Optimization of direct shoot regeneration using cotyledonary explants and true leaves from lettuce cv. Romaine (Lactuca sativa L.) by surface response methodology

Abstract

Lettuce (Lactuca sativa L.) is a commercially important crop and useful platform where different heterologous therapeutic proteins have been expressed. During genetic transformation, sometimes the limiting step is the lack of an efficient and reproducible regeneration protocol. Response surface methodology was successfully applied to find the combination of plant growth regulators inducing the largest number of shoots from cotyledonary explants and true leaves. Two independent variables were studied: concentration of α-naphthaleneacetic acid (NAA) and 6-benzyladenine (BA). A central composite design response surface methodology was developed to generate a quadratic model fitting the experimental data. The best combination of NAA (0.1 mg L−1, 0.54 µM) and BA (0.4 mg L−1, 1.78 µM) averaged 37.4 shoots per explant when using cotyledonary explants and 35.4 shoots when regenerating from true leaves. The surface response method predicted the following optimal concentration of growth regulators: NAA (0.09 mg L−1) which is identical in both explant types, while BA is 0.37 mg L−1 (1.64 µM) for cotyledonary explants and 0.35 mg L−1 (1.55 µM) for true leaves. The desirability value for cotyledonary explants is 0.66 and for true leaves is 0.71. The concentration of growth regulators suggested by the model are comparable to the optima experimentally obtained. Thus, the data from the central composite design response surface methodology model appropriately defines the optimal conditions for regeneration of lettuce in vitro. These findings open the possibility for using response surface methodology to optimize plant regeneration via growth regulators in different lettuce cultivars, as well as in other crops.