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Optimization of Leaf Regeneration and Genetic Transformation System of Populus tomentosa

LI Chun-Li;WANG Xiao-Jing;DING Qiang-Qiang;WU Han*   

  1. College of Horticulture,Nanjing Agricultural University,Nanjing 210095
  • Online:2016-03-15 Published:2016-05-20

Abstract: The experiment was conducted to optimize leaf regeneration and genetic transformation of Populus tomentosa, and performed to select best leaf regenerative method, rooting medium, with an emphasis on hormone combination and compositions, and to select best explants and Agrobacterium concentration. We demonstrated that twenty days were needed for generation of cluster buds through direct differentiation of leaf tissues. With the mixed hormones of 6-BA and TDZ, we observed the 100% leaf differentiation. Further, the propagation coefficient of single leaf was improved by the step of shoot elongation. We also drastically improved root initiation and growth if gelrite was used when compared to commonly used agar, judging with the number and the length of the roots, and the shoots can root for about eight days. For genetic transformation efficiencies, our experiment showed that leaf explants after numerous subcultures can be infected by Agrobacterium but the transformation efficiency was reduced, if the primary cultured leaves were selected as explants and the concentration of bacteria liquid was OD600=0.3, transgenic callus formation was greater than 83.5%, all shoots survived the antibiotic selection were 100% transgenic, and the transformation efficiency was more than 55%. We report a highly efficient genetic transformation method for P.tomentosa, it is significantly different from the previously published methods. The regeneration of leaf discs through direct differentiation, the mixed use of hormone 6-BA and TDZ, and the step of shoot elongation can improve the efficiency of leaf tissue culture, also using gelrite as the coagulant in rooting medium without the addition of hormone promotes the initiation and growth of roots, and improves the plant growth as well. The primary cultured leaves are more suitable for genetic transformation of P.tomentosa.

Key words: Populus tomentosa, leaf regeneration, genetic transformation, TDZ