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| Agrobacterium tumefaciens-mediated Transformation of Larix leptolepis Embryogenic Tissue |
| ZHU Cai-hong1, LI Shui-gen1, QI Li-wang1, HAN Su-ying2 |
1. Research Institute of Forestry, Chinese Academy of Forestry, Beijing 100091 China;
2. Institute of Forest Ecology, Environment and Protection, Chinese Academy of Forestry, Beijing 100091 China |
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Abstract Based on the technique of L. leptolepis somatic embryogenesis, embryogenic tissues were transformed by A. tumefaciens strain GV3101 carrying a binary plasmid pSuper1300+ with the hpt gene as a selectable marker. Factors that influence transformation were investigated and discussed, including the physiological status of plant calli, the concentration of bacteria, the duration of bacterial infection and co-culture. As a result, after infected by 0.4(OD600)bacterial solution for 10min, co-culture for 2d, then washed three times by liquid medium supplemented with 400mg/L cefotaxime, followed by recovery culture for 7d and selection by 5mg/L hygromycin for several times, a total of 54 hygromycin-resistant cell lines had been obtained. The transformation efficiency is 0.94 transgenic cell lines obtained from per gram of plant calli on average. All the resistant cells were positively transgenic confirmed by polymerase chain reaction (PCR). The development of such a robust transformation method not only provides a useful approach for genetic improvement but also allows us to conduct a functional identification of genes in L. leptolepis.
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Received: 08 November 2012
Published: 25 May 2013
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