Whisker-mediated transformation of peanut with chitinase gene enhances resistance to leaf spot disease

Abstract Peanut (Arachis hypogaea) is an important legume and oilseed crop, native to South America and grown in all tropical and temperate regions of the world. A simplified and rapid direct gene delivery system in peanut was developed by vortexing silicon carbide whiskers with callus and with plasmid harboring chitinase and hygromcin genes. The effects of callus age and whisker quantity on transformation efficiency were evaluated. Transformation efficiency (6.88%) was highest when 200 mg of whiskers were used with 5 µg plasmid for 2 g of 20-day-old callus. Hygromcin-resistant calli were regenerated to complete plants which produced seeds normally. Transgene insertion and number of transgene copieswere confirmed by PCR and southern blot analyses, respectively. Transgene expression was evaluated by a pathogenecity test and RT-PCR analysis. In transgenic events, the resistance level to leaf spot disease was far higher than in control plants.

摘要：花生（Arachis hypogaea）是重要的豆科与油料作物，原产于南美洲，目前已在全球所有热带及温带区域规模化种植。本研究开发了一套简便快速的花生直接基因递送体系，通过将碳化硅晶须（silicon carbide whiskers）与愈伤组织、携带几丁质酶（chitinase）基因与潮霉素（hygromcin）抗性基因的质粒共同涡旋振荡，实现外源基因的直接递送。本研究评估了愈伤组织培养时长与晶须用量对转化效率的影响，结果表明：当以2克培养20天的愈伤组织、搭配200毫克晶须与5微克质粒时，转化效率达到最高值6.88%。潮霉素抗性愈伤组织可再生为完整植株，且能正常结出种子。分别通过聚合酶链式反应（PCR）与Southern印迹（Southern blot）分析，验证了外源基因的插入情况与拷贝数；通过致病性试验与逆转录聚合酶链式反应（RT-PCR）对外源基因的表达水平进行了检测。结果显示，转基因株系对叶斑病的抗性水平显著高于对照植株。