Construction of genetic transformation system of Salix mongolica: in vitro leaf-based callus induction, adventitious buds differentiation, and plant regeneration

Abstract

Songnen meadow grassland is a typical saline-alkaline land majorly comprised of carbonate soil. Salix mongolica, a woody species with high adaptability to carbonate soil, is an important supplementary feed in the grassland. Therefore, it is necessary to cultivate new varieties of S. mongolica by using genetic engineering methods to reveal the functions of the plant’s related genes and to construct a plant regeneration and genetic transformation system. In this study, we used leaves of S. mongolica as the explants for induction of leaf-based callus, differentiation of adventitious buds and rooting of adventitious by adding different ratios of 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzyl aminopurine and naphthaleneacetic acid into the Murashige and Skoog medium. Under the screening conditions of 7.5 mg L−1 hygromycin B and transformation period of 2–5 min using a specific Agrobacterium containing pCXSN-gus plasmids infection concentration (ODλ600 = 0.5), we obtained transgenic strains. PCR detected exogenous gus gene integrated into the chromosome of S. mongolica, Southern blot analysed the T0 transgenic strains single copy inserted into the chromosome, Northern hybridization signals indicated that gus gene mRNA was expressed in the five contemporary transgenic strains. The infected callus, adventitious buds, and regenerated plants displayed a blue color through detection by GUS staining, which reflected the activity of s-glucuronidase enzyme. This result demonstrated the successful establishment of an Agrobacterium-mediated genetic transformation system from the callus (S. mongolica leaf as a transformation receptor).