Optimization of Genetic Transformation on Phytochrome A by Directly Differentiated Buds in Alfalfa

Abstract

Background: The study showed that phytochrome A (phyA) is involved in alfalfa fall dormancy regulation. To provide the scientific basis for using fall dormancy to predict new varieties’ dormancy class from the molecular level, cultivate alfalfa varieties with cold solid resistance and weak fall dormancy and improve the fall dormancy level of existing excellent types, we could study its function through gene silencing. But the genetic transformation rate is not high, which affects the research progress. Methods: To optimize the genetic transformation system in alfalfa (Medicago sativa L.), a binary plasmid vector pART27-phyA was transferred into alfalfa in Agrobacterium tumefaciens-mediated way. To improve transformation efficiency, we optimized the mediums by screening different Kan (Kanamycin) concentrations, NAA (a-Naphthylacetic acid) concentrations and adding YE (yeast extracts). Result: The results showed that using cotyledon nodes as explants, the proper concentration of Kan antibiotics resistance screening was 100 mg/L, the appropriate concentration of NAA for inducing cotyledon node differentiation was 0.1 mg/L and 0.2 g/L YE was suitable to add on the root induction medium; The expression of phyA in terminal buds and leaves of transformed plants of T1 and T2 generations of phyA in Reindeer was significantly reduced (P less than 0.05), but the expression trend in roots and stems had decreasing trend with no significant changes.