Articles
EXPRESSION OF A FUNGAL CHITINASE IN VITIS VINIFERA L. ‘MERLOT’ AND ‘CHARDONNAY’ PLANTS PRODUCED BY BIOLISTIC TRANSFORMATION
Article number
528_41
Pages
299 – 306
Language
Abstract
Genetic engineering offers the potential to improve elite cultivars for individual traits such as disease resistance.
For grapevines, resistance to fungal pathogens is highly desirable.
Chitinases from the biocontrol fungus Trichoderma harzianum inhibit spore germination and hyphal elongation of the grapevine pathogens Botrytis cinerea (the causal agent of bunch rot) and Uncinula necator (the causal agent of powdery mildew) in in vitro assays.
Embryogenic cultures of Vitis vinifera L. cultivars ‘Merlot’ and ‘Chardonnay’ were biolistically transformed with the Trichoderma endochitinase gene ThEn42 under the control of a double 35S CaMV promoter and Alfalfa Mosaic Virus leader sequence.
The selectable marker gene neomycin phosphotransferase II (nptII) driven by the nopaline synthase promoter was also used.
A total of 101 ‘Merlot’ and 93 ‘Chardonnay’ putatively transformed plants were regenerated and evaluated for expression of chitinase using a fluorometric assay.
About 41% of the ‘Merlot’ and 55% of the ‘Chardonnay’ selections had 10 to 100-fold higher chitinase activity than non-transformed control plants.
Transformation was further confirmed by PCR and Southern blot analysis.
All of the chitinase-positive plants analyzed by Southern blotting had an intact copy of the ThEn42 gene.
Many of the plants that did not express chitinase contained the nptII gene but were either without or contained small fragments of the ThEn42 gene.
Chitinase expressing lines are being further propagated for disease resistance assays.
For grapevines, resistance to fungal pathogens is highly desirable.
Chitinases from the biocontrol fungus Trichoderma harzianum inhibit spore germination and hyphal elongation of the grapevine pathogens Botrytis cinerea (the causal agent of bunch rot) and Uncinula necator (the causal agent of powdery mildew) in in vitro assays.
Embryogenic cultures of Vitis vinifera L. cultivars ‘Merlot’ and ‘Chardonnay’ were biolistically transformed with the Trichoderma endochitinase gene ThEn42 under the control of a double 35S CaMV promoter and Alfalfa Mosaic Virus leader sequence.
The selectable marker gene neomycin phosphotransferase II (nptII) driven by the nopaline synthase promoter was also used.
A total of 101 ‘Merlot’ and 93 ‘Chardonnay’ putatively transformed plants were regenerated and evaluated for expression of chitinase using a fluorometric assay.
About 41% of the ‘Merlot’ and 55% of the ‘Chardonnay’ selections had 10 to 100-fold higher chitinase activity than non-transformed control plants.
Transformation was further confirmed by PCR and Southern blot analysis.
All of the chitinase-positive plants analyzed by Southern blotting had an intact copy of the ThEn42 gene.
Many of the plants that did not express chitinase contained the nptII gene but were either without or contained small fragments of the ThEn42 gene.
Chitinase expressing lines are being further propagated for disease resistance assays.
Authors
J.R. Kikkert, G.S. Ali, P.G. Wallace, B. Reisch, G.M. Reustle
Keywords
Botrytis cinerea, disease resistance, gene gun, grapevines, microprojectile bombardment, Trichoderma harzianum, Uncinula necator
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