Agrobacterium-mediated transformation of European chestnut somatic embryos with a Castanea sativa (Mill.) endochitinase gene

Abstract

Chestnut blight, caused by Cryphonectria parasitica, is a severe disease that has devastated chestnut stands in North America and Europe. Genes encoding hydrolytic enzymes such as chitinases, which can degrade fungal cell wall components, are attractive candidates for improving disease resistance. This report describes a reliable and efficient protocol for the Agrobacterium-mediated transformation of somatic embryos of European chestnut with the endogenous CsCh3 gene that codes for chitinase. The transformation efficiency, determined on the basis of the fluorescence of surviving explants, was genotype-dependent. Although somatic embryos of all three lines evaluated were transformed, the best results were obtained with somatic embryos derived from line CI-9 (20%). The addition of silver thiosulphate (20 or 40 μM) improved the transformation efficiency of somatic embryos derived from lines CI-3 and CI-9, although the differences were not significant. A total of 88 independent transformed lines were obtained. The presence of transgenes was confirmed by green fluorescent protein (GFP) expression, PCR and Southern blot analysis. Transgenic lines were maintained by secondary embryogenesis or cryopreservation following vitrification procedures. Maturation and germination of transformed somatic embryos yielded transgenic plants. Fluorescence indicating overexpression of the transgenes was observed in somatic embryos and also in shoots and leaves. No phenotypic differences were found relative to control plants, suggesting a lack of any cytotoxic effects of the GFP.

Agrobacterium tumefaciens-mediated transformation of drumstick (Moringa oleifera Lam.)

“…PCR and Southern blot analysis revealed that the gus gene was integrated into the drumstick genome and revealed only one copy of integrated T-DNA. These low copy numbers in drumstick are similar to those in a number of other species, such as centipedegrass [31], European chestnut [33] and Populus [12]. AMT may integrate several copies of the gene of interest into different parts of the plant genome.…”

Breeding and scientific advances in the fight against Dutch elm disease: Will they allow the use of elms in forest restoration?

“…Likewise, the convenience and decreasing cost of obtaining -omics data of nonmodel organisms and the development of new inexpensive tools for gene editing, such as CRISPR-Cas systems (Ledford 2015), will make these technologies available in more laboratories and for more plant species. Cisgenic approaches may gain broader social support than transgenics because they use genes from the same or closely related plant species, whereas transgenic approaches use genes from non-plant organisms or distantly related plants (Hou et al 2014;Corredoira et al 2016). Cisgenics have been considered similar to inter-species backcross breeding (Han et al 2010).…”

Mechanisms and metabolomics of the host–pathogen interactions between Chestnut (Castaneaspecies) and Chestnut blight (Cryphonectria parasitica)

“…Modern genomics and genetic engineering have opened a path to saving C. dentata and other North American Castanea species. Current transformation work with C. sativa has demonstrated promising results, including the production of C. sativa lines overexpressing chitinase (Corredoira et al, , ). With the Hardwood Genomics Project, it may finally be possible to take a close look at the genetics involved in blight tolerance and susceptibility in all species of the Castanea genus.…”

“…Chitinases may also play a role in plants during early embryo development, although this has not been well studied (Collada, Casado, Fraile, & Aragoncillo, ; De Jong et al, ; Kragh et al, ). In Castanea , numerous studies have shown abundant chitinases in nut tissues, particularly in seedling cotyledons (Allona, Collada, Casado, Paz‐Ares, & Aragoncillo, ; Collada, Casado, & Aragoncillo, ; Collada et al, ; Corredoira et al, ; Gomez & Aragoncillo, ). However, it is unclear whether these play any role in blight resistance, as the blight is primarily a stem‐girdling disease.…”

“…In both C. dentata and C. mollissima , transcripts of several compounds expressing chitinase activity accumulated more in canker tissue than healthy stem tissue (Barakat et al, ). Recent transformation work with C. sativa has generated lines which overexpress the CsCh3 gene isolated from the cotyledons of C. sativa (Corredoira et al, ). However, it has yet to be demonstrated if these transformed lines have increased blight tolerance ex vitro, and no further studies have investigated the role of chitinases in the pathogen defence of C. dentata or C. mollissima .…”