Authors: Ruchi Trivedi, Bhupendrasingh Punwar, Jignasha Patel and Tejas Gohil
Crop improvement is an ongoing activity that started many years ago through domestication of crops by farmers’ selections. Plant breeding is developing vary rapidly and new era of modern plant breeding started in eighties and nineties of last century. Nowadays, safe and sufficient food production is important issue worldwide. Development of improved varieties by modern plant breeding is crucial especially when global warming, population growth, environmental stresses, diminishing land resources are associated with increased demand for quality food.
Genetically Modified Organisms (GMO) could be the answer for many relevant problems affecting crops. However, improving crops through GMO is also often associated with safety concerns, environmental risks and health issues due to the presence of foreign DNA. These limitations have prompted the development of alternative technologies. Recently, cisgenesis and intragenesis have been developed as new tools aimed to modify crops. While cisgenesis involves genetic modification using a complete copy of natural genes with their regulatory elements that belong exclusively to sexually compatible plants, intragenesis refers to the transference of new combinations of genes and regulatory sequences belonging to that particular species. So far, application of cisgenesis and intragenesis as alternatives to conventional transgenesis are limited to a few species, mainly due to the lack of knowledge of the regulatory sequences required.
Cisgenesis and Intragenesis:
The concept of intragenesis was put forth by Rommens et al. in 2007. Intragenes refer to genetic elements originating from the crop species itself or from crossable plant species. Genetic elements like promoters, coding regions and sequences that are similar to T-DNA borders from Agrobacterium tumefaciens. These elements can originate from different genes and loci.
The term cisgenesis was introduced in the year 2006 by Schouten, Krens and Jacobsen who defined a cisgenic plant as a crop plant that has been genetically modified with one or more genes isolated from a crossable donor plant. The source of cisgene and an intragene are same i.e. the recipient species itself or a potential crossable species.
Advantages of Cisgenesis and intragenesis:
Ø Great potential to overcome limitations of classical breeding.
Ø The transfer of genes between sexually compatible plants can be speeded up,
Ø ‘Linkage drag’ associated with conventional breeding is avoided i.e. tightly linked ‘inferior traits’ can be completely eliminated with precise exchange of genetic material.
Ø Gene expression can be increased by employing a more efficient promoter and decreased by gene silencing.
Bottlenecks for the practical use of cisgenesis and intragenesis:
- Many important traits in plants constituting major breeding goals result from interaction of several genes, so the effect of inserted gene has to be monitored for its interaction with other genes.
- New resistances are broken rapidly, hence, a combination of genes should be inserted into recipient plant (gene stacking, multigene cassettes).
- Isolated genes and their regulatory elements are introduced into a different genetic background, so, it has to be proven whether they retain their anticipated function in an altered genetic background.
The GM-regulations worldwide do not distinguish between transgenes and cisgenes. This means that the GM-regulations developed for transgenes (representing genes from the new gene pool for plant breeding), are also applied for situations in which only cisgenes and intragenesis are used.
Consideratons for Regulatory Discussions .
Similarity to conventional breeding
• Cisgenesis yields plants that can be also obtained by breeding or via normal reproduction;
• Intragenic plants cannot be obtained by traditional breeding
Similarity to products of natural variation, genome plasticity
• High degree of natural plasticity and variability between genomes;
• Naturally occurring mechanisms for gene duplication, shuffling, and translocation can generate cisgenics and even intragenic plants
• Transferred genes have a history of safe use
• Possible, with prior knowledge from the producer
Rommens et al., 2008 reduced the accumulation of asparagine, the main precursors, in the tubers of potato by silencing two asparagine synthetase genes through ‘all-native DNA’ transformation. Glasshouse-grown tubers of the transformed intragenic plants contained up to 20-fold reduced levels of free asparagines, thus elevating the potential health issues associated with its dietary intake.
Weeks et al., 2008 employed a vortex-mediated seedling transformation method to transform alfalfa with an all-native transfer DNA comprising a silencing construct for the caffeic acid o-methyltransferase (Comt) gene. This resulted in intragenic plants which accumulated reduced levels of the indigestible fiber component lignin that lowers forage quality.
Han et al., 2010 transformed intact genomic copies of PtGA20ox7, PtGA2ox2,Pt RGL1_1, PtRGL1_2 and PtGAI1 genes from Populus trichocarpa clone Nisqually-1 into Populus tremula · alba (clone INRA 717-1B4), and characterized its growth, morphology and xylem cell size in the greenhouse. The PtGA20ox7 cisgene increased rate of shoot regeneration in vitro, accelerated early growth, thus expanding its genetic variance.
Dhekney et al., 2011 used cisgenic engineering for production of fungal-disease resistant cisgenic grapevines. The Vitis vinifera thaumatin-like protein (vvtl-1) gene was reengineered for constitutive expression. Two cisgenic lines VVTL-1 plants exhibited a 7–10 day delay in powdery mildew disease development during greenhouse screening and decreased severity of black rot disease in field tests.
Joshi et al., in 2011 developed intagenic apple by using apple rubisco gene promoter (PMdRbc) for both HcrVf (Homologues of Cladosporium fulvum resistance genes of Vf region) genes which provide good resistance against Vf avirulent isolates to test their effect on expression and phenotype. The scab susceptible cultivar ‘Gala’ was used for plant transformations.
Holme at al., 2011 employed a barley phytase gene (HvPAPhy_a) expressed during grain filling to evaluate the cisgenesis concept in barley using market free transformation. Two potential cisgenic lines with a single extra copy of the HvPAPhy_a insert showed 2.6- and 2.8-fold increases in phytase activities and the activity levels were stable over the three generations analyzed.
Vanblaere et al., in 2011 established marker free system with pMF1 vector in apple cv. Gala which led to the development of three cisgenic line, containing only gene of interest HcrVf2(Homologues of Cladosporium fulvum resistance genes 2 of Vf region), through method based on recombination.
• Cisgenic and intragenic approaches use genetic transformation techniques to introduce new genes having donor from same or sexually compatible species.
• The problem of linkage drag could be elevated with the application of cisgenesis and intragenesis.
• Cisgenesis is particularly efficient method for cross fertilizing heterozygous plants that propogate vegetatively, such as banana, potato and apple.
• Cisgenesis and intragenesis reduce the threat of gene pollution and accepted widely in public domain.
• Cisgenesis and intragenesis have created an opportunity to initiate new dawn for scientists and breeders to produce new group of genetically modified crops which are ‘consumer friendly.
ü Legislation should be framed/laid keeping in view the difference between cisgenic and transgenic product.
ü Wild germplasm should be exploited for gene transfer through cisgenesis and intragenesis.
ü Multigene cassette should be constructed specifically for biotic resistance durability.
Cisgenesis and intragenesis approaches should be focused in cultivated.
1. Dhekney, S. A.; Li,T. Z. and Gray, D. J. (2011) In Vitro Cell.Dev.Biol.- Plant 47:458â€"466
2. Han, K. M.; Dharmawardhana, PArias, S. R.; Ma, C.; Busov, V. and H. Strauss, S. H. (2010) Plant Biotechnol. J. pp. 1â€"17
3. Haverkort, J. A.; Struik, C. P.; R. G. F. Visser, F. G. R. and Jacobsen, E. (2009) Potato Research 52:249â€"264
4. Holme, I. B.; Dionisio, G.; Brinch, P. H.; Wendt, T.; Madsen, C. K.; Vincze, E. and Holm, P. B.(2011) Plant Biotechnol. J. 10, 237-247
5. Holme, I.B.; Wendt, T. and Holm, P. B. (2013) Plant Biotech. J. 11, 395-407
6. Joshi, S. G.; Schaart, J. G.; Groenwold, R.; Jacobsen, E.; Schouten, H. J. and Krens, F. A.; (2011) Plant Mol Biol. 75:579â€"591
7. Nielsen, K. M. (2003) Nature Biotechnology 21 (3): 227â€"228
8. Rommens, C. M.; Haring, M. A.; Swords, K.; Davies, H. V. and Belknap, W. R. (2007) Trends Plant Sci 12:397â€"403
9. Rommens, C. M.; Swords, H. Y.; Richael, C. K. and Ye, J. (2008) Plant Biotechnol. J. 6, 843-853
10. Schouten H. S.; Krens F. A.; Jacobsen E. (2006) EMBO Rep. 7: 750â€"753
11. Vanblaere, T.; Szankowskia, I.; Schaartb, J.; Schoutenb, H.; Flachowskyc, H.; Brogginia, G. A. and Gesslera, C. (2011) Journal of Biotechnology 154: 304â€" 311
12. Weeks, T. J.; Ye, J. and Rommens, C.; M. (2008) Transgenic Res. 17:587â€"597
About Author / Additional Info: