Late Blight resistant Potatoes by Cisgene Stacking

Late Blight resistant Potatoes by Cis element Stacking overcritical fol unhopeful cultivation of former(a) smite resistant white tateres by cis gene stacking BMC Biotechnology, 2014Recombinant DNA and Genomics sarcastic Review of a Scientific PaperBackgroundFood lease from agriculture has never been higher due to the increasing world population. Issues much(prenominal) as climate control and built up overpopu deepd areas of the world touch on the availability and resources to produce crops. The need to enhance the yield of agricultural crops, as considerably as breed crops with correct traits such as viral foe to common maladys which ignore devastate legion(predicate) farmers produce has never been more pressing. As a world population we are a lot more conscious of how we produce are food ethic anyy and need to consider the genetic diversity of crops within agriculture.Over the ago century improvements in sequencing technologies has allowed the mapping of the genome mo re specifically within adjust species that are exploited for agriculture around the world. The first commercialised GM food was the Flavr Savr tomato in 1994, and presently comm altogether rice, maize, potato, soybean and many others (Hamilton et al. 2012 Vanburen et al. 2015). Next generation sequencing technologies have allowed us to target the genome sequencing ge nonypes and identifying specific alleles that designate favourable phenotypes to improve crop populations from their wild-type relatives (Lin et al. 2014 Neves et al. 2013). Our understanding of the structure and act upon of whole shebang genomes within agricultural imbed genetic species has fuelled the information novel biotechnologies and plan of attackes to their finish for life. The isolation of genes that underpin characteristics of interest and how we can target these specific genes and enthrall them into bracing varieties of crops to improve yield or enhance genetic disease metro (He et al. 2014 Gurs kin et al. 2012).Until the last decade the focus has been on transgenic plants in agriculture, where uncaring genes of interest from unrelated species are transferred into the genome of crop plants. Public acceptance of these crops has been arguable and forced the environmental food safety authority for GM organisms to rigorously regulate the commercial use of these crops to protect the environment. Cisgenesis as a inherent biotechnology approach that only utilises genes from the comparable plant or within the aforesaid(prenominal) species has tackled some of the associated problems within GM agriculture. Cisgensis was recently under retread worldwide from the EFSA to be excluded from the strict regulations set for transgenic produced plants, it was finally defined that cisgenic approach to gene transfers is as safe as traditional breeding techniques (low take aim risk) (Hunter et al. 2014). Herewith in, the cisgenesis approach to integrating late molest resistant R genes to potato varieties is reviewed as a biotechnology tool, the ethical advantages it addresses as well as limitations to its application.Outline of methodsAim To use a cisgenesis approach to go in ii broad spectrum potato late blight R genes, Rpi-sto1 and Rpi-vnt1.1 from the crossable species S. stoloniferum and S. venturii, respectively, into 3 different potato varieties (Jo et al.2014). whole kit and caboodle materials Three potato varieties where obtained to introduce late blight underground, a fourth variety potae9 was used as a positive control for its broad spectrum late blight metro sender construction Single R gene constructs, Rpi-sto1 and Rpi-vnt1.1 were cloned into a binary transmitter pBINAW2 (figure 1 below) and were aligned in tandem to form an R gene stack and one sign-free transubstantiation transmitter.Construct containing both cisgenic late blight R genes (Rpi-vnt1.1 and Rpi-sto1), hardly lacking the bacterial kanamycin confrontation plectrum marker (NPTII) w as transformed to the three selected potato varieties using Agrobacterium-mediated shifting.Rooted shoots were analysed by PCR for gene transfer products (desired R genes)Potatoes where analysed for morphological changes and late blight resistance in detached leaf assays.Cisgenic events were selected because they showed broad spectrum late blight resistance due to the activity of both introduced R genes.The marker-free transformation was compared to marker-assisted transformation by the addition of kanamycin in terms of T-DNA and vector rachis consolidation frequencies (Methods as described in Jo et al. 2014).Results/Conclusions of research paperThe study successfully introduced an effective cisgenic marker-free transformation strategy into three varieties of potato. The transformed plants expressed two stacked late blight R genes, through with(predicate) the infiltration of genus Agrobacterium effectors proved the R genes functionality. It was too confirmed that marker-free tr ansformation is slight abandoned to vector backbone integrating compared to marker-assisted transformation (Jo et al. 2014). According to the recent closing of the European Food asylum Authority (EFSA) GMO Panel, cisgenic plants have a low risk level in comparison to transgenic produced plants. therefore, the potatoes produced in this study, are an environmentally friendly alternative to both controversial transgenic agriculture, as well as the use of chemical agents to treat late blight disease (Jo et al. 2014).Critical Appraisal Reviewing the cisgenesis approach in recent literature it has been used to take viral resistance and enhance favourable traits in different crops (Espinoza et al. 2013 Holme et al. 2013). However, not all crops classiffied as cisgenic fully comply with the European Food Safety Authority (EFSA) for GM plants, as they still contain microbial regulative sequences/marker genes (Holme et al. 2013). More recently, molecular characterization of true cisg enic orchard apple tree plants antecedently produced and expressing the Rvi6 scab resistance genes has been treated in detail (Vanblaere et al. 2014), while invigorated cisgene apples with the same trait have been developed using an alternative recombinase organisation (Wurdig et al. 2015). In the study by Jo et al. the focus was on marker-free cisgene potato plants expressing late blight resistance genes from Rpi-sto1 and Rpi-vnt1.1, produced by Agrobacterium-mediated transformation using marker-free gene but PCR for the selection of transformed plants. Due to the activity of both introduced R genes, cisgene plants present broad-spectrum late blight resistance could be selected (Jo et al. 2014).Cisgenesis relies on the transfer of sequences from the same or related species, consequently, intimacy of specific sequences as well as its position and function in the genome is essential. In the future it is expected this knowledge will press forward the application of this technol ogy in place of transgenesis. unmatched advantage of cisgenesis approach over more common techniques is reduced continuance of selection steps and linkage drag (Hou et al. 2014). Although both transgenesis and cisgenesis use the same genetic modification techniques to introduce genes into plants, cisgenesis introduce only genes of interest from the plant itself or from a crossable species. Therefore, cisgenesis is not any different from traditional breeding and there is no environmental risk as associated with transgenic species.The expression of a abroad gene or suppression of a native genes function allowed the movement of DNA between different species, forcing public perceptions of GMO food as unnatural. Although proved to be a promising tool for the enhancement of crops and frugal development, the concern of public acceptance is a major limiting divisor (Espinoza et al. 2013). Therefore, cisgenesis may be the future of plant breeding and crop development as it excludes fore ign genes and strict regulation by the EFSA for GMO transgenes.Despite considering all the advantages of cisgenic approaches there are limitations to its application. Namely the specific requirement of the regulatory sequences outset as well as the type and size of its constructs. For example, although achieving scab resistance in apples the difference in expression level R genes was certified on using operator from the same species or the native promoter of different lengths (Vanblaere et al.2014 Wurdig et al. 2015). Gene expression can also have a lot of variability, through random insertion of cisgenes. Therefore this random admission of cisgenes into a host genome can be similar to that of transgenes, through expiration of functionality of endogenous genes, reverting back to the same ethical issues cisgenesis aims to avoid. Finally, Jo et al. and similar studies report plants produced by cisgenic transformation methods report the integration of vector backbone sequences, th erefore the specific number of genes copied and amount of vector backbone sequence transferred is an classical factor (Vanblaere et al. 2014 Jo et al. 2014). In potato varieties Jo et al. failed to find a difference in vector backbone integration frequencies between different varieties or when marker-assisted only transformations were compared. However, when marker-free and marker-assisted vector backbone integration frequencies were compared, they found that marker-free transformation was associated with less vector backbone integration (Jo et al. 2014). Alternatively now there are new vectors developed containing buffer plant sequences within the backbone that limit the prejudicious affect of unspecific vector backbone integration of cisgenes (Holme et al. 2013).The cisgene stacking method by Jo et al. consisted of two choices to introduce the R gene stack into different potato varieties, marker-assisted transformation or marker-free transformation. By definition cisgenesis excl udes marker-assisted transformation since the genes are derived from foreign species (Jo et al. 2014). Recently there have been methods developed to remove selectable marker genes from the genomes of transgene species. iodin approach exploits a recombinant strategy to extract a marker gene positioned between recombination sites (Zhou et al. 2013). An alternative approach relies on the segregation of independently coordinated T-DNAs similar to those shown within the vector in figure 1 (Hou et al. 2014 Jo et al. 2014). However, Jo et al. chose the marker-free transformation via agrobacterium-based methods that utilize a plant-derived transfer DNA and a novel momentary selection system to insert only native DNA into potato plants similar to earlier cisgene studies (Jo et al. 2014 Rommens et al., 2004).An overlooked issue by many experts in within the field is the implications of large scale introduction of transgenic crops containing resistance genes through the exploitation of anti biotic resistance. Many GMOs have a gene which relates to resistance to antibiotics inserted into their genome to serve as a marker. Bacterial resistance to antibiotics is already a risk associated with animal agriculture through feed being supplemented with antibiotics, and now antibiotic resistance may be farther facilitated by the action of GM crops with these genes integrated in their genome. In potato varieties Jo et al. chose to pursue marker-free transformation of R genes which excluded the use of kanamycin as the marker for gene transfer. However, they also carried out the experiment with kanamycin as a gene marker as a comparison to the effect on vector backbone integration frequencies. Resistance to antibiotics could spread through the use of bacterial genes in the production of transgenic plants, also the genes often remain in the genome with no further use and future transgenic approaches should think about new ways we can remove these genes. It is possible that antibio tic resistance genes will migrate from transgenic plants to bacteria. There is then the possibility that antibiotic resistance bacteria produced from this pipeline could legal injury future human generations. Therefore, the future focus should remain on cisgenic strategies to GM plants that focus on marker-free transformation technologies.ConclusionFinally, according to the EFSA GMO panel cisgenesis is an environmentally safe approach to introducing new disease resistant genes and quality traits into plants. In different potato varieties it has been successful in the regeneration of light blight resistant potatoes by cisgene stacking, it is quicker to achieve than traditional agriculture and less controversial in the publics perception than transgene GM crops. However, there are limitations in particular the unpredictability of cisgene integration and expression from a biotechnology take down of view, but also the impact that may have on its regulatory status and changing public p erceptions. In the future cisgene approaches may take in from being combined with new biotechnologies and GMO techniques, to help reduce the concern with cisgene integration specificity, and develop improved agriculture approaches to disease and crop quality.References Espinoza, C., R. Schlechter, D. Herrera, E. Torres, A. Serrano, C. Medina, and P. Arce-Johnson, 2013 Cisgenesis and intragenesis new tools for modify crops. Biol. Res. 46, 323-331.Gruskin, D., 2012 Agbiotech 2.0. Nat. Biotechnol. 30, 211-214.Hamilton, J. P., and C. R. Buell, 2012 Advances in plant genome sequencing. Plant J. 70, 177-190.He, J., X. Zhao, A. Laroche, Z. X. Lu, H. Liu, and Z. Li, 2014 Genotyping-by-sequencing (GBS), an ultimate marker-assisted selection (MAS) tool to accelerate plant breeding. Front. Plant Sci. 5, 484.Holme, I. B., T. Wendt, and P. B. Holm, 2013 Intragenesis and cisgenesis as alternatives to transgenic crop development. Plant Biotechnol. J. 11, 395-407.Hou H, Atlihan N, Lu ZX. 2014. innovative biotechnology enhances the application of cisgenesis in plant breeding. Front Plnt Sci. 5 389.Hunter, P., 2014 genetically Modified Lite placates public but not activists new technologies to curb plant genomes could help to overcome public concerns about GM crops. EMBO Rep. 15, 138- 141.Jo, K. R., C. J. Kim, S. J. Kim, T. Y. Kim, M. Bergervoet, M. A. Jongsma, R. G. Visser, E. Jacobsen, and J. H. Vossen, 2014 Development of late blight resistant potatoes by cisgene stacking. BMC Biotechnol. 14, 50.VanBuren, 2015 Progress, challenges and the future of crop genomes. Curr. Opin. Plant Biol. 24, 71-81.Neves, L. G., J. M. Davis, W. B. Barbazuk, and M. Kirst, 2013 Wholeexome targeted sequencing of the uncharacterized pine genome. Plant J. 75, 146-156.Podevin, N., Y. Devos, H. V. Davies, and K. M. Nielsen, 2012 Transgenic or not? No simple perform New biotechnology-based plant breeding techniques and the regulatory landscape. EMBO Rep. 13, 1057-1061.Rommens, C. M., J. S. Ye, C. Richael, and K. Swords, 2004 Improving potato warehousing and processing characteristics through all-native DNA transformation. J. Agric. Food Chem. 54, 9882-9887.Vanblaere, T., I. Szankowski, J. Schaart, H. Schouten, H. Flachowsky, G. A. L. Broggini, and C. Gessler, 2011 The development of a cisgenic apple plant. J. Biotechnol. 154, 304-311.Wurdig, J., H. Flachowsky, A. Sa, A. Peil, and M.-V. Hanke, 2015 Improving resistance of different apple cultivars using the Rvi6 scab resistance gene in a cisgenic approach based on the Flp/FRT recombinase system. Mol. Breeding. 35, 95.