的基因序列一致，阳性植株在荧光显微镜下可以检测到红色荧光，表明ABD2-mCherry已经导入多芽，并得到正确表达。该方法整个试验周期仅需3个月，只需2次转移培养即可获得转基因多芽。具有时间周期短，省工省力的优势，有望应用于棉花快速、高通量转基因研究。
目录
中文摘要Ⅰ
关键词Ⅰ
AbstractⅡ
引言
引言1
1材料与方法2
1.1 实验材料2
1.2 试验方法2
1.2.1 种子消毒及培养无菌苗2
1.2.2 外植体制备2
1.2.3 陆地棉子叶节再生体系的建立2
1.2.4 农杆菌的培养及活化3
1.2.5 农杆菌侵染3
1.2.6 *景先生毕设|www.jxszl.com +Q: ￥351916072$ 
探究影响陆地棉多芽再生转化的因素3
1.2.7 转基因植株的红色荧光蛋白检测3
1.2.8 筛选转基因植株对卡那霉素的敏感浓度3
1.2.9 转化再生植株的分子检测3
2 结果与分析3
2.1 培养条件的建立3
2.2 在MSB培养基中添加不同激素或物质对子叶节多芽诱导的影响4
2.2.1 6BA对子叶节多芽诱导的影响4
2.2.2 KT对子叶节多芽诱导的影响4
2.2.3 不同6BA和KT浓度配比对子叶节多芽诱导的影响5
2.3 AgNO3对子叶节多芽诱导的影响6
2.3.1 不同浓度AgNO3浓度对子叶节多芽诱导的影响6
2.3.2 AgNO3能促进外植体的生根8
2.4 不同基因型子叶节多芽的诱导8
2.5 多芽的伸长9
2.6 激素对棉花生根的影响9
2.7 再生植株的移栽10
2.8 农杆菌侵染陆地棉多芽再生转化的影响11
2.8.1不同农杆菌浓度对多芽再生能力的影响11
2.8.2 不同侵染时间对多芽再生能力的影响11
2.8.3 不同共培养时间对多芽再生能力的影响12
2.9 转基因植株的鉴定13
2.9.1 体视镜下观察红色荧光蛋白表达情况13
2.9.2 转基因植株对卡那霉素的敏感性13
2.9.3 转基因植株的PCR分子检测14
3 讨论16
3.1 激素对陆地棉子叶节多芽诱导的影响16
3.2 AgNO3对陆地棉子叶节多芽诱导的影响16
3.3 农杆菌侵染和共培养条件16
3.4 转基因植株的鉴定16
致谢17
参考文献17陆地棉子叶节多芽再生体系与遗传转化方法的探究
Study on multibud regeneration system and genetic transformation method of cotyledonary node in terrestrial cotton
Seed Science and Engineering Student Shi Xian
Instructor Professor Liu Kang
Abstract: Cotton tissue culture and transgenic systems with hypocotyls as explants have been widely used in cotton transgenic research, but the experimental process of this system requires repeated transfer culture, which is easy to pollute and consume long. In order to explore a transgenic system with short timeconsuming and simplified experimental procedures, this study explored the use of cotyledonary nodes as explants to induce the production of multiple shoots in adventitious embryos. At the same time, the establishment of genetic transformation system was explored by Agrobacteriummediated method. Four land cotton products (species) of Xinluzao No.15, Yanmian No.3, W0 and Coker 315 were selected as experimental materials, and cotyledonary leaves were used as explants, and different hormone or hormone ratios, AgNO3, etc. were analyzed. To induce the effect of multibud production by the organogenesis pathway, the effect of introducing ABD2mCherry fluorescent marker gene into cotton plants by Agrobacterium infecting cotyledonary nodes was explored. The results showed that the addition of 6BA or KT alone in the medium could not induce multibud production, while the hormone was combined with 1.5 mg/L 6BA and 1.5 mg/L KT or 2 mg/L 6BA and 2 mg/L. The efficiency of KT in inducing multiple shoots is higher than when used alone. The use of AgNO3 alone is more efficient than hormoneinduced multibud, up to 88.9%. The addition of GA3 or zeatin to the medium did not significantly induce the elongation of the multiple buds, while the bud elongation was highest in the MSB medium without hormones, and the average bud length was 2.3 cm. The induction rates of different buds were different, and the multiple bud induction rate was affected by genotype. However, the multibud rooting rate is low and is limited by genotype. With AgNO3, the rooting rate was the highest in Xinluzao 42, which was 42.9%. ABD2mCherry was introduced into Xinluzao No.15, Yumian No.3 and W0, 25% multibud anti500mg/L kanamycin by Agrobacteriummediated method, and the target gene was positive by PCR, and PCR amplification was carried out. The fragment was sequenced and proved to be consistent with the target gene sequence. The positive plants could detect red fluorescence under the fluorescence microscope, indicating that ABD2mCherry had introduced multiple buds and was correctly expressed. The method only takes 3 months for the whole test period, and only 2 times of transfer culture can obtain the transgenic multiple shoots. It has the advantages of short time period, saving labor and labor, and is expected to be applied to rapid, highthroughput transgenic research of cotton.

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