An Improved Method for Agrobacterium-Mediated Genetic Transformation of Three Types of Lettuce

Lettuce genetic transformation is genotype-dependent. In the present study, we have successfully developed an optimized Agrobacterium-mediated transformation protocol for elite lettuce cultivars, which belong to the romaine, leaf, and butterhead cultivar types. We optimized the type and concentration of plant growth regulators (PGRs) and selection antibiotics and found that the use of 1-naphthaleneacetic acid (NAA; 0.10 mg/L) and 6-benzyladenine (BA; 0.25 mg/L) as plant growth regulators, the use of hygromycin (15 mg/L) for transgenic plant selection, and the use of cotyledons and the first true leaf as explants efficiently yielded transformed plants for seven out of the eleven tested cultivars, achieving a 24.3-100% transformation efficiency. These seven cultivars include two romaine-type cultivars, three leaf-type cultivars, and two butterhead-type cultivars, and mark the first successful genetic transformation of the romaine cultivars 'Kahu' and 'Rosalita', the leaf cultivars 'Red Sails' and 'Royal Oak Leaf', and the butterhead cultivar 'Lollo Biondo'. We also observed that substituting hygromycin selection with kanamycin selection (40 mg/L) resulted in a 64.3% transformation efficiency in the butterhead-type 'Mariska', one of the remaining four cultivars. Our newly optimized protocols are applicable in elite lettuce cultivars for Agrobacterium-mediated genetic transformation and regeneration, enabling hygromycin or kanamycin selection.