| Populus L. is an important genus globally planted in commercial forest plantations and some species are used as model plants in the study of forest biotechnology. Up to now, research into genetic transformation of poplars has been made significant achievement, especially in improving the biotic stress and abiotic stress. But most of them were transformed by single gene and ordinarily single trait was improved. In order to obtain transgenic plants with multiple resistant traits, a possible method is to transfere multi-genes simaltaneously.In this study, regeneration systems with high regenerate frequency and biolistic transformation systems were established, and genes of Vgb, SacB, JEF36# and CryIII+Oc-1, were transfered into poplar plants of P.×euramericana'Guariento', P.×euramricana(Dode)Guineir cv.'zhonglin-46', P.detoides ×P.nigra cv.Chile, P.alba×P.berolinensis and P.alba×P.glandulosa via biolistic transformation; Many transgenic plants were obtained containing three exotic genes, double exotic genes and single exotic gene, as confirmed by PCR amplification. Meanwhile, the environmental conditions were optimized too. The results were as follows:In vitro environmental conditions were optimized. The photoperiod, culture media and pH values of the media were studied respectively. It was found that the photoperiod and content of active carbon in the media have little effects on the growth of the in vitro explants, and the optimum pH of the subculture medium for in vitro explants was 7.0.The establishment of high frequency regeneration systems. The regeneration systems for explants from leaves of P.detoides ×P.nigra cv.Chile of were established. The best regeneration media were as follows:MS + 0.8mg/L BA + 0.08mg/L NAA for P.×euramricana (Dode) Guineir cv. 'zhonglin-46'; MS + 1.0mg/L BA + 0.05mg/L NAA for P.×euramericana'Guariento'; MS + 0.8mg/L BA + 0.08mg/L NAA for P.detoides ×P.nigra cv.Chile.The best rooting media were as follows:MS + 0.02 mg/L NAA + 0.02 mg/L IBA for P.×euramricana (Dode) Guineir cv. 'zhonglin-46'; 1/2MS + 0.02 mg/L NAA+ 0.02 mg/L IBA for P.×euramericana'Guariento'; 1/2MS + 0.02 mg/L NAA for P.detoides ×P.nigra cv.Chile ,too.The selected Kanamycin pressures were also determined, 40mg/L and 35 mg/L for the pressures of P.×euramricana (Dode) Guineir cv. 'zhonglin-46'and P.deltoides ×P.nigra cv.Chile and P.×euramericana'Guariento' respectively.The optimization of factors affecting biolistic transformation. It was found that the distance between the stopping screen and target was 9cm, the helium pressure was 1100Psi, and the vacuum level was 25inchHg. The treatment of leaves in mannitol had no effects on the transformation efficiency.4. Multiple genes transformation. Vgb, JEF36# and CryIII+Oc-1 were transfered to P.×euramericana'Guariento',P.×euramricana(Dode)Guineir cv.'zhonglin-46', P.detoides×P.nigra cv.Chile, P.alba×P.berolinensis and P.alba×P.glandulosa simultaneously, and 116 kan-resistant plants were obtained. Transgenic nature was confirmed by PCR amplification. 6 plants with three exotic genes (Vgb, JEF36# and CryIII+Oc-1), 58 plants with double exotic genes (Vgb and JEF36#), and 27 plants with single gene (Vgb or JEF36#) were obtained. |
