Establishment of genetic transformation system mediated by Agrobacterium in muskmelon

Abstract:【Objective】Melon (Cucumis melo L.) is one of the world’s top ten fresh fruits and is loved by consumers all over the world. With the rapid development of biotechnology, plant breeding technology is changing from domestication breeding, hybrid breeding and molecular marker-assisted selection to artificial intelligence breeding relying on transgenic technology. Genetic transformation has been an important method for gene function verification. At present, the genetic transformation system of melon is not perfect, and the genetic transformation methods and efficiency between different melon genotypes or materials vary differently. To construct a genetic transformation system for muskmelon with a strong stability, good reproducibility and high efficiency, the present experiment was carried out, so as to provide technical support and theoretical basis for the verification of gene function and the improvement of germplasm resources.【Methods】In this study, the binary expressed vector pQY002005-GFP (Green Fluorescent Protein) was used to infect the explant from the cotyledons of B8 genotype (C. melo L. subsp. melo), and all explants that infected the Agrobacterium were used to induce buds regeneration.Key factors affecting the whole genetic transformation process, including the culture of seedlings, seedlings ages, treatment of explants, infection mode and positive buds screening way, were explored to establish the genetic transformation system based on B8.【Results】The explants were first cultured darkly and then treated by photoculture, which was not suitable for transformation. Fluorescent buds could be obtained by dark culture treatment alone, but with D2L0, D3L0 and D4L0 treatments, the metamorphosis rate of fluorescent buds reached 42.9%, 61.5% and 66.7%, respectively, which was significantly higher than that of other treatments and was not suitable for transformation. With D1L0 and D0L3 treatments, the fluorescent bud acquisition rate was relatively high up to 25.6% and 26.7%, respectively, and the fluorescent bud metamorphosis rate was relatively low. The above results showed that the longer the dark culture time, the higher the metamorphosis rate of regenerated buds. D0L3 or D1L0 was a suitable sterile seedling culture method under normal photoperiod. When the seedling age was consistent, the number of bud bushes, bud fluorescence rate and fluorescence bud rate in the non-invasive treatment group were significantly lower than those in other groups, indicating that trauma could promote Agrobacterium infection and increase the number of adventitious buds. The fluorescence bud rate increased significantly by ultrasound and microbrush + sonication treatment of D1L0 explants, but there were no significant differences in the number of bud bushes and fluorescent bud rates of 1.6, 18.4%, 1.8 and 23.3%, respectively, indicating that although microbrushing increased the trauma area of plants and promoted the infection of Agrobacterium infection, it may not improve the fluorescence bud rate due to cell damage. The number of bud plexuses of D0L3 were significantly higher than D1L0, so the best treatment for explants was microbrush + sonication. Furthermore, the degree of infection was high after 25 min immersion, but the number of bud plexes, the fluorescence rate of the bud plexus and the fluorescent bud rate were significantly lower than those of the vacuum pump 85 kPa for 5 min and the vacuum pump 85 kPa for 5 min twice with an interval of 1 min. While using the needle vacuum infection for 30 s, Agrobacterium could reach the deep cells, but the number of bud bushes and the acquisition rate of fluorescent buds were the lowest, being only 1.4 and 4.3%, which was significantly lower than that of the vacuum pump 85 kPa for 5 min, so the vacuum of the needle for 30 s was not suitable for infection. In comparison, vacuum pump 85 kPa for 5 min twice with an interval of 1 min treatment had the highest (64.8%) fluorescent bud rate, and the fluorescence area of explants was also greater than that of one vacuum treatment. Besides, the effect of Basta on budding status was observed in order to obtain a more suitable concentration. The analysis showed that without adding Basta, B8 had strong budding ability. When the concentration of Basta was 2 mg·L^-1 , the germination time was later and the budding amount was less than that without Basta, but the germination rate was still high up to 78.7%, so 2 mg·L-1 Basta could not strongly inhibit negative bud plexes. When the concentration of Basta was 4 mg·L^-1 , the budding rate of explants was late, the number of bud bushes was small, and the budding rate was 14.3%, which was lower than that of 2 mg ·L^{- 1} treatment, indicating that the addition of 4 mg ·L^{- 1} Basta could play a role in screening resistant adventitious buds. When the Basta concentration was much more than 6 mg · L^{- 1} , the effloration rate of explants was 5.5% lower, and it even caused death of explants. The above results showed that 4 mg · L^{- 1} of Basta was a suitable concentration for screening resistant buds. 【Conclusion】The results revealed that cotyledons from sterile seedling cultured for 3 days (under light condition) with microbrush and 10 seconds ultrasonic treatment, could improve the efficiency of Agrobacterium infection. The acquisition rate of fluorescence bud was 26.2%; the best infection system was vacuumed for 5 min twice with an interval of 1 min under the pressure of 85 kPa. The suitable concentration for screening resistant buds was 4 mg·L^-1 Basta. Thirty-one regenerated fluorescent buds and 17rooting seedlings were obtained in a single transformation of 120 explants, and 8 positive seedlings were identified by PCR reaction, with a positive transformation rate and seedlings rate of 58.8% and 6.7%, respectively. This study successfully established a relatively complete melon genetic transformation system on B8, which provided technical support and theoretical basis for key gene function verification and germplasm improvement.