Relationship Between Seed Coat Color and Cytokinin Concentration in Efficiently Regenerating Leaf Lettuce Shoots: In Vitro Experimental Study

Abstract Background Lettuce ( Lactuca sativa L.) is an economically important leafy vegetable that is cultivated worldwide. Advances in plant biotechnology have enabled the development of transgenic and transplastomic lettuce lines with specific agronomic traits that produce pharmaceutical proteins and biological compounds. Plant regeneration efficiency is a critical and highly cultivar-dependent step in plant genetic transformation. No morphological markers have been identified that predict the regeneration ability or cytokinin requirement of lettuce cultivars, hindering the establishment of efficient regeneration systems. Objective This study aimed to optimize the direct shoot regeneration efficiency of leaf lettuce cultivars and identify a morphological trait that predicts the optimal cytokinin concentration for each cultivar. Methods The direct shoot regeneration of two cultivars (Chima-sanchi and Chirimen-chisya) was tested on media containing various concentrations of the cytokinin 6-benzylaminopurine (BAP). Four additional cultivars with different seed coat colors were analyzed to determine the relationship between seed coat color and the optimal BAP concentration. Statistical significance was evaluated using the Student t test, with significance set at P <.01. Results The highest regeneration efficiencies in Chima-sanchi (80.5%, SE 3.0%; 103 of 128 explants) and Chirimen-chisya (50%, SE 4.4%; 64 of 128 explants) were obtained with 0.05 and 0.5 mg/L BAP, respectively. Therefore, the optimal BAP concentration differed significantly between the cultivars ( P <.01). The seed coat color and the optimal BAP concentration required for efficient direct shoot regeneration were strongly correlated among the six cultivars. Conclusions Seed coat color is a useful morphological marker for predicting the optimal BAP concentration required for efficient direct shoot regeneration in leaf lettuce cultivars. These findings contribute to optimizing lettuce shoot regeneration systems for specific cultivars.