Obtainment of fertile and genetically stable transplastomic sugar beet plants over three generations

There are still some obstacles to be overcome in order to improve the plastid genome transformation technology. The most important one is to increase the number of species whose plastid genome can be engineered, and develop for these species reproducible transformation protocols to produce stable and fertile transplastomic plants. We recently produced transplastomic sugar beet (Beta vulgaris L. ssp. vulgaris) plants expressing the aadA and gfp genes, but the primary transformants did not have viable pollen and showed a dwarf phenotype. In this study we obtained transplastomic sugar beet plants of the T2 generation with a normal phenotype by crossing the primary transformants with wild-type plants. The stability of plastid transgenes was demonstrated in the T2 plants because they inherited functional aadA and gfp genes. In addition, to confirm that sugar beet plastids were only maternally inherited, seeds were collected from wild-type plants after their cross-pollination with T1 plants and, as expected, transgenes were not detected in the 1360 progeny seedlings analysed. Moreover, we showed that the sugar beet plastome transformation procedure was reproducible since other transplastomic plants had been obtained with a vector harboring the bar gene, even if afterwards the transgene was lost due to homologous recombination.