Articles
EVALUATION OF CAULIFLOWER TRANSGENIC FOR RESISTANCE TO XANTHOMONAS CAMPESTRIS PV. CAMPESTRIS
Article number
539_17
Pages
137 – 143
Language
Abstract
Fourteen independent lines of transgenic cauliflower from four cultivars were transformed with binary vectors containing two types of broad spectrum antibacterial peptides.
The Shiva protein is a synthetic analogue of cecropin B from the giant silk moth, while the magainin II peptide was derived from the African clawed frog.
Both chimeric genes were modified for plant codon usage and include a 35S promoter, the 5′ leader sequence from alfalfa mosaic virus (AMV), the signal peptide from the tobacco PR-S gene to target protein export to the intercellular space and a NOS terminator.
In addition, both vectors contained a NPTII (NOS-NPTII-NOS) gene for selection of transgenic cells.
Both Agrobacterium rhizogenes and A. tumefaciens were used to obtain transgenic plants.
Transgene incorporation was confirmed by PCR and Southern analyses, while RT-PCR detected a RNA transcript in three magainin-containing lines.
Southern analysis on 13 lines indicated NPTII copy number ranged from 1-3. Plants from 14 independent transgenic lines from four cultivars were transferred to a contained greenhouse.
In vitro segregation assays on selfed seed from 10 lines indicated a 3:1 ratio for kanamycin resistance to sensitivity in eight lines, as expected for a single active insertion site of the Ti T-DNA. Three A. rhizogenes-derived lines have a mixture of kanamycin resistant seedlings with normal and Ri phenotypes indicating independent segregation of the Ti and Ri T-DNAs.
In vitro bacterial assays using crude leaf extracts confirmed increased resistance to Xanthomonas campestris pv. campestris. Screening of T1 seedlings of one Shiva-containing line in the glasshouse failed to show any increased resistance compared to controls.
The Shiva protein is a synthetic analogue of cecropin B from the giant silk moth, while the magainin II peptide was derived from the African clawed frog.
Both chimeric genes were modified for plant codon usage and include a 35S promoter, the 5′ leader sequence from alfalfa mosaic virus (AMV), the signal peptide from the tobacco PR-S gene to target protein export to the intercellular space and a NOS terminator.
In addition, both vectors contained a NPTII (NOS-NPTII-NOS) gene for selection of transgenic cells.
Both Agrobacterium rhizogenes and A. tumefaciens were used to obtain transgenic plants.
Transgene incorporation was confirmed by PCR and Southern analyses, while RT-PCR detected a RNA transcript in three magainin-containing lines.
Southern analysis on 13 lines indicated NPTII copy number ranged from 1-3. Plants from 14 independent transgenic lines from four cultivars were transferred to a contained greenhouse.
In vitro segregation assays on selfed seed from 10 lines indicated a 3:1 ratio for kanamycin resistance to sensitivity in eight lines, as expected for a single active insertion site of the Ti T-DNA. Three A. rhizogenes-derived lines have a mixture of kanamycin resistant seedlings with normal and Ri phenotypes indicating independent segregation of the Ti and Ri T-DNAs.
In vitro bacterial assays using crude leaf extracts confirmed increased resistance to Xanthomonas campestris pv. campestris. Screening of T1 seedlings of one Shiva-containing line in the glasshouse failed to show any increased resistance compared to controls.
Authors
R.H. Braun, J.K. Reader, M.C. Christey
Keywords
Agrobacterium, black rot, Brassica oleracea, magainin, Shiva
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