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.

Pig edema disease is a bacterial disease caused by enterohemorrhagic Escherichia coli. E. coli produces Shiga toxin 2e (Stx2e), which is composed of one A subunit (Stx2eA) and five B subunits (Stx2eB). We previously reported production of Stx2eB in lettuce plants as a potential edible vaccine (Matsui et al. in Biosci Biotechnol Biochem 73:1628-1634, 2009). However, the accumulation level was very low, and it was necessary to improve expression of Stx2eB for potential use of this plant-based vaccine. Therefore, in this study, we optimized the Stx2eB expression cassette and found that a double repeated Stx2eB (2× Stx2eB) accumulates to higher levels than a single Stx2eB in cultured tobacco cells. Furthermore, a linker peptide between the two Stx2eB moieties played an important role in maximizing the effects of the double repeat. Finally, we generated transgenic lettuce plants expressing 2× Stx2eB with a suitable linker peptide that accumulate as much as 80 mg per 100 g fresh weight, a level that will allow us to use these transgenic lettuce plants practically to generate vaccine material.

Fragaria •~ Ananassa Duch. cv. Toyonoka is a main variety of strawberry in Japan, but it is susceptible to a pathogenic fungus, Sphaerotheca humuli. Rice chitinase gene under the control of cauliflower mosaic virus (CaMV) 35S promoter was introduced into the strawberry plants using Agrobacterium tumefaciens. The transgenic plants showed an increased resistance to the powdery mildew, S. humuli. 1.

Pig edema disease is a bacterial disease caused by Shiga toxin 2e-producing Escherichia coli belonging mainly to serotypes O138, O139, and O141. The B subunit of Shiga toxin 2e (Stx2eB) is a candidate protein for use in a vaccine against edema disease. We produced this protein in transgenic lettuce (Lactuca sativa), an edible plant that can be cultivated in a factory setting. In a transient expression system, we found that NtADH 5'-untranslated region (5'-UTR) functions as a translational enhancer in lettuce cells, and that Stx2eB accumulates most efficiently in the endoplasmic reticulum (ER) of lettuce cells. Stx2eB was produced in stable transgenic lettuce plants expressing a modified Stx2eB gene fused with the NtADH 5'-UTR and sequence encoding ER localization signals.