Optimization and Improvement of Agrobacterium-Mediated Genetic Transformation System for Lactuca sativa L.

Objective Lactuca sativa L. genetic transformation was optimized by reformulating the hormones used in culture medium.

Methods BINFEN-3 was used as the receptor to construct the pKSE401-MYB75 knockout vector to be introduced into Agrobacterium GV3101 for the transformation. Using MS as the basic medium, different types and concentration ratios of plant growth regulators, including naphthylacetic acid (NAA), 6-benzylaminopurine (6-BA), kinetin (KT), and 2,4-dichlorophenoxyacetic acid (2,4-D), were designed for pre-culture/co-culture media.The specific designs are as follows: 0.05 mg·L⁻¹ NAA + 0.5 mg·L⁻¹ KT, 0.1 mg·L⁻¹ NAA + 0.05 mg·L⁻¹ 6-BA, 0.1 mg·L⁻¹ NAA + 0.05 mg·L⁻¹ 6-BA + 0.5 mg·L⁻¹ 2,4-D, and 0.1 mg·L⁻¹ NAA + 0.1 mg·L⁻¹ 6-BA + 0.5 mg·L⁻¹ NAA. Under the constant antibiotic concentrations of 300 mg·L-1 timentin (Tim) and 50 mg·L-1 kanamycin (Kan) in the differentiation and selective medium, four transformation systems were established corresponding to the four plant growth regulator combinations mentioned above, labeled as Scheme A (0.05 mg·L⁻¹ NAA + 0.5 mg·L⁻¹ KT + 300 mg·L⁻¹ Tim + 50 mg·L⁻¹ Kan), B (0.1 mg·L⁻¹ NAA + 0.05 mg·L⁻¹ 6-BA + 300 mg·L⁻¹ Tim + 50 mg·L⁻¹ Kan), C (0.1 mg·L⁻¹ NAA + 0.05 mg·L⁻¹ 6-BA + 0.5 mg·L⁻¹ 2,4-D + 300 mg·L⁻¹ Tim + 50 mg·L⁻¹ Kan), and D (0.1 mg·L⁻¹ NAA + 0.1 mg·L⁻¹ 6-BA + 0.5 mg·L⁻¹ NAA + 300 mg·L⁻¹ Tim + 50 mg·L⁻¹ Kan).

Results For efficient lettuce bud induction, a pre-culture/co-culture medium of MS + 0.1mg NAA·L⁻¹ + 0.05mg 6-BA·L⁻¹ and a differentiation/selective medium of MS + 0.1mg NAA·L⁻¹ + 0.05mg 6-BA·L⁻¹ + 300mg Tim·L⁻¹ + 50mg Kan·L⁻¹ were used. A callus rate of 99%, a budding rate of 50%, and a conversion efficiency of positive seedlings reaching 6.4% were obtained on the culture.

Conclusion The genetic transformation of BINFEN-3 was achieved with the optimized plant growth regulators ratio for the Agrobacterium-mediated callus regeneration.