Studies on the N_2-laser microbeam system for efficient gene delivery and the utilization of this system for plant protection

波长为337.1 nm、脉宽为3ns的n_2激光在选定的照射条件（1.5 ~ 6 muj/点）下，在甜瓜或烟草培养细胞的细胞壁和细胞膜上灼烧出直径约为0.5 μ m的局部孔。在这些条件下，没有检测到辐射造成的明显损伤。采用荧光-异thyocyanate （FITC）测定培养细胞周围介质的渗透值，0.3 mol / l浓度可获得66.3%的细胞显示荧光。用N_2激光处理植物细胞，测试了基因的传递效率。在含有0.3 M甘露醇和含有β -葡萄糖醛酸酶的质粒PBI221的培养基中，以3muj/点激光照射香仙花、白莲花和石蒜的成熟花粉。瞬时基因表达的最高频率为24%。结果表明，n_2激光微束在表达GUS基因时，花粉粒周围介质的渗透性对GUS基因的表达至关重要，可以将任意基因准确地逐一导入花粉中。在黄瓜花叶病毒RNA存在的情况下用激光处理甜瓜子叶，24小时后在表皮和细胞周围观察到病毒的增殖。这些结果表明，这种激光方法对植物细胞的转化是有用的。非洲野生植物黄瓜（Cucumis figarei Des.et.Noude）对巨细胞病毒的抗性与病毒的受限运动有关，而C.figarei的细胞间和长距离运动速率受巨细胞病毒RNA 3序列的调控。利用n_2激光微束将花菜花叶病毒35S启动子连接的CMV （pepo株）3a和外壳蛋白基因cDNA克隆导入烟草，使表达CP基因的转基因烟草植株免受CMV感染。

An N_2-laser with a 337.1 nm wavelength and 3 ns pulse width burned localized hole ca.0.5 mum in diameter on the cell wall and cell membrane of cultured cells of melon or tobacco under selected irradiation conditions (1.5-6 muj/spot). No visible damage due to the irradiation was detected under these conditions. Fluotescent-isothyocyanate (FITC) was used to determine the osmotic value of the medium surrounding cultured cells and 0.3 Molarity was the best to get 66.3 % of cells which showed fluorescence. Efficiency of gene delivery into plant cells was tested using this N_2 laser treatment. Mature pollen of lmpatiens balsamina, I.walleriana and Lycoris albiflora was irradiated with a laser at 3muj/spot in medium containing 0.3 M mannitol and plasmid PBI221 harboring beta-glucuronidase. The maximum frequency of transient gene expression was 24% in these treatments. It is concluded that the osmotic value of medium surrounding the pollen grains is extremely important for the GUS gene epression to use N_2-laser microbeam and it is able to introduce any gene into pollen exactly one by one. When the melon cotyledons were treated with a laser in the presence of cucumber mosaic virus RNA,virus multiplication was observed in the epidermal and around the cells 24 hr after irradiation. These results suggest that the present laser method is useful for the transformation of plant cells. Resistance in the African wild plant, Cucumis figarei Des.et.Noude to CMV was found to related to restricted virus movement, and the rates of cell-to-cell and long distance movement in C.figarei is regulated by sequences in CMV RNA 3. A cDNA clone encoding the 3a and coat protein gene of CMV (pepo strain), linked to the cauliflower mosaic virus 35S promotor, was introdeced into tobacco by N_2-laser microbeam and the transgenic tobacco plants expressing the CP gene were protected from infection with CMV.