Article | 02. 2016 Vol. 34, Issue. 1
Stable Inheritance of an Integrated Transgene and Its Expression in Phenylethylisothiocyanate-Enriched Transgenic Chinese cabbage

Department of Horticultural Biotechnology, Kyunghee University1
Korea Institute of Science and Technology Gangneung Institute2

2016.02. 112:121


Development of genetically-modified (GM) crops enables the introduction of new traits to the plant to confer characteristics such as disease resistance, herbicide resistance and human healthpromoting bioactivity. Successful commercialization of newly developed GM crops requires stable inheritance of integrated T-DNA and newly introduced traits through the multiple generations. This study was carried out to confirm the stable inheritance of the integrated T-DNA in T1 and T2 transgenic Chinese cabbage (Brassica rapa ssp. pekinensis) that was genetically modified to increase concentrations of phenylethylisothiocyanate (PEITC), which is a potential anti-carcinogenic phytochemical. For this purpose, the IGA 1-3 (T1 generation) and IGA 1-3-5 (T2 generation) lines were selected by PCR and a IGA 1-3 transgenic plant (T1 generation) was analyzed to confirm the T-DNA insertion site in the Chinese cabbage genome by VA-TAIL PCR. The results of this study showed that the introduced T-DNA in IGA 1 line was stably inherited to the next generations without any variations in terms of the structure of the transgenes, and this line also showed the expected transgene function that resulted in increased concentration of PEITC through the multiple generations. Finally, we confirmed the increased QR activity in IGA 1 T1 and T2 transgenic lines, which indicates an enhanced potential anti-carcinogenic bioactivity and its stable inheritance in IGA1 T1 and T2 transgenic lines.

1. Benson, A.M., M.J. Hunkeler, and P. Talalay. 1980. Increase of NAD(P)H:quinone reductase by dietary antioxidants: possible role in protection against carcinogenesis and toxicity. Proc. Natl. Acad. Sci. USA. 77:5216-5220.  

2. Bhalla, P.L. Genetic engineering of wheat–current challenges and opportunities. 2006. Trends biotechnol. 24:305-311.  

3. Brenda A.L., N.S. Prakash, M. Way, M.T. Mann, T.M. Spencer, and R.S. Boddupalli. 2009. Enhanced single copy integration events in corn via particle bombardment using low quantities of DNA. Transgenic Res. 18:831-840.  

4. Chen, S., J. Zhang, H. Pu, A. Shen, X, Zhou, W. Long, M. Hu, and C. Qi. 2012. Analysis of insertion copy number and integration site of T-DNA in the genome of transgenic high oelic rapeseed (L.). Plant Gene Trait 2:15-22.  

5. Dai, S., P. Zheng, P. Marmey, S. Zhang, W. Tian, W., S. Chen, R.N. Beachy, and C. Fauquet. 2001. Comparative analysis of transgenic rice plants obtained by -mediated transformation and particle bombardment. Mol. Breed. 7:25-33.  

6. Iyer, L.M., S.P. Kumpatla, M.B. Chandrasekharan, and T.C Hall. 2000. Transgene silencing in monocots. Springer Netherlands 43:323-  

7. Kim, J.S., J. Kim, T.H. Lee, K.M. Jun, T.H. Kim, Y.H. Kim, H.M. Park, J.S. Jeon, G. An, U.H. Yoon, B.H. Nahm, and Y.K. Kim. 2012. FSTVAL: a new web tool to validate bulk flanking sequence tags. Plant Methods 8:19.  

8. Ku, K.M., E.H. Jeffery, and J.A. Juvik. 2014. Optimization of methyl jasmonate application to broccoli florets to enhance health‐promoting phytochemical content. J. Sci. Food Agriculture. 94:2090-2096.  

9. Kumpatla, S.P., M.B. Chandrasekharan, L.M. Iyer, G. Li, T.C. Hall. 1998. Genome intruder scanning and modulation systems and transgene silencing. Trends Plant Sci. 3:97-104.  

10. Liu, Y. and R. Whittier. 1995. Thermal asymmetric interlaced PCR: automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking. Genomics 25:674-681.  

11. Liu, Y.G. and Y. Chen. 2007. High-efficiency thermal asymmetric interlaced PCR for amplification of unknown flanking sequences. Biotechniques 43:649-656.  

12. Lee, G.H, J.G. Yu, and Y.D. Park. 2015. Development of an effective PCR technique for analyzing T-DNA integration sites in Brassica  

13. Matzke, M. A. and A.J.M. Matzke. 1995. How and why do plants inactivate homologous (trans) genes?. Plant Physiol. 107:679-685.  

14. Park, J.H., S.J. Lee, B.R. Kim, E.T. Woo, J.S. Lee, E.H. Han, Y.H. Lee, and Y.D. Park. 2011. Isolation of myrosinase and genes and transformation of these genes to develop phenylethylisothiocyanate enriching Chinese cabbage. Korean J. Hortic. Sci. Technol. 29:623-632.  

15. Pla, M., J.L. La Paz, and C. Vicient. 2012. Insert stability and transgenic plant risk. Encyclopedia Biotechnol. Agriculture Food.  

16. Qin, Y., H.I. Ahn, S.Y. Park, M.H. Lim, H.J. Woo, K.S. Shin, J.H. Lee, H.S. Cho, S.H. Baek, S.K. Park and S.J. Kweon. 2014. T-DNA inheritance stability of resveratrol rice Iksan526 over multi-generations. Plant Breed. Biotechnol. 2:268-275.  

17. Stoger, E., J.K. Ma, R. Fischer, and P. Christou. 2005. Sowing the seeds of success: pharmaceutical proteins from plants. Curr. Opin. Biotechnol. 16:167-173.  

18. Vain, P., B. Worland, A. Kohli, J.W. Snape, P. Christou, G.C. Allen, and W.F. Thompson. 1999. Matrix attachment regions increase transgene expression levels and stability in transgenic rice plants and their progeny. Plant J. 18:233–242.  

19. Vaucheret, H., C. Béclin, T. Elmayan, F. Feuerbach, C. Godon, J.B. Morel, P. Mourrain, J.C. Palauqui, and S. Vernhettes. 1998. Transgene‐induced gene silencing in plants. Plant J. 16:651-659.  

20. Wang, X., H. Wang, J. Wang, R. Sun, J. Wu, S. Liu, Y. Bai, J.H. Mun, I. Bancroft, F. Cheng, S. Huang, X. Li, W. Hua, J. Wang, X. Wang'G. Conant, G. Lassalle, G.J. King, G. Bonnema, H. Tang, H. Wang, H. Belcram, H. Zhou, H. Hirakawa, H. Abe, H. Guo, H. Wang,and H. Abe. 2011. The genome of the mesopolyploid crop species Brassica rapa . Nat. Genet. 43:1035-1039.  

21. Yu, J.G., G. H. Lee, J.S. Kim, and Y.D. Park. 2010. An insertional mutagenesis system for analyzing the Chinese cabbage genome using T‐DNA. Molecules and Cells 29:267-275.  

22. Yu, J.G., G.H. Lee, J.H. Park, and Y.D. Park. 2014. Characterization and gene co-expression network analysis of a salt tolerance-related gene, BrSSR, in Brassica rapa . Korean J. Hortic. Sci. Technol. 32:845-852.