Agrobacterium-mediated transformation of Nicotiana glauca and Nicotiana sylvestris

Agrobacterium-mediated transformation is the most popular approach for obtaining transgenic plants nowadays. There are plenty of protocols developed for different plant species. These protocols usually include the medium composition, the technology for preparing plant explants and cultivation conditions, as well as the choice of agrobacteria strains. Nicotiana tabacum, or cultivated tobacco, was one of the first successfully transformed plant species. Nicotiana tabacum is a model object in plant genetics, particularly due to its ability for transformation and regeneration. N. tabacum is a naturally transgenic plant since its genome contains a cellular T-DNA acquired from Agrobacteria. The significance of cT-DNA for plants has not yet been established. Some assume that cT-DNA can increase the ability of plants to regenerate due to some of the genes they contain. For example, rolC has been shown to affect the hormonal balance of plants, but the molecular mechanisms underlying this have yet to be found. RolC is also somehow involved in the secondary metabolism of plants. Like N. tabacum, Nicotiana glauca produces a wide range of secondary metabolites and contains an intact rolC gene in its genome. At the same time, unlike N. tabacum, N. glauca is a diploid species, which makes it more suitable for genetic engineering approaches. Nicotiana sylvestris is one of the ancestral species of N. tabacum and does not contain cT-DNA. The aim of this work was to develop a protocol for transformation and regeneration of N. glauca and N. sylvestris. We managed to find an optimum ratio of auxins and cytokinins that promotes both active callus formation and organogenesis in N. glauca and N. sylvestris leaf explants. The developed technique will be useful both for fundamental research that includes the N. glauca and N. sylvestris species, and for practical application in the pharmaceutical industry and biosynthesis.

1. Ali G., Hadi F., Ali Z., Tariq M., Ali Khan M. Callus induction and in vitro complete plant regeneration of different cultivars of tobacco (Nicotiana tabacum L.) on media of different hormonal concentrations. Biotechnology. 2007;6(4):561-566. DOI:10.3923/biotech.2007.561.566.

2. Chen K., Dorlhac de Borne F., Szegedi E., Otten L. Deep sequencing of the ancestral tobacco species Nicotiana tomentosiformis reveals multiple T-DNA inserts and a complex evolutionary history of natural transformation in the genus Nicotiana. Plant J. 2014;80(4):669-682. DOI:10.1111/tpj.12661.

3. Chen K., Otten L. Natural Agrobacterium transformants: recent results and some theoretical considerations. Front. Plant Sci. 2017;8:1600. DOI:10.3389/fpls.2017.01600.

4. Cheng M., Lowe B.A., Spencer T.M., Ye X., Armstrong C.L. Factors influencing Agrobacterium-mediated transformation of monocotyledonous species. In Vitro Cell. Dev. Biol. Plant. 2004;40(1):31-45. DOI:10.1079/ivp2003501.

5. Cheng Z.J., Wang L., Sun W., Zhang Y., Zhou C., Su Y.H., Li W., Sun T.T., Zhao X.Y., Li X.G., Cheng Y., Zhao Y., Xie Q., Zhang X.S. Pattern of auxin and cytokinin responses for shoot meristem induction results from the regulation of cytokinin biosynthesis by AUXIN RESPONSE FACTOR3. Plant Physiol. 2013;161(1):240-251. DOI:10.1104/pp.112.203166.

6. Clarkson J.J., Knapp S., Garcia V.F., Olmstead R.G., Leitch A.R., Chase M.W. Phylogenetic relationships in Nicotiana (Solanaceae) inferred from multiple plastid DNA regions. Mol. Phylogenet. Evol. 2004;33(1):75-90. DOI:10.1016/j.ympev.2004.05.002.

7. Draper J., Scott R., Armitage Ph., Dury G., Jacob L., Walden R., Kumar A., Jefferson R., Hamil J. Genetic Engineering of Plants. Laboratory guide. Moscow, 1991. (Russian translation)

8. Edwards K.D., Fernandez-Pozo N., Drake-Stowe K., Humphry M., Evans A.D., Bombarely A., Allen F., Hurst R., White B., Bromley J.R., Sanchez-Tamburrino J.P., Lewis R.S., Mueller L.A. A reference genome for Nicotiana tabacum enables map-based cloning of homeologous loci implicated in nitrogen utilization efficiency. BMC Genom. 2017;18:448. DOI:10.1186/s12864-017-3791-6.

9. Furze J.M., Hamill J.D., Parr A.J., Robins R.J., Rhodes M.J.C. Variations in morphology and nicotine alkaloid accumulation in protoplast-derived hairy root cultures of Nicotiana rustica. J. Plant Physiol. 1987;131(3-4):237-246. DOI:10.1016/s0176-1617(87)80163-3.

10. Gill R., Rashid A., Maheshwari S.C. Isolation of mesophyll protoplasts of Nicotiana rustica and their regeneration into plants flowering in vitro. Physiol. Plant. 1979;47(1):7-10. DOI:10.1111/j.1399-3054.1979.tb06502.x.

11. Hasan M.M., Kim H.-S., Jeon J.-H., Kim S.H., Moon B., Song J.-Y., Shim S.H., Baek K.-H. Metabolic engineering of Nicotiana benthamiana for the increased production of taxadiene. Plant Cell Rep. 2014;33(6):895-904. DOI:10.1007/s00299-014-1568-9.

12. Herrera-Estrella L., Depicker A., Van Montagu M., Schell J. Expression of chimaeric genes transferred into plant cells using a Ti-plasmidderived vector. Nature. 1983;303(5914):209-213. DOI:10.1038/303209a0.

13. Horsch R.B., Fry J.E., Hoffmann N.L., Wallroth M., Eichholtz D., Rogersan S.G., Fraley R.T. A simple and general method for transferring genes into plants. Science. 1985;227(4691):1229-1231. DOI:10.1126/science.227.4691.1229.

14. Ichikawa T., Ozeki Y., Syöno K. Evidence for the expression of the rol genes of Nicotiana glauca in genetic tumors of N. glauca × N. langsdorflli. Mol. Gen. Genet. 1990;220(2):177-180. DOI:10.1007/BF00260478.

15. Intrieri M.C., Buiatti M. The horizontal transfer of Agrobacterium rhizogenes genes and the evolution of the genus Nicotiana. Mol. Phylogenet. Evol. 2001;20(1):100-110. DOI:10.1006/mpev.2001.0927.

16. Khafizova G.V., Matveeva T.V. The rolC gene of agrobacteria: towards the understanding of its functions. Biotegnologiâ i Selekciâ Rastenij = Plant Biotechnology and Breeding. 2021;4(1):36-46. DOI:10.30901/2658-6266-2021-1-o4. (in Russian)

17. Loiseau J., Marche C., Le Deunff Y. Effects of auxins, cytokinins, carbohydrates and amino acids on somatic embryogenesis induction from shoot apices of pea. Plant Cell Tissue Organ Cult. 1995;41:267-275. DOI:10.1007/BF00045091.

18. Long N., Ren X., Xiang Z., Wan W., Dong Y. Sequencing and characterization of leaf transcriptomes of six diploid Nicotiana species. J. Biol. Res. (Thessalon). 2016;23(1):1-12. DOI:10.1186/s40709-016-0048-5.

19. Lutova L.A., Bondarenko L.V., Buzovkina I.S., Levashina E.A., Tikhodeev O.N., Khodjaiova L.T., Sharova N.V., Shishkova S.O. The influence of plant genotype on regeneration processes. Genetika = Soviet Genetics. 1994;30(8):928-936.

20. Matveeva T.V. Agrobacterium-mediated transformation in the evolution of plants. Curr. Top. Microbiol. Immunol. 2018;418:421-441. DOI:10.1007/82_2018_80.

21. Matveeva T.V. New naturally transgenic plants: 2020 update. Biol. Commun. 2021;66(1):36-46. DOI:10.21638/spbu03.2021.105.

22. Matveeva T.V., Sokornova S.V. Biological traits of naturally transgenic plants and their evolutional roles. Fiziologiya Rastenii = Russ. J. Plant Physiol. 2017;64(5):635-648. DOI:10.1134/S1021443717050089.

23. Mohajjel-Shoja H., Clément B., Perot J., Alioua M., Otten L. Biological activity of the Agrobacterium rhizogenes-derived trolC gene of Nicotiana tabacum and its functional relation to other plast genes. Mol. Plant-Microbe Interact. 2011;24(1):44-53. DOI:10.1094/MPMI-06-10-0139.

24. Murashige T., Skoog F. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol. Plant. 1962;15(3): 473-497.

25. Murray M.G., Thompson W.F. Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res. 1980;8(19):4321-4325. DOI:10.1093/nar/8.19.4321.

26. Narasimhulu S.B., Chopra V.L. Species specific shoot regeneration response of cotyledonary explants of Brassicas. Plant Cell Rep. 1988; 7:104-106. DOI:10.1007/BF00270115.

27. Ockendon D.J., Sutherland R.A. Genetic and non-genetic factors affecting anther culture of Brussels sprouts (Brassica oleracea var. gemmifera). Theor. Appl. Genet. 1987;74(5):566-570. DOI:10.1007/BF00288853.

28. Otten L. The Agrobacterium phenotypic plasticity (Plast) genes. In: Gelvin S. (Ed.) Agrobacterium Biology. Current Topics in Microbiology and Immunology. Vol. 418. Springer, 2018. DOI:10.1007/82_2018_93.

29. Otten L., Helfer A. Biological activity of the rolB-like 5′ end of the A4-orf8 gene from the Agrobacterium rhizogenes TL-DNA. Mol. Plant Microbe Interact. 2001;14(3):405-411. DOI:10.1094/mpmi.2001.14.3.405.

30. Pang E.C.K., Croser J.S., Imberger K.T., McCutchan J.S., Taylor P.W.J. Tissue culture and protoplast fusion of cool-season pulses: pea (Pisum sativum L.) and chickpea (Cicer arietinum L.). In: Knight R. (Ed.) Linking Research and Marketing Opportunities for Pulses in the 21st Century. Current Plant Science and Biotechnology in Agriculture. Vol. 34. Dordrecht: Springer, 2000;429-436. DOI:10.1007/978-94-011-4385-1_40.

31. Saschenko M.N. Role of pea genotype in tissue culture propagation. Vestnik Altayskogo Gosudarstvennogo Agrarnogo Universiteta = Bulletin of the Altai State Agricultural University. 2014;11(121): 20-25. (in Russian)

32. Sawahel W.A., Cove D.J. Gene transfer strategies in plants. Biotechnol. Adv. 1992;10(3):393-412. DOI:10.1016/0734-9750(92)90302-p.

33. Schmulling T., Schell J., Spena A. Single genes from Agrobacterium rhizogenes influence plant development. EMBO J. 1988;7:2621-2629. DOI:10.1002/j.1460-2075.1988.tb03114.x.

34. Su Y.H., Zhang X.S. The hormonal control of regeneration in plants. Curr. Top. Dev. Biol. 2014;108:35-69. DOI:10.1016/B978-0-12-391498-9.00010-3.

35. Tinland B., Fournier P., Heckel T., Otten L. Expression of a chimaeric heat-shock-inducible Agrobacterium 6b oncogene in Nicotiana rustica. Plant Mol. Biol. 1992;18(5):921-930. DOI:10.1007/bf00019206.

36. Wang K. Agrobacterium Protocols. Methods in Molecular Biology. Humana Press, 2015. DOI:10.1007/978-1-4939-1658-0.

37. White F.F., Garfinkel D.J., Huffman G.A., Gordon M.P., Nester E.W. Sequence homologous to Agrobacterium rhizogenes T-DNA in the genomes of uninfected plants. Nature. 1983;301:348-350. DOI:10.1038/301348a0.

38. Xing H.L., Dong L., Wang Z.P., Zhang H.Y., Han C.Y., Lui B., Wang X.C., Chen Q.J. A CRISPR/Cas9 toolkit for multiplex genome editing in plants. BMC Plant Biol. 2014;14:327. DOI:10.1186/s12870-014-0327-y.

39. Yukawa M., Tsudzuki T., Sugiura M. The chloroplast genome of Nicotiana sylvestris and Nicotiana tomentosiformis: complete sequencing confirms that the Nicotiana sylvestris progenitor is the maternal genome donor of Nicotiana tabacum. Mol. Genet. Genom. 2006; 275:367-373. DOI:10.1007/s00438-005-0092-6.