ORIGINAL ARTICLES   

Minerva Biotecnologica 2014 December;26(4):247-55

Copyright © 2014 EDIZIONI MINERVA MEDICA

language: English

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

De Marchis F., Pompa A., Bellucci M.

Istituto di Bioscenze e Biorisorse, Consiglio Nazionale delle Ricerche, Perugia, Italy

AIM: The aim of this study was to investigate how to obtain stable transplastomic sugar beet plants with a normal flowering and growth phenotype, and to verify if they maternally inherited functional transgenes over three generations.
METHODS: T0 (primary transformants) transplastomic sugar beet plants, with the aadA and gfp genes inserted in their chloroplast DNA, which did not have viable pollen and showed a dwarf phenotype, were back-crossed with wild-type plants as pollen donors. To confirm that sugar beet plastids were maternally inherited, seeds were collected from wild-type plants after their cross-pollination with T1 plants and the presence of transgenes was investigated by PCR. PCR analysis was also used in T2 plants to evaluate the inheritance of transgenes, while their functionality was tested by spectinomycin resistance for the aadA gene and by both western blot and green fluorescence emission for the gfp gene. Transplastomic sugar beet plants expressing the bar gene, encoding resistance to the herbicide phosphinothrycin, were generated by particle bombardment.
RESULTS: Back-crossing with wild-type plants was the right way to obtain fertile transplastomic sugar beet T2 plants with a normal growth phenotype and maternally inherited functional aadA and gfp genes. Paternal transmission of the transgenes was not detected in the 1360 progeny seedlings analysed. Our sugar beet plastome transformation procedure was also reproducible since transplastomic plants harboring the bar gene had been obtained, even if afterwards the transgene was lost due to homologous recombination.
CONCLUSION: This transformation procedure, joined to the back-cross with wild-type plants, can be used in the future to obtain genetically and phenotypically stable sugar beet plastid transformants expressing genes of interest.