High-efficiency biolistic co-transformation and regeneration of 'Chardonnay' (Vitis vinifera L.) containing npt-II and antimicrobial peptide genes.
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 15 mg/l kanamycin; and (3) embryo induction at 27 degrees C. Embryogenic cell cultures were either bombarded with pBI426, which contains the reporter gene gus (uidA) coding for beta-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 95 days 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.
Vidal
J R
JR
Department of Horticultural Sciences, New York State Agricultural Experiment Station, Cornell University, Geneva, NY 14456, USA.
Kikkert
J R
JR
Wallace
P G
PG
Reisch
B I
BI
D000900:Anti-Bacterial Agents / Q000378:metabolism*; D001483:Base Sequence; D019470:Biolistics; D017931:DNA Primers; D010455:Peptides; D014170:Transformation, Genetic; D019076:Transgenes; D027843:Vitis / Q000235:genetics*
D000900:Anti-Bacterial Agents; D017931:DNA Primers; D010455:Peptides