Plastid Transformation in Lettuce ( Lactuca sativa L.) by Polyethylene Glycol Treatment of Protoplasts

: A detailed protocol for PEG-mediated plastid transformation of Lactuca sativa cv. Flora, using leaf protoplasts, is described. Successful plastid transformation using protoplasts requires a large number of viable cells, high plating densities, and an efficient regeneration system. Transformation was achieved using a vector that targets genes to the trnI/trnA intergenic region of the lettuce plastid genome. The aadA gene, encoding an adenylyltransferase enzyme that confers spectinomycin resistance, was used as a selectable marker. With the current method, the expected transformation frequency is 1-2 spectinomycin-resistant cell lines per 10(6) viable protoplasts. Fertile, diploid, homoplasmic, plastid-transformed lines were obtained. Transmission of the plastid-encoded transgene to the T1 generation was demonstrated.