Fulltext

High-efficiency biolistic co-transformation and regeneration of 'Chardonnay' ( Vitis vinifera L.) containing npt-II and antimicrobial peptide genes

Vidal, J. ; Kikkert, J. ; Wallace, P. ; Reisch, B.

In: Plant Cell Reports, 2003, vol. 22, no. 4, p. 252-260

Aggiungi alla tua lista
    Titre
    • High-efficiency biolistic co-transformation and regeneration of 'Chardonnay' ( Vitis vinifera L.) containing npt-II and antimicrobial peptide genes
    Auteur
    • Vidal, J.. Department of Horticultural Sciences, New York State Agricultural Experiment Station, Cornell University, NY 14456, Geneva, USA
    • Kikkert, J.. Department of Horticultural Sciences, New York State Agricultural Experiment Station, Cornell University, NY 14456, Geneva, USA
    • Wallace, P.. Department of Horticultural Sciences, New York State Agricultural Experiment Station, Cornell University, NY 14456, Geneva, USA
    • Reisch, B.. Department of Horticultural Sciences, New York State Agricultural Experiment Station, Cornell University, NY 14456, Geneva, USA
    Type de document
    • Postprint
    Identifiant OAI-PMH
    • oai:doc.rero.ch:322519
    Summary
    A reliable and efficient system for transformation and regeneration of 'Chardonnay' (Vitis vinifera L.) plants via microprojectile bombardment was developed. Improvements over the previous biolistic transformation system included: (1) the use of gold particles for bombardment; (2) step-wise selection at 10 then 15mg/l kanamycin; and (3) embryo induction at 27°C. Embryogenic cell cultures were either bombarded with pBI426, which contains the reporter gene gus (uidA) coding for β-glucuronidase (GUS), or were co-bombarded with pSAN237 carrying the npt-II (neomycin phosphotransferase II) selectable marker gene, and a second plasmid with an antimicrobial peptide gene. A large number of transient (7,883±1,928) and stable (46±32) blue spots per plate at 2 and 95days after bombardment, respectively, were obtained according to GUS expression analyses. A total of 447 putative transgenic embryos was harvested from 84 bombarded plates. From these embryos, 242 (54%) were regenerated into plants within the first year of the experiment. Southern blot analyses confirmed integration of the transgenes into the grape genome. Co-transformation was tested with four separate antimicrobial constructs. The co-transformation frequency of unlinked genes was 48% as measured by polymerase chain reaction (PCR), and 56% as estimated by dot blot hybridization. Expression of the gus gene, and PCR and Southern blot analyses of npt-II and antimicrobial genes from regenerated plants document stable transformation of 'Chardonnay' and establish the parameters for highly-efficient biolistic transformation in V. vinifera