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 'Chardonna...

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Bibliographic Details
Published in:	Plant cell reports Vol. 25; no. 8; pp. 807 - 814
Main Authors:	Vidal, J.R, Kikkert, J.R, Donzelli, B.D, Wallace, P.G, Reisch, B.I
Format:	Journal Article
Language:	English
Published:	Berlin Springer 01-08-2006 Springer Nature B.V
Subjects:	Bacteria Biolistics Biological and medical sciences Biotechnology Blotting, Southern cell suspension culture Cultivars cultured cells Deoxyribonucleic acid DNA DNA, Plant - metabolism Fundamental and applied biological sciences. Psychology gene expression regulation Genes, Plant - genetics Genetic engineering Genetic technics genetic transformation Genome, Plant - genetics grapes Methods. Procedures. Technologies Microbiology minimal gene cassettes Mutagenesis, Insertional neomycin phosphotransferase II open reading frames Phenotype Phenotypes plant regeneration promoter regions Regeneration Reverse Transcriptase Polymerase Chain Reaction terminator regions transcription (genetics) Transcription, Genetic Transformation, Genetic Transgenes Transgenic animals and transgenic plants Transgenic plants Vitaceae Vitis - genetics Vitis - physiology Vitis vinifera Microprojectile bombardment Genetic transformation Transcription Transgene-transcription Integration events Vitis vinifera Cassette Minimal cassette Gene Magainin genes Vitidaceae Dicotyledones Angiospermae Spermatophyta Transgenic plant
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Summary:	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.
Bibliography:	http://dx.doi.org/10.1007/s00299-006-0132-7 ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	0721-7714 1432-203X
DOI:	10.1007/s00299-006-0132-7