Transgenic silver birch (Betula pendula) expressing sugarbeet chitinase 4 shows enhanced resistance to Pyrenopeziza betulicola

Abstract

A sugar beet chitinase gene driven by the (4×) CaMV 35S promoter was introduced into silver birch (Betula pendula) through Agrobacterium-mediated transformation. Transgenic shoots were regenerated and grown on WPM medium supplemented with 150 mg/ml kanamycin. From a total of 220 explants, 52 transgenics were obtained and 13 transgenic lines were randomly taken for molecular analysis to confirm the presence of the introduced sugar beet chitinase 4 cDNA by polymerase chain reaction and Southern hybridisation. All 13 transgenic lines were confirmed to contain the gene and further characterised. Northern blot analysis of total RNA indicated that the transgenic lines differed with respect to the steady-state levels of chitinase mRNA. Transgenic lines with high levels of mRNA of chitinase 4 cDNA consistently showed higher levels of resistance to Pyrenopeziza betulicola than transgenics with intermediate or low mRNA levels or a non-transgenic control plant. This report demonstrates that the constitutive expression of this gene in transgenic birch lines increased the resistance of birch against the leaf spot fungus P. betulicola.

Field performance of chitinase transgenic silver birches (Betula pendula): resistance to fungal diseases

“…In the transformation process, foreign DNA is randomly integrated into the plant genome, and the integration site, configuration and copy number of the integrated transgene along with metabolic and environmental factors may all influence the stability of transgene expression (Fladung 1999;LevØe et al 1999;Cervera et al 2000;Kumar and Fladung 2001). In chitinase transgenic birches, the copy numbers of the sugar beet chitinase IV gene were studied before the seedlings were transferred to the field and found to vary between two and four (Pappinen et al 2002). Contrary to some other studies (Cervera et al 2000;Kumar and Fladung 2001), no association between copy number and the stability of the transgene expression was detected in this study.…”

“…Expression of the chitinase IV gene was studied by Northern analyses of the leaves of the transgenic lines grown in the greenhouse before they were transferred to the field (Pappinen et al 2002) and before harvesting the field trial. The same four lines that showed a high level of sugar beet chitinase IV transcript accumulation in the greenhouse also showed high sugar beet chitinase IV expression after 3 years in the field.…”

“…Leaf explants from an elite Finnish birch clone (JR 1/4) in tissue culture were used as the recipient plant material in gene transformations. Pre-culturing the plant tissue, co-culturing with Agrobacterium and selection of transgenic tissue have been described in more detail in Pappinen et al (2002). Fifteen transgenic lines were selected for further analyses and transferred after in vitro propagation to the greenhouse.…”

“…Total RNA was extracted from each sample as described by Chang et al (1993). The RNA was blotted and hybridised as described in Church and Gilbert (1984) (see more details in Pappinen et al 2002). Northern analysis was repeated twice.…”

“…The expression of a basic chitinase IV gene from sugar beet in transgenic silver birch (Betula pendula Roth) improved the resistance of birch against the leaf spot fungus Pyrenopeziza betulicola (Fuckel) in a greenhouse experiment (Pappinen et al 2002). Pyrenopeziza betulicola has recently been identified as the major causative agent of leaf spot disease in silver and pubescent (Betula pubescens Ehrh.)…”