Assessing zygosity in progeny of transgenic plants: current methods and perspectives
Main Article Content
Keywords
Digital PCR, homozygous line, LAMP assay, real-time PCR, transgenic plants
Abstract
Homozygosity is highly desirable in transgenic plants research to ensure the stable integration and inheritance of transgene(s). Simple, reliable and high-throughput techniques to detect the zygosity of transgenic events in plants are invaluable tools for biotechnology and plant breeding companies. Currently, a number of basic techniques are being used to determine the zygosity of transgenic plants in T1 generation. For successful application of any technique, precision and simplicity of approach combined with the power of resolution are important parameters. On the basis of simplicity, resolution and cost involved, the available techniques have been classified into three major classes which are conventional methods, current methods and next generation methods. Conventional methods include antibiotic marker-based selection and the highly labour intensive Southern blot analysis. In contrast, methods such as real time PCR, TAIL PCR and competitive PCR are not only cost effective but rapid as well. Moreover, methods such as NGS, Digital PCR and Loop-mediated isothermal amplification also provide cost effective, fast and not so labour-intensive provide substitute of current methods. In this review we have attempted to compare and contrast all the available efficient methods to distinguish the homozygous plant in progeny of transgenics. This review also provides plenty of information regarding the various techniques available for determining zygosity in plants so as to permit the scientists to make informed choices of techniques that best suit their analyses. More importantly, detection and subsequent selection of homozygous individuals is central for facilitating the movement of transgenic plant from the laboratory to the field.
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References
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6. Frame BR, Zhang H, Cocciolone SM, Sidorenko LV, Dietrich CR, Pegg SE, et al. Production of transgenic maize from bombarded type II callus: Effect of gold particle size and callus morphology on transformation efficiency. In Vitro CellDevBiol-Plant. 2000;36(1):21-9. doi: 10.1007/s11627-000-0007-5.
7. Nath M, Yadav S, Kumar Sahoo R, Passricha N, Tuteja R, Tuteja N. PDH45 transgenic rice maintain cell viability through lower accumulation of Na+, ROS and calcium homeostasis in roots under salinity stress. Journal of Plant Physiology. 2016;191:1-11. doi: 10.1016/j.jplph.2015.11.008.
8. Rani T, Yadav RAMC, Yadav NR, Rani A, Singh D. Genetic transformation in oilseed brassicas - A review. 2013;83(April):367-73.
9. Rizvi I, Choudhury NR, Tuteja N. Arabidopsis thaliana MCM3 single subunit of MCM2–7 complex functions as 3′ to 5′ DNA helicase. Protoplasma. 2015. doi: 10.1007/s00709-015-0825-2.
10. Bechtold N, Pelletier G. In planta Agrobacterium-mediated transformation of adult Arabidopsis thaliana plants by vacuum infiltration. Methods in molecular biology. 1998;82:259-66. PubMed PMID: 9664431.
11. Clough SJ, Bent AF. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 1998;16(6):735-43. PubMed PMID: 10069079.
12. Sahoo RK, Tuteja N. Development of Agrobacterium-mediated transformation technology for mature seed-derived callus tissues of indica rice cultivar IR64. GM Crops & Food. 2012;3(2):123-8. doi: 10.4161/gmcr.20032.
13. Khatodia S, Kharb P, Batra P, Chowdhury VK. Development and characterization of transgenic chickpea ( Cicer arietinum L .) plants with cry1Ac gene using tissue culture independent protocol. 2014;2(8):323-31.
14. Supartana P, Shimizu T, Shioiri H, Nogawa M, Nozue M, Kojima M. Development of simple and efficient in planta transformation method for rice (Oryza sativa L.) using Agrobacterium tumefaciens. Journal of Bioscience and Bioengineering. 2005;100(4):391-7. doi: 10.1263/jbb.100.391.
15. Fujimura T, Sakurai M, Akagi H, Negishi T, Hirose A. Regeneration of Rice Plants from Protoplasts. Plant tissue culture letters. 1985;2(2):74-5. doi: 10.5511/plantbiotechnology1984.2.74.
16. Kikkert JR, Vidal JR, Reisch BI. Stable Transformation of Plant Cells by Particle Bombardment/Biolistics. Transgenic Plants: Springer Science + Business Media; 2004. p. 061-78.
17. Kaur A, Gosal SS. Optimization of Gamma Radiation Dose for Induction of Genetic Variation in Sugarcane (Saccharum spp) Callus and Regenerated Shoot Cultures. Journal of Plant Biochemistry and Biotechnology. 2008;18(1):117-20. doi: 10.1007/bf03263308.
18. Dai N, Schaffer A, Petreikov M, Shahak Y, Giller Y, Ratner K, et al. Overexpression of Arabidopsis hexokinase in tomato plants inhibits growth, reduces photosynthesis, and induces rapid senescence. The Plant cell. 1999;11(7):1253-66. PubMed PMID: 10402427; PubMed Central PMCID: PMC144264.
19. KHATODIA S, BHATOTIA K, PASSRICHA N, KHURANA SMP, TUTEJA N. The CRISPR/Cas genome-editing tool: application in improvement of crops. Frontiers in Plant Science. 2016;7. doi: 10.3389/fpls.2016.00506.
20. Li T, Liu B, Chen CY, Yang B. TALEN utilization in rice genome modifications. Methods. 2014;69(1):9-16. doi: 10.1016/j.ymeth.2014.03.019.
21. Van Duyne GD. A Structural View of Cre- loxP Site-Specific Recombination. Annual Review of Biophysics and Biomolecular Structure. 2001;30(1):87-104. doi: 10.1146/annurev.biophys.30.1.87.
22. Tuteja N, Verma S, Sahoo RK, Raveendar S, Reddy INBL. Recent advances in development of marker-free transgenic plants: Regulation and biosafety concern. Journal of biosciences. 2012;37(1):167-97. doi: 10.1007/s12038-012-9187-5.
23. Christou P, Swain WF, Yang NS, McCabe DE. Inheritance and expression of foreign genes in transgenic soybean plants. Proceedings of the National Academy of Sciences. 1989;86(19):7500-4. doi: 10.1073/pnas.86.19.7500.
24. Theuns I, Windels P, De Buck S, Depicker A, Van Bockstaele E, De Loose M. Euphytica. 2002;123(1):75-84. doi: 10.1023/a:1014415619527.
25. Srivastava V, Vasil V, Vasil IK. Molecular characterization of the fate of transgenes in transformed wheat (Triticum aestivum L.). Theoret Appl Genetics. 1996;92(8):1031-7. doi: 10.1007/bf00224045.
26. Perret SJ, Valentine J, Mike Leggett J, Morris P. Integration, expression and inheritance of transgenes in hexaploid oat (Avena sativaL.). Journal of Plant Physiology. 2003;160(8):931-43. doi: 10.1078/0176-1617-00880.
27. Shrawat AK, Becker D, Lörz H. Agrobacterium tumefaciens-mediated genetic transformation of barley (Hordeum vulgare L.). Plant Science. 2007;172(2):281-90. doi: 10.1016/j.plantsci.2006.09.005.
28. Fritsch L, Fischer R, Wambach C, Dudek M, Schillberg S, Schröper F. Next-generation sequencing is a robust strategy for the high-throughput detection of zygosity in transgenic maize. Transgenic Res. 2015;24(4):615-23. doi: 10.1007/s11248-015-9864-x.
29. Southern EM. Detection of Specific Sequences Among DNA Fragments Separated by Gel Electrophoresis. Molecular Biology: Elsevier BV; 1989. p. 605-22.
30. Sridevi G, Sabapathi N, Meena P, Nandakumar R, Samiyappan R, Muthukrishnan S, et al. Transgenic indica Rice Variety Pusa Basmati 1 Constitutively Expressing a Rice Chitinase Gene Exhibits Enhanced Resistance to Rhizoctonia solani. Journal of Plant Biochemistry and Biotechnology. 2003;12(2):93-101. doi: 10.1007/bf03263168.
31. McGarvey P, Kaper JM. A simple and rapid method for screening transgenic plants using the PCR. BioTechniques. 1991;11(4):428-32. PubMed PMID: 1793572.
32. Khatodia S, Kharb P, Batra P, Chowdhury VK. Real time PCR based detection of transgene copy number in transgenic chickpea lines expressing Cry1Aa3 and Cry1Ac. Int J Pure App Biosci. 2014;2(4):100-5.
33. German MA, Kandel-Kfir M, Swarzberg D, Matsevitz T, Granot D. A rapid method for the analysis of zygosity in transgenic plants. Plant Science. 2003;164(2):183-7. doi: 10.1016/s0168-9452(02)00381-3.
34. Mieog JC, Howitt CA, Ral J-P. Fast-tracking development of homozygous transgenic cereal lines using a simple and highly flexible real-time PCR assay. BMC Plant Biol. 2013;13(1):71. doi: 10.1186/1471-2229-13-71.
35. Heid CA, Stevens J, Livak KJ, Williams PM. Real time quantitative PCR. Genome Research. 1996;6(10):986-94. doi: 10.1101/gr.6.10.986.
36. Jiang L, Yang L, Zhang H, Guo J, Mazzara M, Van den Eede G, et al. International Collaborative Study of the Endogenous Reference Gene, Sucrose Phosphate Synthase ( SPS ), Used for Qualitative and Quantitative Analysis of Genetically Modified Rice. J Agric Food Chem. 2009;57(9):3525-32. doi: 10.1021/jf803166p.
37. Elliott KJ, Butler WO, Dickinson CD, Konno Y, Vedvick TS, Fitzmaurice L, et al. Isolation and characterization of fruit vacuolar invertase genes from two tomato species and temporal differences in mRNA levels during fruit ripening. Plant Mol Biol. 1993;21(3):515-24. doi: 10.1007/bf00028808.
38. Gadaleta A, Giancaspro A, Cardone M, Blanco A. Real-time PCR for the detection of precise transgene copy number in durum wheat. Cellular and Molecular Biology Letters. 2011;16(4). doi: 10.2478/s11658-011-0029-5.
39. Li Z, Hansen JL, Liu Y, Zemetra RS, Berger PH. Using real-time PCR to determine transgene copy number in wheat. Plant Molecular Biology Reporter. 2004;22(2):179-88. doi: 10.1007/bf02772725.
40. Shou H, Frame BR, Whitham SA, Wang K. Assessment of transgenic maize events produced by particle bombardment or Agrobacterium-mediated transformation. Molecular Breeding. 2004;13(2):201-8. doi: 10.1023/b:molb.0000018767.64586.53.
41. Yang L, Ding J, Zhang C, Jia J, Weng H, Liu W, et al. Estimating the copy number of transgenes in transformed rice by real-time quantitative PCR. Plant cell reports. 2004;23(10-11):759-63. doi: 10.1007/s00299-004-0881-0.
42. Casu RE, Selivanova A, Perroux JM. High-throughput assessment of transgene copy number in sugarcane using real-time quantitative PCR. Plant cell reports. 2011;31(1):167-77. doi: 10.1007/s00299-011-1150-7.
43. Hindson CM, Chevillet JR, Briggs HA, Gallichotte EN, Ruf IK, Hindson BJ, et al. Absolute quantification by droplet digital PCR versus analog real-time PCR. Nature methods. 2013;10(10):1003-5. doi: 10.1038/nmeth.2633.
44. Metzker ML. Sequencing technologies - the next generation. Nature reviews Genetics. 2010;11(1):31-46. doi: 10.1038/nrg2626. PubMed PMID: 19997069.
45. Yockteng R, Almeida AMR, Yee S, Andre T, Hill C, Specht CD. A Method for Extracting High-Quality RNA from Diverse Plants for Next-Generation Sequencing and Gene Expression Analyses. Applications in Plant Sciences. 2013;1(12):1300070. doi: 10.3732/apps.1300070.
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Jul 18, 2016
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Passricha N, Saifi S, Khatodia S, Tuteja N. Assessing zygosity in progeny of transgenic plants: current methods and perspectives. J Biol Methods [Internet]. 2016 Jul. 18 [cited 2024 Apr. 20];3(3):e46. Available from: https://jbmethods.org/index.php/jbm/article/view/114
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Author Biography
Narendra Tuteja, Amity Institute of Microbial Technology, Amity University Uttar Predesh, Sector 125, Noida, UP, India
Amity Institute of Microbial Technology Professor & Director
