Cisgenesis: an Approach for Crop Improvement

Available online at www.ijpab.com Rameshraddy et al Int. J. Pure App. Biosci. 5 (3): 245-250 (2017) ISSN: 2320 – 7051 DOI: http://dx.doi.org/10.18782/2320-7051.4041 ISSN: 2320 – 7051 Int. J. Pure App. Biosci. 5 (3): 245-250 (2017) Research Article

Cisgenesis: An Approach for Crop Improvement

Rameshraddy1, Khan, J. A.2, Salimath Mahesh1 and A. G. Shankar1* 1Department of Crop Physiology UAS, GKVK Bengaluru 2 Department of Plant Biotechnology UAS, GKVK Bengaluru *Corresponding Author E-mail: [email protected] Received: 13.06.2017 | Revised: 21.06.2017 | Accepted: 22.06.2017

ABSTRACT Cisgenesis is a precise science where in genetic modification is by transferring beneficial alleles from crossable species into recipient plant it has additional advantages over traditional breeding / conventional breeding. Cisgenesis can avoid linkage drag, enhance the use of existing alleles. It saves the time to develop variety by combining traditional breeding techniques and modern biotechnology and hence speeds the breeding processes. There are several successful study the utilization of cisgenesis in developing disease resistant crop, quality control, value added to the economic products of many crops perhaps, this technique has its own demerits, hoping that modern techniques will overcome these demerits. Cisgenesis allows plant genome to be modified while remaining plants within the genepool therefore cisgenic plants should not be assessed as transgenic plants.

Key words: Cisgenesis, Allels, Crop, Linkage.

INTRODUCTION quality for varied health benefits as well as for The agricultural production has seen various major abiotic stress like drought with higher revolutions from the green revolution of nitrogen use efficiency. One of the typical 1960’s to the production of transgenic examples for abiotic tolerant GM crop is (genetically modified, GM) crops. This has led ‘Drought Gourd’. However, other trait to unprecedented increase in the cultivation of introgressions are in process of being GM crops which was introduced 15 years ago. implemented1, 2. Keeping the pace, cultivation of GM now The impact of GM technology was accounts for 185 million hectares across globe loud and clear by its success in scientific covering 26 countries GM technology in crop arena. It is unfortunate that the potential of production has been phased into two GM crops is not being realized, primarily generations, while first generation GM crops because of perception of public about mostly account for tolerance to herbicide and transgenic crops being contaminated and insect larvae. Currently, so-called second mixed with DNA of other organisms that generation GM crops aims at enhancing cannot be crossed by natural means.

Cite this article: Rameshraddy, Khan, J.A., Mahesh, S. and Shankar, A.G., Cisgenesis: An Approach for Crop Improvement , Int. J. Pure App. Biosci. 5(3): 245-250 (2017). doi: http://dx.doi.org/10.18782/2320- 7051.4041

Rameshraddy et al Int. J. Pure App. Biosci. 5 (3): 245-250 (2017) ISSN: 2320 – 7051 This had led to prejudice of GM crops to be sexually compatible species. Hence, the unnatural and against the will of God3. This concept and process of cisgenesis and reservation is often linked to a notion of intragenesis are different and regulatory respect for nature and also appears to be framework of these need to be different as interlinked with fears for potential health risks well. and for the spreading of new gene In the process to develop a cisgenic combinations in the environment. plant, which contains one or more genes from With the aim of overcoming major problem of its sexually compatible species, any suitable transgenic i.e., genes from foreign organism, a technique from transgenic organism can be novel scientific intervention came to rescue utilized. Gene isolation, cloning and known as cisgenesis. With promise of transformation are necessarily important step environmental safety4, the transformation for cisgenesis also. Sometimes the term concept of cisgenesis was developed as an cisgenesis is also used to describe an alternative transgenic process. The main Agrobacterium-mediated transfer of a gene objective of cisgenesis is based on exclusive from a crossable – sexually compatible – plant use of genetic material from the same species where T-DNA borders may remain in the or genetic material from closely related species resulting organism after transformation capable of sexual hybridization5, 6. On contrary cisgenesis with T-DNA borders10. to transgenics, where the genes or DNA During the last few decades, a variety of sequence from unrelated species are indigenous genes, coding for valuable traits transferred. Hence, the gene pool exploited by like disease resistance and quality, from crop cisgenesis mimics that of gene pool exhibited plants and their wild relatives have been by traditional breeding 7. Another key point of isolated, characterized and introduced into the cisgenesis is the elimination of selection genetic background of elite germplasm. These marker genes and vector backbone genes from native genes, isolated from the crop plant itself the transformants. Similarity between or from other cross compatible species, are transgenics, Cisgenesis and conventional currently referred as cisgenesis to distinguish breeding is integration of the gene into the such group of genes from the transgenes7 (as genome requires chromosome breaks and in cisgenic approach as there is no introduction DNA repair mechanism. of new gene class from cross incompatible Concept of cisgenesis/ what are cisgenesis species). Hence, exploiting genetic variation A concept named cisgenesis was introduced by symbolize an important application of Jochemsen and Schouten in 2000 in the book cisgenesis which are applied in conventional ‘Toetsen en begrenzen. Een ethische en breeding programme that has been safely used politieke beoordeling van de moderne since decades. biotechnologie’ and made famous by Schouten The worthiness of GM for promising and colleagues8. They claimed cisgenesis as a high quality and quantity of food has got a transformation process wherein, genetic major setback about the concerns of safety in modifications of recipient organism done with terms of growing and eating. The matter is a gene from sexually compatible organisms. further aggregated by some misconceptions of Genes from sexually compatible species which ‘newspaper reports’ for the probable could be same species or closely related unpredictable hazards to biodiversity and includes introns and is also flanked by its confer toxicity, allergy and genetic threat to native promoter and terminator in sense human nutrition. These arguments are based orientation. Even though there has been on a misreading of the world's real problems various definitions as to how a cisgenesis and are driven not by rationality. People would be defined,7,9 as more complexity arises mostly tend to rely on intuitive feelings to give from intragensis where P-border and vector judgement on GMO’s 11. GMO liberalization backbone sequence do not originate from the within a larger framework and to examine how Copyright © June, 2017; IJPAB 246

Rameshraddy et al Int. J. Pure App. Biosci. 5 (3): 245-250 (2017) ISSN: 2320 – 7051 governments deal with this kind of policy recombination and thus resulted in marker-free through their communication is also a driving lines. Presence of HcrVf2, absence of trfA force and needs to be assessed12. Analysing the (responsible for initiation of replication) and gravity of the situation scientists considered nptIII as part of the backbone, and the fusion developing ‘publicly accepted’ technology marker gene nptII/codAwas demonstrated by which can be perceived as safe as conventional PCR. The author considers this as the first breeding preferably lacking the linkage drag. report of generation of true cisgenic plant. Hence, the cisgenesis was conceived with a Cisgenic barley with improved phytase main objective of transferring only desired activity18 was demonstrated with barley genes and furnished no unnecessary hazard phytase gene (HvPAPhy_a) which is expressed compared to induced translocation or mutation during grain filling. Marker gene elimination breeding. method was employed to obtain marker free Application of cisgenesis in crop plant lines. Both the gene of interest and the improvement selection gene were flanked by their own T- Cisgenesis precludes linkage drag, and hence, DNA to allow unlinked integration of two prevents hazards from unidentified hitch genes. Homozyous plants for (HvPAPhy_a) hiking genes13. Due to this reason, cisgenesis were slected which showed an increase of 2.6 is normally safe than traditional breeding to 2.8 fold increase in phytase activity which programmes and various biotic and abiotic was evaluated till three generations. The lines stress resistance genes can be pyramided to with absence of truncated vector-backbone provide wider and long lasting forms of sequences linked to the borders were classified resistance. Common people are found to be as cisgenic. more satisfied with cisgenesis compared to Cisgenic inhibition of the potato cold transgenic crop. In Mississippi, ananalysis induced phosphorylase L gene expression revealed that 81% of public favored to eat and decrease in sugar contents19 uses RNA cisgenic vegetables while only 14 – 23% for silencing construct controlled by 35S promoter transgenic vegetables14. Also the notion and the OCS terminator and selected putative towards transgenic has brought firm transgenic shoots on kanamycin-containing regulations worldwide which made it medium. Here, removal of the selection necessary to differentiate between these two marker was not reported. However, silencing technologies. Although in Indian scenario15, no of starch phosphorylase L gene reduced starch significantly different (P = 0.16), 76% and breakdown during cold storage conditions. 73% of respondents stated a willingness-to- Expression of KxhKN4 and KxhKN5 genes consume GM and cisgenic foods, respectively. in Kalanchoë blossfeldiana 'Molly' results in Pertinently, a recent study in Denmark novel compact plant phenotypes20. This is a indicated that higher level of scientific novel approach towards a cisgenesis knowledge generally makes people less likely alternative to growth retardants. To control the to care about the technology involved elongated habit of potted plants, growth (cisgenesis or transgenic)16. regulators are used. It is expected that in near Success stories of Cisgenesis future these regulatory may be banned and The development of cisgenic Apple plant17 there should be an alternative for growth employed the use of ORF of endogenous apple regulators. Authors in this study have used two scab resistance gene HcrVf2 from the wild constructs with the coding sequence of the relative Malus floribunda under the control of class I and class II homeobox KN genes, its own regulatory sequence into scab KxhKN5 and KxhKN4, respectively, were susceptible cultivar ‘Gala’. The segment overexpressed in the commercially important between the recombination sites that contains ornamental Kalanchoë blossfeldiana 'Molly'. the nptII gene for kanamycin selection was Furthermore, a post-transcriptional gene removed through dexamethasone-induced silencing construct was made with a partial Copyright © June, 2017; IJPAB 247

Rameshraddy et al Int. J. Pure App. Biosci. 5 (3): 245-250 (2017) ISSN: 2320 – 7051 sequence of KxhKN5 and also transformed into the gene expression25 and phenotypic 'Molly'. Several transgenic plants exhibited differences26. Development of marker free compact phenotypes and some lines had a transgenics requires implementation of new relative higher number of inflorescences. A technologies, and such technologies may not positive correlation between gene expression be available for crop to be engineered. Thus, levels and the degree of compactness was considerable efforts have to be given to found. These transgenic plants show that a produce high numbers of transformants, cisgenesis approach towards production of especially for crops with low transformation compact plants with improved quality as an efficiencies alternative to chemical growth retardants may Safety and regulatory issues regarding be feasible. cisgenesis Gibberellin-associated cisgenes modify There are different views emanated regarding growth, stature and wood properties in cisgenesis in recent past. Cisgenesis may be Populus21 by inserting five cisgenes that safer than conventional breeding, wherein encode proteins involved in gibberellin linkage drag can be prevented27. However, the metabolism or signalling. All cisgenes were issue of any endogenous gene silencing need isolated and transformed along with their to be considered. The donor sequence does not promoter and terminator regions (in this case replace an allelic sequence, but is added to the 1-2-kb of 5’ and 1-kb of 3’ flanking DNA), recipient species’ genome. Owing to the and as contiguous sequences including all process of gene transfer, it is possible that the exons and introns. Basta (glufosinate- new sequence is inserted several times in one ammonium) was used for selection during genome, which might affect gene expression plant regeneration (bar gene with NOS and, therefore, phenotype. However, gene promoter and terminator), no removal of the duplication is a common natural occurrence, selectable marker gene was demonstrated. The for instance in the case of resistance genes or genes used in the study were expressed in the other multigene families28. Further, the xylem and phloem and identified by cisgenic plant might contain some small, non- microarray expression data. They observed a coding sequences from the vector such as T- great variation in the large number of DNA borders, which are 25-base-pair independent events they analysed. The imperfect repeats that delimit the DNA successful insertion of the cisgene was PCR segment transferred to plant cells when using verified using primers directed at flanking T- Agrobacterium mediated gene transfer. DNA sequence that was not present in wild Similarly as transgenics, similar safety issue type plants. should be concerned for cisgenesis, since they A partial cisgenic event was done for contain new gene / protein (s)29. late blight resistance in potato using resistant gene RB from the wild species Solanum CONCLUSION bulbocastanum Dun22. However, transformed By definition, cisgenesis is a form of genetic plants contained kanamycin resistance gene modification, as it transfers a gene and its NPTII with normal RB construct. Hence, this promoter to a recipient species. However, the even is considered in partial cisgenesis. product is clearly different from transgenic Drawbacks of cisgenesis plants, which are derived by transferring Unlike transgenics, the gene(s) from sexually ‘foreign’ or artificial genes, or artificial incompatible cannot be introduced and the combinations of genes and promoters. generation of cisgenesis is time consuming23. Cisgenesis therefore respects species barriers, Moreover, the gene of interest or fragments of and in this sense differs fundamentally from genes may not be readily available but need to transgenesis. Cisgenic plants are similar to be isolated from the sexually compatible gene traditionally bred plants, because the pool24. Position effect may lead to alteration of transferred genes come from the same gene Copyright © June, 2017; IJPAB 248

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