Cisgenesis and intragenesis as new strategies for crop improvement

Cisgenesis and intragenesis are emerging plant breeding technologies which offer great promise for future acceptance of genetically engineered crops. The techniques employ traditional genetic engineering methods but are confined to transferring of genes and genetic elements between sexually compatible species that can breed naturally. One of the main requirements is the absence of selectable marker genes (such as antibiotic resistance genes) in the genome. Hence the sensitive issues with regard to transfer of foreign genes and antibiotic resistance are overcome. It is a targeted technique involving specific locus; therefore, linkage drag that prolongs the time for crop improvement in traditional breeding does not occur. It has great potential for crop improvement using superior alleles that exist in the untapped germplasm or wild species. Cisgenic and intragenic plants may not face the same stringent regulatory assessment for field release as transgenic plants which is a clear added advantage that would save time. In this chapter, the concepts of cis/intragenesis and the prerequisites for the development of cis/intragenesis plants are elaborated. Strategies for marker gene removal after selection of transformants are discussed based on the few recent reports from various plant species

Green Genetic Engineering Technology: The Use of Endogenous Genes to Create Fungal Disease-Resistant Grapevines

Use of genetic engineering technology to add disease resistance to otherwise desirable varieties is an attractive approach to establishing a Vitis vinifera L.-based agriculture in the Caribbean. Endogenous genes isolated directly from grapevine were engineered for enhanced expression and transformed into V. vinifera 'Merlot', 'Shiraz' and 'Thompson Seedless', plus Vitis hybrid 'Seyval Blanc'. Transgenic plant lines were selected via greenhouse screening based on resistance to powdery mildew. Outstanding lines were vegetatively propagated and established, along with non-transgenic controls, into USDA APHIS-approved field test sites at UVI St. Croix and UF/IFAS Florida in 2007. Vines grew rapidly and began to fruit in 2008. Based on preliminary results, certain transgenic lines exhibited significant resistance to black rot and anthracnose diseases compared to non-transgenic control lines. Because these are three of the most serious tropical/subtropical fungal diseases of V. vinifera, availability of resistant lines will provide new opportunities for production in the region. Our adaptation of endogenous genes to modulate disease resistance is a first step in creating "green transgenic plants" that contain only genetic elements from grapevine, thus eliminating concerns about incorporation of foreign genes in GMOs

Enhanced resistance to Botrytis cinerea in genetically-modified Vitis vinifera L. plants over-expressing the grapevine stilbene synthase gene

Grapevine (Vitis vinifera L.) was genetically modified with a construct containing a cDNA insert encoding the stilbene synthase gene (Vst1) from grapevine, under the control of the cauliflower mosaic virus 35S promoter in order to test the potential of over-production of resveratrol to protect plants from fungal attack. Southern blot hybridization and quantitative real-time PCR analysis demonstrated the presence and integration of one copy of exogenous DNA sequences in two grapevine-modified lines. Relative expression of the Vst1 gene in different modified lines was confirmed by using gene-specific quantitative real-time PCR. Compared to the control, the concentration of trans-resveratrol quantified by HPLC was up to 7.5 fold higher in the modified plants. The necrotic lesion size of leaves of intact modified plants inoculated by Botrytis cinerea B05.10 strain was consistently smaller and significantly different (p B 0.05) than in control plants, showing that modified grapevine plants were more resistant to the pathogen than the control plants