Abstract:
Objective To optimize an Agrobacterium-mediated transient transformation system with Larix kaempferi embryogenic callus.
MethodsThe embryogenic callus of Larix kaempferi cultured in liquid medium for 7 days was used as the receptor material, and pCAMBIA1305.1 vector carrying β-glucuronidase (GUS) was used for transient transformation. Based on the expression level and enzyme activity of GUS, the optimal infection solution concentration, infection time and co-culture time were screened. The activity of Larix kaempferi scarecrow-like 6 (LaSCL6) promoter was analyzed with the screened transformation system.
ResultsAfter transient transformation, the expression of GUS was obvious. When the concentration of infection solution was 0.2, the infection lasted for 5 minutes, and the co-culture time was 72 hours, GUS expression was the highest, with -2.274 2. When the concentration of infection solution was 0.05, the infection lasted for 5 minutes, and the co-culture time was 72 hours, GUS enzyme activity was the highest with 25.728 6 U/L. The activity of LaSCL6 promoter was 1.55 times higher than that of CaMV35S promoter
Conclusion In view of the expression level and enzyme activity of GUS, transformation efficiency is high when the concentration of infection solution is 0.05, the infection time is 5 minutes, and the co-culture time is 24 hours, which can be used for efficient transformation of embryogenic callus of Larix kaempferi.
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