Direct Gene Introduction for Woody plants

木本植物样品制备：首先，对木本植物组织进行灭菌处理。幼嫩枝条是灭菌所必需的，而1个月以上的幼嫩枝条不能与真菌或细菌一起去污。实验用琼脂上培养的无菌芽，然后进行薄层培养，以将基因导入组织中。用手工切片机从嫩枝上切出2 mm厚的圆盘，刀片间隔2 mm，间隔2 mm。该切片机能够在几分钟内切出500-1000片2毫米厚的圆盘。另一台手工切片机也用于从杨树嫩枝中制备薄片(500微米厚)，在将基因导入圆盘或薄片之前，对2 mm厚的圆盘进行再生优化。培养1个月后，在含有BA(2ppm)和NAA(0.01PPm)的杨树圆盘上形成2-5个芽。基因的直接导入：用pBI221进行细胞化学瞬时检测。在切片的杨树组织中观察到相当低的瞬时表达。效率为1-40个斑点/片。组织的预处理提高了效率(200-500个点/皿)。这是足够高的获得转基因杨树植株。为了弄清哪些因素与高瞬时表达有关，研究正在进行中。

Sample Preparation of Woody Plants :First, tissues from woody plants were sterilized. Young shoots were essential for the sterilization while the shoots older than one month failed the decontamination with fungi or bacteria. The sterilized shoots cultured on agar medium were used for the experiments.Thin layr culture was then examined for introduction of genes into the tissue. Two mm thick discs from the shoots were obtained by a handmade slicer, which was razor blades arranged 2mm interval with spacers. The slicer was able to slice 500-1000 pieces of 2mm-thick discs within several minutes. Another hand-made slicer was also used for the preparation of thin section (500um thick) from aspen shoots.Before introduction of genes into the discs or thin sections, 2mm thick discs were optimized for regenaration. The discs were cultured on the medium containing various concentration of cytokinin with NAA.After one month culture, 2-5 shoots in an aspen disc were formed with BA(2ppm) and NAA(0.01PPm).Direct introduction of genes :pBI221 was used for cytochemical transient assay. Rather low transient expressions were observed in the aspen tissues sliced. The efficiency was 1-40 spot/plate. A pretreatment of the tissues improved the efficiency(200-500 spots/plate). It is high enough to obtain transgenic aspen plants. In order to clarify which factor is concerned with the high transient expression, studies are going on.