SONICATION AND VACUUM INFILTRATION ENHANCED AGROBACTERIUM RHIZOGENES MEDIATED TRANSFORMATION IN SOYBEAN
Objective: The present study involved the formulation of protocol of Agrobacterium rhizogenes-mediated transformation for the detailed study of isoflavones metabolism in soybean.
Methods: Cotyledons were separated from 4-day-old soybean seedlings and infected with three different A. rhizogenes strains under various time durations of sonication, vacuum infiltration and co-cultivated on MS medium supplemented with various concentrations of acetosyringone. The induced hairy roots were established as a culture with the selection agent hygromycin B. Transgenes integrated in hairy roots were analysed at molecular level by PCR assay.
Results: A. rhizogenes strain R1000 harbouring pCAMBIA1301 resulted in better transformation efficiency when compared with other strains. The optimum duration of sonication (2 min) and vacuum infiltration (2 min) enhanced the transformation efficiency up to 76.47 %. Â PCR analyses revealed the integration of transgene in hairy roots lines.
Conclusion: Â From the present study, we could conclude that, sonication and vacuum infiltration techniques could be employed to produce genotype independent transgenic soybean hairy root lines and which could be used to study for the improved production of potent anti-cancer compounds, isoflavones in soybean.Keywords: Agrobacterium, isoflavones, soybean, sonication, vacuum infiltration
1. Omoni AO, Aluko RE. Soybean foods and their benefits: potential mechanisms of action. Nutrition reviews, 2005; 63: 272-283.
2. Graham TL. Flavonoid and isoflavonoid distribution in developing soybean seedling tissues and in seed and root exudates. Plant physiol 1991; 95: 594-603.
3. Jeong GT, Park DH. Enhanced secondary metabolite biosynthesis by elicitation in transformed plant root system. In Twenty-Seventh Symposium on Biotechnology for Fuels and Chemicals 2006 (pp. 436-446). Humana Press.
4. Chandra S, Chandra R. Engineering secondary metabolite production in hairy roots. Phytochemistry Reviews 2011;10: 371-395.
5. Mariashibu TS, Ganapathi A, Jiang SY, Ramachandran S, Anbazhagan VR. In vitro regeneration and genetic transformation of soybean: Current status and future prospects. INTECH Open Access Publisher 2013.
6. Cho HJ, Farrand SK, Noel GR, and Widholm JM. High-efficiency induction of soybean hairy roots and propagation of the soybean cyst nematode. Planta 2000; 210: 195-204.
7. Li J, Todd TC, Trick HN. Rapid in planta evaluation of root expressed transgenes in chimeric soybean plants. Plant cell rep 2010; 29:113-123.
8. Preiszner J, VanToai T, Huynh L, Bolla R, Yen H. Structure and activity of a soybean Adh promoter in transgenic hairy roots. Plant Cell Rep 2001; 20: 763-769.
9. Owens LD, Cress DE. Genotypic variability of soybean response to Agrobacterium strains harboring the Ti or Ri plasmids. Plant physiol 1985; 77: 87-94.
10. Le Flem-Bonhomme V, Laurain-Mattar D, Fliniaux MA. Hairy root induction of Papaver somniferum var. album, a difficult-to-transform plant, by A. rhizogenes LBA 9402. Planta 2004; 218: 890-893.
11. Georgiev MI, Ludwig-MÃ¼ller J, Alipieva K, Lippert A. Sonication-assisted Agrobacterium rhizogenes-mediated transformation of Verbascum xanthophoeniceum Griseb. for bioactive metabolite accumulation. Plant cell rep 2011; 30: 859-866.
12. Ishida JK, Yoshida S, Ito M, Namba S, Shirasu K. Agrobacterium rhizogenes-mediated transformation of the parasitic plant Phtheirospermum japonicum. PLoS One 2011; 6: e25802.
13. Mariashibu TS, Subramanyam K, Arun M, Mayavan S, Rajesh M, Theboral J, Ganapathi A. Vacuum infiltration enhances the Agrobacterium-mediated genetic transformation in Indian soybean cultivars. Acta Physiol Plantarum 2013; 35: 41-54.
14. Arun M, Subramanyam K, Mariashibu TS, Theboral J, Shivanandhan G, Manickavasagam M, and Ganapathi A. Application of sonication in combination with vacuum infiltration enhances the Agrobacterium-mediated genetic transformation in Indian soybean cultivars. Applied Biochem Biotech 2015; 175: 2266-2287.
15. Theboral J, Sivanandhan G, Subramanyam K, Arun M, Selvaraj N, Manickavasagam M, Ganapathi A. Enhanced production of isoflavones by elicitation in hairy root cultures of Soybean. Plant Cell Tiss Org Cult 2014; 117: 477-481.
16. Jefferson RA. Assaying chimeric genes in plants: the GUS gene fusion system. Plant molecular biology rep 1987; 5: 387-405.
17. Owczarzy R, Tataurov AV, Wu Y, Manthey JA, McQuisten KA, Almabrazi HG, et al., IDT SciTools: a suite for analysis and design of nucleic acid oligomers. Nucleic acids res 2008; 36: W163-W169
18. Cao D, Hou W, Song S, Sun H, Wu C, Gao Y, Han T. Assessment of conditions affecting Agrobacterium rhizogenes-mediated transformation of soybean. Plant Cell Tiss Org Cult 2009; 96: 45-52.
19. Olhoft PM, Bernal LM, Grist LB, Hill DS, Mankin SL, Shen Y, et al., A novel Agrobacterium rhizogenes-mediated transformation method of soybean [Glycine max (L.) Merrill] using primary-node explants from seedlings. In vitro Cellular and Developmental Biology-Plant 2007; 43: 536-549.
20. Srivastava S, Adholeya A, Conlan XA, Cahill DM. Elite hairy roots of Ocimum basilicum as a new source of rosmarinic acid and antioxidants. Plant Cell Tiss Org Cult 2016; 126: 19â€“32
21. Mazarei M, Ying Z, Houtz RL. Functional analysis of the Rubisco large subunit Ã‚N-methyltransferase promoter from tobacco and its regulation by light in soybean hairy roots. Plant cell rep 1998; 17: 907-912.
22. Lozovaya VV, Lygin AV, Zernova OV, Li S, Hartman GL, Widholm JM. Isoflavonoid accumulation in soybean hairy roots upon treatment with Fusarium solani. Plant Physiol Biochem 2004; 42:671-679.
23. Weber RLM, Bodanese Zanettini MH. Induction of transgenic hairy roots in soybean genotypes by Agrobacterium rhizogenes mediated transformation. Pesquisa AgropecuÃ¡ria Brasileira 2011; 46:1070-1075.
24. Nourozi E, Hosseini B, Hassani A. A reliable and efficient protocol for induction of hairy roots in Agastache foeniculum. Biologia 2014; 69: 870-879.
25. Tiwari RK, Trivedi M, Guang ZC, Guo GQ, Zheng GC. Genetic transformation of Gentiana macrophylla with Agrobacterium rhizogenes: growth and production of secoiridoid glucoside gentiopicroside in transformed hairy root cultures. Plant Cell Rep 2007; 26: 199-210.
26. Khatodia S, Biswas K, Bhatotia K. Induction and establishment of hairy root culture of Solanum xanthocarpum using Agrobacterium rhizogenes. J Pharm BioSci 2013; 1:59-63.
27. Park NI, Tuan PA, Li X, Kim YK, Yang TJ, Park SU. An efficient protocol for genetic transformation of Platycodon grandiflorum with Agrobacterium rhizogenes. Molecular biol rep 2011; 38: 2307-2313.
28. Bansal M, Kumar A, Reddy MS. Influence of Agrobacterium rhizogenes strains on hairy root induction and â€˜bacoside Aâ€™production from Bacopa monnieri (L.) Wettst. Acta Physiol Plantarum 2014; 36: 2793-2801.
29. Wahby I, Caba JM, Ligero F. Agrobacterium infection of hemp (Cannabis sativa L.): establishment of hairy root cultures. Journal of Plant Interactions 2013; 8:312-320.
30. Savka M, Ravillion B, Noel GR, Farrand SK. Induction of hairy roots on cultivated soybean genotypes and their use to propagate the soybean cyst nematode. Phytopathol 1990; 80: 503-508.
31. BeranovÃ¡ M, Rakousky S, VÃ¡vrovÃ¡ Z, Skalicky T. Sonication assisted Agrobacterium-mediated transformation enhances the transformation efficiency in flax (Linux usitatissimumL.). Plant Cell Tiss Org Cult 2008; 94:253â€“259
32. Gonzalez A, Jimenez A, Vazquez D, Davies JE, Schindler D. Studies on the mode of action of hygromycin B, an inhibitor of translocation in eukaryotes. Biochimica et Biophysica Acta (BBA)-Nucleic Acids and Protein Synthesis 1978; 521: 459-469.
33. Guo BF, Guo Y, Wang J, Zhang LJ, Jin LG, Hong HL. Co-treatment with surfactant and sonication significantly improves Agrobacterium-mediated resistant bud formation and transient expression efficiency in soybean. J. Integr. Agric. 2015; 14:1242â€“1250