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作 者:何玮毅[1,2] 陈晓静[1,2] 叶一江[1,2] 申艳红[1,2] 卢秉国[1]
机构地区:[1]福建农林大学园艺学院 [2]福建农林大学园艺植物遗传育种研究所,福州350002
出 处:《果树学报》2010年第3期397-403,共7页Journal of Fruit Science
基 金:福建省科技厅重点项目(编号:2008N0008)
摘 要:番木瓜采后期间的迅速软化现象,导致果实的货架期十分短暂。利用RNAi-β-Gal双T-DNA植物表达载体p2301/TTRG,通过农杆菌介导,探索了液体振荡转化法和浸泡转化法对番木瓜胚性愈伤组织共转化效率的影响。经抗生素敏感性测定,将卡那霉素筛选质量浓度确定为150mg·L-1。在该质量浓度下,2种方法均获得了转基因再生植株,但转化效率仍较低。正交试验结果表明,浸泡转化法中各处理因素的作用大小为:AS质量浓度>共培养时间=菌液光密度值=侵染时间,最佳共转化组合为:100μmol·L-1AS+菌液光密度值0.2+侵染20min+共培养3d。经PCR和Southern杂交检测,所获得的5株再生植株中有2株为共转化的结果,诱导了其中1株生根并移栽。为进一步选育无选择标记基因的抗软化转基因番木瓜奠定了基础。The rapid softening of fruit during postharvest storage has led to the limited shelf life of papaya. Transformation methods of liquid-shaking and soaking, mediated by Agrobacterium tumefaciens harboring an RNAi-β-Ga/two-T-DNA plant expression vector p2301/TTRG, were compared and optimized for the efficiency in co-transformation of embryogenic cafli of papaya. The suitable kanamycin concentration for the selection of transformants was 150 mg. L^-1 based on the antibiotic sensitivity measurements. Under this concentration, the transgenic plantlets were obtained by both the two methods, but the transformation efficiency was low. The results of orthogonal experiments indicated that AS concentration was most important among the factors which influenced the transformation efficiency in soaking method. The co-cultivation time, bacterial culture OD and infection time functioned equally. The calculated best combination consisted of 100 μmol. L^-1 AS, bacterial cuhure OD 0.2, 20 min for infection and 3 d for co-cultivation. Two of the five regenerated plantlets were proved to be cotransformed by PCR assay and southern blot, and one of them was induced to root and transplanted. The study will facilitate the breeding of transgenic papaya lines, which are marker-free and softening-resistant.
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