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Transgenic plantlets of ‘Chancellor’ grapevine (Vitis sp.) from biolistic transformation of embryogenic cell suspensions

Abstract

Transgenic plantlets of ‘Chancellor’ grapevine (Vitis L. complex interspecific hybrid) were produced via biolistic transformation. Embryogenic cell suspensions were bombarded with 1 μm tungsten particles coated with pBI426 which encodes a fusion peptide between β-glucuronidase (GUS) and neomycin phosphotransferase II (NPTII). The fusion peptide is under the control of a double 35S Cauliflower Mosaic Virus promoter and a leader sequence from Alfalfa Mosaic Virus. The cells were placed on kanamycin-containing media (10, 25 or 50 mg/l) 2 d after bombardment. Activated charcoal reduced cell browning. Embryos were first observed on selective media 14–29 weeks after bombardment. More than 1600 clusters of embryos were germinated and/or assayed for GUS. Of 621 embryos assayed for GUS expression, 182 (29.3%) were positive. PCR confirmed the presence of the NPTII gene in all 5 GUS-positive and 2 GUS-negative (bombarded) embryos tested. In germination experiments, 15% of the embryo clusters produced at least one plant with normal shoot growth. Of 164 normal plants assayed for GUS expression, 37 (22.6%) were positive. The NPTII gene was amplified by PCR in 1 (of 1) GUS-positive and 4 (of 5) GUS-negative bombarded plants, but not in non-bombarded control plants. Southern blotting confirmed integration of the NPTII gene in all 3 of the GUS and PCR-NPTII positive plants tested. Biolistics is an efficient method for transformation of ‘Chancellor’ and should be applicable to other important grape cultivars.

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Abbreviations

AC:

activated charcoal

GUS:

β-glucuronidase

2,4-D:

2,4-dichlorophenoxyacetic acid

BA:

6-benzylaminopurine

NAA:

α-naphthalene acetic acid

TDZ:

thidiazuron

NPTII:

neomycin phosphotransferase II

Km:

kanamycin

MS:

Murashige and Skoog (1962) medium

WPM:

Woody Plant Medium of Lloyd and McCown (1980)

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Communicated by G. C. Phillips

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Kikkert, J.R., Hébert-Soulé, D., Wallace, P.G. et al. Transgenic plantlets of ‘Chancellor’ grapevine (Vitis sp.) from biolistic transformation of embryogenic cell suspensions. Plant Cell Reports 15, 311–316 (1996). https://doi.org/10.1007/BF00232362

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Keywords

  • Activate Charcoal
  • NPTII Gene
  • Fusion Peptide
  • Tungsten Particle
  • Grape Cultivar