摘要
享有“绿色产品”、“生态珍品”的天然彩色棉因其独特的优势和特性，成为目前研究的热点之一。运用转基因技术提高植物抗性，为植物遗传育种提供了一条新的途径。作者在实验中对彩色棉再生体系建立中的一些影响因子作了探讨，并对利用农杆菌介导法将抗病基因兔防御素NP-1导入彩色棉的前期工作进行了初步研究。
选用彩色棉优良品种(棕色)：新彩1号、新彩2号。以下胚轴和子叶作为外植体，确立了5～6日苗龄的彩色棉下胚轴是较好的愈伤组织诱导外植体。不同激素组合及浓度对愈伤组织的诱导有重要作用，2，4-D是愈伤组织诱导中重要的激素，在附加有0.1mol／L 2，4-D+0.1mol／L KT的MSB培养基上，诱导形成的愈伤组织质地疏松、生长旺盛，有利于分化为胚性愈伤。比较了不同糖源在愈伤组织诱导中的效应，在蔗糖和葡萄糖为糖源的培养基中，愈伤组织诱导率均为100％，但葡萄糖更有利于愈伤组织的形成和生长，蔗糖的诱导效果次之；麦芽糖和乳糖对愈伤组织的诱导效应不及蔗糖和葡萄糖；甘露醇作为糖源则抑制了愈伤组织的诱导和生长。在胚性愈伤组织的诱导培养中，KNO_3加倍和NH_4NO_3减半的组合利于胚性愈伤组织的发生。
实验中，利用石蜡切片法和扫描电镜技术对愈伤组织的发生及细胞整体形貌进行了观察。结果表明，存在不同的两类愈伤组织，第一类愈伤组织多呈淡黄色、黄绿色或灰色，质地疏松，生长旺盛，易于分化为胚性愈伤，且该类愈伤组织来源于皮层细胞；第二类愈伤多呈深绿色或白色致密状，生长缓慢，不利于分化为胚性愈伤。
在根癌农杆菌介导的转化过程中，对选择性抗生素Kan的最适浓度、外植体与农杆菌的感染时间、共培养时间以及乙酰丁香酮的效应等进行了初步研究。外植体经农杆菌浸染10min，共培养48h较为合适，在添加头孢霉素500mg／L、卡那霉素50mg／L的培养基中筛选抗性愈伤组织。
Naturally colored cotton, is considered as a "green product" and "ecological treasure". Because of its unique advantage and characteristic, colored cotton becomes one of the fields of research at present. The use of transgenic technology to raise the disease-resistance of the plant, has offered a new way for breeding of plant. We discussed some factors influencing the callus induction and growth in a regeneration system. Preliminary studies on gene transformation of colored cotton via Agrobacterium tumefaciens carrying NP-1 was done, in order to enhance the disease-resistance.
Explants derived from hypocotyls and cotyledons segments of 5-6 day seedlings , and hypocotyls were superior to cotyledons in the induction of callus. Different hormones and concentrations play an important role in the callus induction and growth. 2,4-D was an important hormone of induced callus. State of the callus growth was better on basic medium MSB medium containing 0.1mol/L2,4-D and 0.1mol/L KT. The effects of various sugar sources were different on the formation and growth of cotton callus. The results indicated that glucose was advantageous to the induction and proliferation of cotton callus, sucrose came second. Maltose and lactose were not advantageous to the induction and growth of callus. The addition of mannitol restrained the induction of callus. The medium MSB containing twice of KNO3 and NH4NO3 reduced by half could induce a number of embryogenic callus.
At the same time, the callus occurrence and cell configuration of the callus were observed via paraffin slice method and scanning electron microscope technology. Two kinds of callus formed in cultures. One was light yellow or grey and loose, while the other was hard and white or dark green. The former grew rapidly and eventually developed into embryogenic callus. On the contrary, the latter grew slowly. And the former was derived from the cortex tissue.
Furthermore, we discussed Agrobacterium-mediated transformation and
some factors affecting the transformation frequency. For example, the optimum concentration of selective antibiotic kanamycin, infection time, coculture time of explants and Agrobaclerium, and effect of AS. It were appropriate for Agrobacterium infecting explants 10min and coculturing 48h, then transferred into the medium supplemented with 500mg/L Cefotaxime and 50mg/L kanamycin to select resistant callus.
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