Biolistic transformation of grapevine using minimal gene cassette technology.
The use of minimal gene cassettes (MCs), which are linear DNA fragments (promoter+open reading frame+terminator) lacking the vector backbone sequence, was compared to the traditional use of whole circular plasmids (CPs) for transformation of grapevine. Embryogenic cell suspensions of 'Chardonnay' (Vitis vinifera L.) were transformed via particle co-bombardment using two nonlinked genes in either MCs or CPs. One construct contained the npt-II selectable marker and the second construct contained the MSI99 antimicrobial peptide gene. A total of five lines each from MC and CP treatments that showed positive signals by PCR for both the npt-II and MSI99 genes were selected. Southern blot analyses revealed up to five integration events in the DNA treatments. Transcription levels determined by semi-quantitative RT-PCR varied among transgenic lines. No significant differences were found in transgene transcription between lines from MC and CP transformation. The correlation between npt-II and MSI99 transcription levels was positive (P<0.05), however, no correlation between the transcription level and the number of integration events was observed. Transgenic lines presented a similar phenotype in leaf morphology and plant vigor compared to non-transgenic lines. Moreover, transgenic lines from both MC and CP DNA treatments produced fruit as did the non-transgenic lines in the third year of growth in the greenhouse. Our data confirm the effectiveness of the minimal cassette technology for genetic transformation of grapevine cultivars.
Vidal
Jose R
JR
Department of Horticultural Sciences, New York State Agricultural Experiment Station, Cornell University, Geneva, NY, 14456, USA.
Kikkert
Julie R
JR
Donzelli
Bruno D
BD
Wallace
Patricia G
PG
Reisch
Bruce I
BI
D019470:Biolistics; D015139:Blotting, Southern; D018744:DNA, Plant / Q000378:metabolism; D017343:Genes, Plant / Q000235:genetics; D018745:Genome, Plant / Q000235:genetics; D016254:Mutagenesis, Insertional; D010641:Phenotype; D012038:Regeneration; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic; D014170:Transformation, Genetic; D019076:Transgenes; D027843:Vitis / Q000235:genetics* / Q000502:physiology
D018744:DNA, Plant