Establishment of genetic transformation system for Baihuayushizi pomegranate

Abstract:【Objective】Pomegranate is a favorate among consumers because of its high economic, nutritional and medicinal values. The rapid development of molecular biology has made genetic transformation an important means to obtain new excellent germplasm of crops and carry out gene function verification, as well as apply an effective supplement to traditional breeding. At present, the genetic transformation system of pomegranate is incomplete, resulting in the lagging of gene function research and molecular breeding in pomegranate compared with other fruit crops. This experiment aimed to build a stable and efficient genetic transformation system for 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 early stage, the redifferentiation system of pomegranate and the concentrations of kanamycin and timentin were screened, followed by a discussion on the related influencing factors concerning genetic transformation such as pre- culture time, concentration of Agrobacterium, infection time and socking time of antibacterial agents. Finally, the optimal genetic transformation system for 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 into the WPM medium significantly improved the redifferentiation of pomegranate implants, which was manifested as higher differentiation rates of leaves and tender stems compared to other treatment portfolios. The differentiation rates were 83.93%±2.52% and 96.30%±5.20%, respectively, and 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 treatments 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 stems was severely browned or even died with a differentiation rate of zero, which indicated that this concentration was 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 treatments. When the concentration of timentin increased by 200 mg·L^-1 , while the growth of Agrobacterium was basically inhibited, the differentiation rates 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 showed that the transformation rate varied greatly among different treatment portfolios, and their effect on the genetic transformation rate was 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 for 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 into the WPM medium significantly improved 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 were optimal for screening resistant bud. Pre-culture for 3 d, concentration of Agrobacterium OD600=0.8, 10 min infection time and 15 min immersion in 200 mg·L^-1 timentin were 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 that were Agrobacterium-mediated was successfully established, laying the foundation for verifying pomegranate gene function.