Bhatnagar Somika, Kapur Anita, Khurana Paramjit
Department of Plant Molecular Biology, University of Delhi South Campus, Benito Juarez Road, New Delhi 110021, India.
Indian J Exp Biol. 2002 Dec;40(12):1387-92.
Particle bombardment is a popular method of direct gene delivery into cell, tissue and organs since it requires minimum pre- and post-bombardment manipulation. In addition, this technique is much easier and fast to perform with intact tissue/organ and reduces the period of in vitro culture. Genetic transformation of mulberry, Morus indica cv. K2 was attempted by particle bombardment using hypocotyl, cotyledon, leaf and leaf callus explants. The effect of various physical and biological parameters during bombardment were studied by the histochemical localization of GUS reporter gene following two days of bombardment and by assessing the number of blue spots per explant. p35SGUSINT was used for optimization of different parameters. The percentage of GUS positive explants was very low with tungsten (20%) as compared to gold particles (36%) indicating tungsten toxicity to the tissue. Maximum GUS activity was observed at 1100 psi helium pressure and 9 cm target distance for hypocotyl, cotyledon and leaf. Double bombardment of explants with 10 microg of DNA loaded on macrocarriers clearly yielded a better (up to 56%) result as compared to a single bombardment (30%). Amongst the various plasmids tested, pBI221 gave the highest (100%) GUS positive explants in the leaf callus.
粒子轰击是一种将基因直接导入细胞、组织和器官的常用方法，因为它在轰击前后所需的操作最少。此外，该技术对完整的组织/器官进行操作更加简便快捷，还缩短了体外培养时间。本研究尝试利用粒子轰击技术，以印度桑（Morus indica cv. K2）的下胚轴、子叶、叶片和叶片愈伤组织为外植体进行遗传转化。通过轰击两天后GUS报告基因的组织化学定位以及评估每个外植体上蓝色斑点的数量，研究了轰击过程中各种物理和生物学参数的影响。使用p35SGUSINT对不同参数进行优化。与金颗粒（36%）相比，钨（20%）处理的GUS阳性外植体百分比非常低，表明钨对组织具有毒性。在下胚轴、子叶和叶片中，在1100 psi氦气压力和9 cm靶距下观察到最大GUS活性。与单次轰击（30%）相比，用加载有10 μg DNA的大载体对外植体进行两次轰击明显产生了更好的结果（高达56%）。在所测试的各种质粒中，pBI221在叶片愈伤组织中产生的GUS阳性外植体比例最高（100%）。
