Kumar Pankaj, Gambhir Geetika, Gaur Ayesh, Sharma Krishan C, Thakur Ajay K, Srivastava Dinesh K
1Dr. Y.S. Parmar University of Horticulture and Forestry, Solan, Himachal Pradesh 173 230 India.
2ICAR-Indian Agricultural Research Institute, New Delhi, 110 012 India.
3 Biotech. 2018 Jul;8(7):299. doi: 10.1007/s13205-018-1316-x. Epub 2018 Jun 27.
Transgenic broccoli ( L. var. cv. 'Solan Green Head' has been developed by using strain harboring gene for resistance against diamondback moth (). By combining the best treatments of 72 h pre-culturing and 48 h co-cultivation time period, a transformation frequency of 4.92 and 13.83% was obtained from cotyledon and hypocotyl explants, respectively. Supplementing the co-cultivation medium with acetosyringone in a concentration of 100 µM enhanced the transformation frequency to 17.92 and 32.11% in cotyledon and hypocotyl explants, respectively. The transgene () integration was confirmed by polymerase chain reaction using gene-specific primers and Southern blot analysis using digoxigenin nonradiolabelled DNA probe. Gene expression in the PCR-positive transgenic events had been confirmed by reverse transcriptase-PCR and quantitative real time-PCR. Insect bioassay proved the effectiveness of the transgene against infestation by diamondback moth () larvae. To the best of our knowledge, this is the first report of optimization of a highly efficient transformation system and transgenic development in broccoli using gene for insect resistance.
通过使用携带抗小菜蛾基因的菌株，已培育出转基因西兰花（L. var. cv. 'Solan Green Head'）。通过结合72小时预培养和48小时共培养的最佳处理时间段，子叶和下胚轴外植体的转化频率分别为4.92%和13.83%。在共培养基中添加浓度为100µM的乙酰丁香酮，子叶和下胚轴外植体的转化频率分别提高到17.92%和32.11%。使用基因特异性引物通过聚合酶链反应以及使用地高辛非放射性标记DNA探针的Southern印迹分析证实了转基因（）整合。通过逆转录聚合酶链反应和定量实时聚合酶链反应证实了PCR阳性转基因事件中的基因表达。昆虫生物测定证明了转基因对小菜蛾（）幼虫侵袭的有效性。据我们所知，这是关于利用抗虫基因优化西兰花高效转化系统和转基因培育的首次报道。
