Efficient and Stable Transformation of Lactuca sativa L. cv. Cisco (lettuce) Plastids

Abstract

Transgenic plastids offer unique advantages in plant biotechnology, including high-level foreign protein expression. However, broad application of plastid genome engineering in biotechnology has been largely hampered by the lack of plastid transformation systems for major crops. Here we describe the development of a plastid transformation system for lettuce, Lactuca sativa L. cv. Cisco. The transforming DNA carries a spectinomycin-resistance gene (aadA) under the control of lettuce chloroplast regulatory expression elements, flanked by two adjacent lettuce plastid genome sequences allowing its targeted insertion between the rbcL and accD genes. On average, we obtained 1 transplastomic lettuce plant per bombardment. We show that lettuce leaf chloroplasts can express transgene-encoded GFP to approximately 36% of the total soluble protein. All transplastomic T0 plants were fertile and the T1 progeny uniformly showed stability of the transgene in the chloroplast genome. This system will open up new possibilities for the efficient production of edible vaccines, pharmaceuticals, and antibodies in plants.

High efficiency plastid transformation in potato and regulation of transgene expression in leaves and tubers by alternative 5′ and 3′ regulatory sequences

“…A further dramatic increase of the transformation efficiency (equal to approximately one shoot per bombardment) was, however, obtained when tobacco flanking sequences in transformation vectors were replaced with homologous potato sequences. Results similar to those reported for potato in this study, and for tobacco elsewhere, have been obtained in carrot, cotton, soybean and lettuce, where, in biolistic approaches, the use of species-specific vectors allowed the improvement of plastid transformation frequencies up to 1-2 shoots per bombardment (Dufourmantel et al 2004;Kumar et al 2004a, b;Kanamoto et al 2006). Conversely, the transformation efficiency in tobacco decreased by more than tenfold when flanking sequences derived from the petunia plastome were used for recombination (DeGray et al 2001).…”

“…Overall, the vectors with the tobacco regulatory sequences used in this study gave comparable results in potato and tobacco leaves Tangphatsornruang et al 2003), confirming that regulatory sequences are sufficiently conserved at the structural and functional level to be used across species (Sidorov et al 1999;Heifetz 2000;Kumar et al 2004b;Kanamoto et al 2006).…”

The complete nucleotide sequence of the cassava (Manihot esculenta) chloroplast genome and the evolution of atpF in Malpighiales: RNA editing and multiple losses of a group II intron

“…These examples emphasize the advantages of species-specific vectors for efficient homologous recombination within the intergenic spacer regions of chloroplast genomes. Species-specific chloroplast vectors were successfully employed in developing plastid transformants in carrot, cotton, and lettuce (Kumar 2007(Kumar , 2004aKanamoto et al 2006;Ruhlman et al 2007). In addition to intergenic spacer regions, regulatory elements like promoters, 5′-UTRs and 3′-UTRs are important for expression of transgenes in plastids.…”

“…In addition to intergenic spacer regions, regulatory elements like promoters, 5′-UTRs and 3′-UTRs are important for expression of transgenes in plastids. Recently, high-level GFP expression was achieved in lettuce using endogenous regulatory elements (Kanamoto et al 2006). Therefore, it is important to obtain complete genome sequences of crop plants for various biotechnology applications ; http://www.ncbi.nlm.nih.gov/genomes/ORGANELLES/plastids_tax.html).…”

Transgene containment by maternal inheritance: Effective or elusive?

“…Multigene engineering is also feasible in a single transformation step (23,26). Chloroplast genomes of several major crops have been transformed, including soybean (27) and cotton (28), tree species such as poplar (29), and vegetables or fruits, including tomato (20), potato (30), and lettuce (21). Further research is required to achieve similar success in monocots.…”