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

Isolation and functional characterisation of banana phytoene synthase genes as potential cisgenes

Carotenoids occur in all photosynthetic organisms where they protect photosystems from auto-oxidation, participate in photosynthetic energy-transfer and are secondary metabolites. Of the more than 600 known plant carotenoids, few can be converted into vitamin A by humans and so these pro-vitamin A carotenoids (pVAC) are important in human nutrition. Phytoene synthase (PSY) is a key enzyme in the biosynthetic pathway of pVACs and plays a central role in regulating pVAC accumulation in the edible portion of crop plants. Bananas are a major commercial crop and serve as a staple crop for more than 30 million people. There is natural variation in fruit pVAC content across different banana cultivars, but this is not well understood. Therefore, we isolated PSY genes from banana cultivars with relatively high (cv. Asupina) and low (cv. Cavendish) pVAC content. We provide evidence that PSY in banana is encoded by two paralogs (PSY1 and PSY2), each with a similar gene structure to homologous genes in other monocots. Further, we demonstrate that PSY2 is more highly expressed in fruit pulp compared to leaf. Functional analysis of PSY1 and PSY2 in rice callus and E. coli demonstrate that both genes encode functional enzymes, and that Asupina PSYs have approximately twice the enzymatic activity of the corresponding Cavendish PSYs. These results suggest that differences in PSY enzyme activity contribute significantly to the differences in Asupina and Cavendish fruit pVAC content. Importantly, Asupina PSY genes could potentially be used to generate new cisgenic or intragenic banana cultivars with enhanced pVAC content

Armillaria root rot on highbush blueberry in northern Italy: monitoring, identification and inoculum sources

Highbush blueberry plants infected by Armillaria spp. were reported in north-eastern Italy (Province of Trento). After inspection, 13 blueberry orchards were found to be infected in the Valsugana valley. Armillaria sp. samples were collected from blueberry plants, from bark spread on the blueberry rows and from infected trees and stumps in the orchard surroundings. The species determination was performed using a species-specific multiplex PCR approach. Efficacy trials with potential biocontrol agents against Armillaria sp. were carried out on young blueberry plants. The average percentage of stunted plants in the infected fields was 11%, while the percentage of dead plants was generally very low (average of 1.5%). The most frequent species infecting blueberries were A. gallica and A. mellea: in each field one species largely dominated the other. The tested Trichoderma strains, especially T. atroviride SC1, were the most effective biocontrol agents against A. gallica and A. mellea

The development of a cisgenic apple plant

Cisgenesis represents a step toward a new generation of GM crops. The lack of selectable genes (e.g. antibiotic or herbicide resistance) in the final product and the fact that the inserted gene(s) derive from organisms sexually compatible with the target crop should rise less environmental concerns and increase consumer's acceptance. Here we report the generation of a cisgenic apple plant by inserting the endogenous apple scab resistance gene HcrVf2 under the control of its own regulatory sequences into the scab susceptible apple cultivar Gala. A previously developed method based on Agrobacterium-mediated transformation combined with a positive and negative selection system and a chemically inducible recombination machinery allowed the generation of apple cv. Gala carrying the scab resistance gene HcrVf2 under its native regulatory sequences and no foreign genes. Three cisgenic lines were chosen for detailed investigation and were shown to carry a single T-DNA insertion and express the target gene HcrVf2. This is the first report of the generation of a true cisgenic plan