Establishment of a high-efficiency genetic transformation system for papaya using embryogenic cell suspensions as genetic transformation receptors

Abstract: 【Objecive】Papaya (Carica papaya L.) is a wildly cultivated tropical and subtropical fruit with a high nutritional and medicinal value. However, papaya disease is very serious, which is an important problem restricting the development of papaya industry. The key to solve this problem is to use molecular breeding to breed disease-resistant varieties of papaya, and the establishment of efficient genetic transformation system is an important premise of molecular breeding. Therefore, the aim of this study was to establish an efficient genetic transformation system, and provide new technical support for the study of important gene functions and molecular genetic improvement of papaya.【Methods】In thisstudy, immature zygote embryos of Zihui papaya were used as explants to obtain embryogenic cell suspensions (ECS) by induction, proliferation, screening and liquid oscillation culture. Using ECS as the receptors for genetic transformation, and the plant expression vector pCAMBIA1301 was transformed by Agrobacterium- mediated method. This study explored the suitable conditions of antibiotic concentration, infection time, subculture, induction, maturation and germination of resistant embryos, and finally obtained resistant regenerated plants.【Results】First, ECS were treated with different concentrations of cefotaxime sodium (0, 100, 200, 300, 400 mg·L^-1) and hygromycin (0, 3, 5, 7, 10 mg·L^-1 ) respectively, and then the growth state and cell morphology of suspended cells were observed. The results showed that the optimal concentrations of cefotaxime sodium and hygromycin were 200 mg·L^-1 and 5 mg·L^-1 , respectively. After that, the engineered bacteria containing target genes were prepared, and the prepared ECS were co-cultured with the engineered bacteria for 2 days and transferred to the liquid screening medium containing cefotaxime sodiums and hygromycin for subculture. The subculture period was 14 days. GUS staining showed that after 3 subgenerations, almost all the suspended cells were transformed cells. These suspended cells were transferred to a liquid embryo induction medium for culture. After 2 months, a large number of spherical somatic embryos were obtained, and GUS tissue staining was blue. The spheroidal somatic embryos were transferred to a mature medium for culture, and the mature somatic embryos at cotyledon stage were obtained after 2 months. The average resistance somatic embryos rate was 23.2×10³ mature somatic embryos per 1 mL packed cell volume (PCV) of ECS. The germination rate of resistant somatic embryos was 73.26% when cotyledon stage embryos were cultured on a germination medium for 30 days. The result of GUS staining showed that any regenerated bud could be dyed blue. By promoting root culture, the resistant regenerated buds were successfully regenerated, and the regeneration rate was 80.55%. Simultaneously, PCR amplification of the regenerated plants indicated that GUS gene had been integrated into the papaya genome.【Conclusion】In this study, an Agrobacterium-mediated genetic transformation system using papaya ECS as transformation receptor was successfully established. In this technology system, almost all of the ECS infected by Agrobacterium were transformed cells after three times of subselection, and these transformed cells could regenerate plants successfully through somatic embryo induction, maturation, germination and rooting. The germination rate of resistant somatic embryos was 43.65%, the germination rate of resistant somatic embryos was 73.26%, and the plant regeneration rate was 80.55%. The efficiency of genetic transformation of papaya was greatly improved. This system could provide a new approach for gene function study and molecular breeding of papaya.