Abstract:【Objective】Pomegranate is favored among consumers because of its high economic, nutritional and medicinal value. The rapid development of molecular biology has made genetic transformation an important means for obtainment of excellent new germplasm of crops and for gene function verification, as well as an effective supplement to traditional breeding. At present, the genetic transformation system of pomegranate is incomplete, resulting in gene function research and molecular breeding of pomegranate is lagged compared with other fruit trees. This experiment aims to build a stable and efficient genetic transformation system of ‘Baihuayushizi’ pomegranate, with a view to providing satisfactory technical support for gene function verification of pomegranate and the improvement of its germplasm resources.【Methods】In this study, sterile seedlings of Baihuayushizi pomegranate were used. On the basis of the tissue culture system in the early stage, the redifferentiation system of pomegranate and the concentration of kanamycin and timentin for screening was screened, followed by a discussion on the related influencing factors of genetic transformation such as pre-culture time, concentration of agrobacterium, infection time, and socking time of antibacterial agents. Finally, the optimal genetic transformation system of Baihuayushizi pomegranate mediated by agrobacterium was established.【Results】The addition of 0.22 mg·L-1 6-BA and 0.60 mg·L-1 IBA to the WPM medium significantly improves the redifferentiation of pomegranate implants, which is manifested as higher differentiation rate of leaves and tender stems compared to other treatment portfolios. The differentiation rates were 83.93%±2.52% and 96.30%±5.20%, respectively, the differentiation rate of tender stems increased by 14.74% compared with that of leaves. In addition, tender stems with a high differentiation rate were used as the receptor, and both induction rate and differentiation rate were significantly higher than other treatment without addition of kanamycin. When kanamycin was 50 mg·L-1, the induction rate of the callus of tender stem was reduced from 80.00% to 56.67%, and the differentiation of adventitious buds was as weak as only 8.33%±0.02%. When kanamycin was >60 mg·L-1, the callus induced by tender stem was severely browned or even died with a differentiation rate of 0, which indicates that this concentration is not suitable for screening pomegranate seedlings transformed from tender stems. When timentin was used as the antibacterial agent, if the concentration was 50 mg·L-1, the differentiation rate of tender stems was the highest at 67.22%±0.03%, but the contamination rate of the implant was also significantly higher than that of other treatment groups. When the concentration of timentin was increased to 200 mg·L-1, while the growth of agrobacterium was basically inhibited, the differentiation rate of tender stems and buds could reach more than 50%. Although 250~300 mg·L-1 timentin completely inhibited the growth of agrobacterium, the excessively high concentration was also had a certain inhibitory effect on the growth of implants, and the differentiation rate was less than 50%. In addition, the study on the four important factors of agrobacterium-mediated genetic transformation of pomegranate has showed that the transformation rate varies greatly among different treatment portfolios, and their effect on the genetic transformation rate is manifested as follows: pre-culture time>concentration of agrobacterium>infection time>socking time of antibacterial agents. Further single-factor analysis of variance showed that the genetic transformation rate with pre-culture of 3 d was the highest at 19.33%, which was significantly higher than that of other treatment portfolios. When concentration of agrobacterium OD600=0.7, the transformation rate was 12.17%, which was not much different from that when OD600=0.6 and 0.8 but was significantly different from that when OD600=0.5. The genetic conversion rate with 10 min infection and 15 min immersion in antibacterial agent was higher than that of other treatments.【Conclusion】The addition of 0.22 mg·L-1 6-BA and 0.60 mg·L-1 IBA to the WPM medium significantly improves the redifferentiation of pomegranate implants, with a differentiation rate of tender stems of 96.30%±5.20%. 50 mg·L-1 kanamycin and 200 mg·L-1 timentin are optimal for screening resistant bud. Pre-culture of 3 d, concentration of agrobacterium OD600=0.8, 10 min infection time, and 15 min immersion in 200 mg·L-1 timentin are the most suitable portfolio for genetic transformation of pomegranate. GFP fluorescence detection was performed for verification on the plants obtained under the above-mentioned genetic transformation system, and the positive plant acquisition rate was 26.00%. In this study, genetic transformation system of tender stems agrobacterium-mediated pomegranate stems is successfully established, laying the foundation for verifying pomegranate gene function.
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