发根农杆菌在植物细胞壁上受体位点的研究

刚分离的烟草和元麦叶肉原生的南体及用植物细胞壁再生抑制剂处理的上述原质体分别与发根农杆菌共培养，未发现农杆菌的附着；随着原生质体壁再生时间的延长，农杆菌的附着率逐渐的增加；用植物细胞主要组分的水解酶和ＴｒｉｔｏｎＸ－１００分别处理３日龄烟草叶肉原生质体后，进行随着菌数的放射性分析，结果表明，细菌对经过胰蛋白酶和ＴｒｉｔｏｎＸ－１００处理的细胞的附着率显著下降，ＴｒｉｔｏｎＸ－１００提取液中含有糖蛋     

本生烟组织培养及遗传转化体系的建立

本文研究了通过农杆菌侵染获得本生烟(Nicotiana benthamiana)转基因植株的方法。将本生烟中上部叶片消毒后切成5mm×5mm左右的小块作为外植体,在含有6-BA(3mg/L)和NAA(0.2mg/L)的MS培养基中培养2~3d,用含有表达载体的农杆菌侵染后共培养3~4d,转入含有6-BA(3mg/L)、NAA(0.2mg/L)和卡那霉素(100mg/L)的MS培养基中培养2~3周。将外植体边缘产生的愈伤组织和芽点转移到只含有卡那霉素(100mg/L)的MS培养基中继续培养,2周后分化出大量不定芽。继续培养2周,当芽长1~3cm时,将芽转移到含IBA(0.1mg/L)的MS培养基中分化生根,得到再生植株。当再生植株高8~10cm、根长4~5cm以上时,移栽到经过灭菌的营养土中。通过实时荧光PCR扩增,从获得的植株中检测35S启动子和NOS终止子外源基因片段,判定所得的植株的确为转基因植株。最后对本生烟组织培养中应该注意的问题进行了讨论。     

烟草维管束发育相关基因Nvas启动子顺式作用元件鉴定

为研究烟草维管束发育相关基因Nvas启动子顺式作用元件,将克隆得到的Nvas启动子ATG+1~-463区域分为10个不同长度的片段与GFP报告基因融合构建了植物表达载体并完成了5'端缺失分析.利用根癌农杆菌介导转化W38型烟草叶盘,得到了含有不同长度Nvas启动子片段的转基因烟草植株.体视显微镜下观察到含Nvas启动子-216~-463区域的转基因烟草均有绿色荧光蛋白的表达,表达部位集中在维管束组织.结果表明Nvas启动子能使外源基因在维管束组织中特异表达.该启动子活性高于Ca MV35S,在ATG+1~-216区域存在核心启动子元件,在-337~-379区域存在能增强启动子活性的元件.     

三种植物叶肉原生质体分离的比较

以四季樱草(Primula obconica),矮牵牛(Petunia hybrida)及黄花烟草(Nicotiana rustical)叶片为材料,用纤维素酶液分离原生质体,分离结果:三种植物叶肉原生质体数量,随酶液浸泡时间增长和浓度升高而增多;在同一浓度,同一时间酶解下,三种植物原生质体数量,以矮牵牛酶解数量最多,质量最好,四季樱草次之,黄花烟草较差。     

烟草脱外壁花粉的制备

烟草脱外壁花粉的制备夏惠君，周嫦，杨弘远（武汉大学生命科学学院，武汉，４３００７２）关键词脱外壁花粉，制备，烟草中图法分类号Ｑ９４４．４２花粉粒由外壁、内壁与原生质体３部分组成．有关花粉生物学的研究已有报道［‘·‘ｊ．近年来，花粉原生质体的研究也取得...     

Visualizing endomembranes with confocal microscopy in protoplasts and cells of tobacco

The distribution and redistribution of the endoplasmic reticulum (ER) was studied with Confocal Laser Scanning Microscopy (CLSM) during the regeneration of tobacco protoplasts. In freshly isolated protoplasts the ER is cisternal in appearance, forming aggregates of plate-like structures clustered around the chloroplasts. During the cell's development, the ER profiles first lose the clustered appearance, then gradually tubular forms develop. Finally mixed population of some cisternal and tubular shapes and a lot of discrete spherical or cylindrical vesicles is spread through the cell's cytoplasm. The probe 3,3'-dihexyloxacarbocyanine iodide (DiOC₆(3)) proved to be a very efficient tool, allowing observations with a resolution of 0.2 μm.     

烟草脱外壁花粉与完整花粉电激导入效果的比较研究

利用膜不通透的荧光染料钙黄素作为指示剂，比较了烟草脱外壁花粉与完整的未萌发花粉和萌发花粉电激导入的效果．研究了电激过程中的电场强度、脉冲持续时间、电激液的介质成分和浓度对三者的导入率及电激后花粉萌发率的影响．结果表明，从未萌发花粉、萌发花粉到脱外壁花粉，导入率逐渐升高，证实了脱外壁花粉由于摆脱了外壁的阻碍而有利于外源物质的电激导入．但电激后花粉萌发率的变化规律相反．荧光指示剂的应用便于优化选择适宜电激参数，为外源基因的导入实验提供了参考依据．     

Preparation of exine-detached pollen inNicotiana tabacum

A method of preparing exine-detached pollen inNicotiana tabacum was established. Anthers containing early-middle binucleate pollen were cold-pretreated at 4–6°C for 7–14 days, and were suspended in 0.3 mol/L sucrose solution for 2 days. During this process, the exine of most pollen grains dehisced. Then they were transferred into an enzyme solution containing 1% cellulase, 1% pectinase, 0.1% pectolyase, 1 mol/L mannitol, 0.3 mol/L sorbitol, 0.5% potassium dextran sulphate and K3 medium macro elements. After 15–20 min enzymatic maceration, the exine was detached resulting in the release of exine-detached pollen. Factors affecting preparation of exine-detached pollen were investigated, including cold-pretreatment, osmoticum concentration and enzymes used. Xia Huijun: born in Oct. 1963, Ph.D. Current research interest is in plant reproductive biology Supported by the National Natural Science Foundation of China     

三生烟和心叶烟对TMV挑战接种的响应差异

用TMV溶液分别感染三生烟和心叶烟,36h,24h和12h后挑战接种同一浓度的TMV溶液,对其产生枯斑的数目和大小进行了分析比较．结果表明：在感染病毒后同一间隔时间挑战接种TMV,三生烟各处理叶片上形成的全叶枯斑数显著少于心叶烟;两品种烟草感染病毒36h后挑战接种TMV产生的全叶平均枯斑数与其他各处理组的差异都极为显著．     

烟草光能兼养型愈伤组织的诱导和培养条件

以烟草SR1品种的4周龄叶片为外植体,使用MS＋2 mg/L 2,4 D（2,4-二氯苯氧乙酸）＋0.25mg/L KT（6-糖基氨基嘌呤）＋3%蔗糖＋0.8%琼脂的固体培养基成功诱导了叶片的愈伤组织.通过改变蔗糖浓度和激素配比,对兼性愈伤组织的培养基进行了筛选.结果表明：在500 lxs光照下,蔗糖浓度由3%降至1.5%时,愈伤组织有变绿趋势;蔗糖浓度〈1.5%时,愈伤组织容易出现褐化.2 mg/L NAA（α-萘乙酸）＋0.25 mg/L KT的激素组合可有效诱导愈伤组织的光合兼养,而单一植物激素如NAA和6-BA（6-苄氨基腺嘌呤）,不是理想的诱导配方.