Inducing of direct shoot regeneration during the genetic transformation of tobacco with Agrobacterium rhizogenes strain K599

E.A. Baimukhametova*, Z.A. Berezhneva**, Kh.G. Musin***, D.Yu. Shvets****, A.V. Knyazev*****, B.R. Kuluev******

Institute of Biochemistry and Genetics – Subdivision of the Ufa Federal Research Centre of the Russian Academy of Sciences, Ufa, 450054 Russia

E-mail: *elvina.baimuhametova@yandex.ru, **berezhneva-z@yandex.ru, ***khalit.musin@yandex.ru, ****shvetsdasha99@yandex.ru, *****knyazev@anrb.ru, ******kuluev@bk.ru

Received December 27, 2021

 

ORIGINAL ARTICLE

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DOI: 10.26907/2542-064X.2022.2.249-262

For citation: Baimukhametova E.A., Berezhneva Z.A., Musin Kh.G., Shvets D.Yu., Knya­zev A.V., Kuluev B.R. Inducing of direct shoot regeneration during the genetic transformation of tobacco with Agrobacterium rhizogenes strain K599. Uchenye Zapiski Kazanskogo Universiteta. Seriya Estestvennye Nauki, 2022, vol. 164, no. 2, pp. 249–262. doi: 10.26907/2542-064X.2022.2.249-262. (In Russian)

Abstract

This article is devoted to the study of the phenotypic manifestations of transformation induced by the Agrobacterium rhizogenes strain K599 in Nicotiana tabacum L., a model plant sensitive to agrobacteria. For this purpose, we carried out a genetic transformation of tobacco leaf disks that resulted in spontaneous callus and shoot formation on hormone-free media. On average, 6 regenerated shoots per leaf explant were obtained. Overall, 2 out of 87 transplanted shoots spontaneously formed roots, and 21 shoots died. In the remaining shoots, rooting was observed only when the growth regulator IAA was added to the medium. Using the above protocol, a total of 39 transgenic plantlets adapted to the given soil conditions were produced from spontaneous points of regeneration. The transformation also initiated the formation of hairy roots, but there were very few of them – 0.77 roots per explant on average. The isolated cultures of tobacco hairy roots generated using strain K599 did not phenotypically differ from those induced by strains A4 and 15834. On the hairy roots, 10 shoots transgenic for rol genes were regenerated by the induction of shoots using growth regulators, 8 of them were later rooted and acclimated to the given soil conditions. The PCR analysis showed the presence of rol genes in all transgenic plants that regenerated spontaneously or were induced on roots by growth regulators. Thus, strain K599, unlike other strains of A. rhizogenes, has a potential to be used to grow transgenic tobacco plants on hormone-free nutrient media by direct regeneration of shoots on explants.

Keywords: Agrobacterium-mediated transformation, hairy roots, regeneration, callus formation, rhizogenesis, transgenic plants

Acknowledgements. This study was performed as part of state assignment no. 122030200143-8 and supported by the grant of the President of the Russian Federation no. MD-2304.2020.

Figure Captions

Fig. 1. Results of the transformation of tobacco leaf explants with the A. rhizogenes strain K599: a – the beginning of shoot regeneration on leaf explants following 17 days of inoculation with agrobacteria; b – a hairy root following 28 days of inoculation with agrobacteria (marked with an arrow); c – the cultures of hairy roots following 20 days of isolation from leaf explants; d – the regenerated shoots following 14 days of isolation from leaf explants.

Fig. 2. Electropherogram of the PCR results with the primer RolAB1: 1–66 – the regenerated shoots; M – the DNA size marker 1kb DNA Ladder; -K – negative PCR control. PCR product size – 1112 bps.

Fig. 3. Electropherogram of the PCR results with the primer RolAB2: M – the DNA size marker 1kb DNA Ladder; 1–66 – the directly regenerated shoots; -K – negative PCR control. PCR product size – 1127 bps.

Fig. 4. Electropherogram of the PCR results with the primer RolС1: M – the DNA size marker 1kb DNA Ladder; 1–66 – the directly regenerated shoots; +K – positive PCR control. PCR product size – 267 bps.

Fig. 5. Spontaneous regeneration of the shoots on the isolated cultures of hairy roots: a – the regeneration of shoots on the isolated cultures of hairy roots on the medium without phytohormones; b – rhizogenesis on the regenerant shoots on the medium without phytohormones; c, d – the plagiotropic growth of roots on the regenerant shoots on the media with IAA.

Fig. 6. Electropherogram of the PCR results with the following primers: a – RolC1, PCR product size 267 bps; b – RolAB1, PCR product size 1112 bps; c – RolAB2, PCR product size 1127 bps; M – the DNA size marker 1kb DNA Ladder; 1–7 – the shoots that spontaneously regenerated on the roots; -K – negative PCR control; +K – positive PCR control.

Fig. 7. Callus and shoot formation on the hairy roots of the tobacco plants when they were transferred to the media containing 6-BAP and IAA: a – callus formation on hairy roots on the media with     6-BAP and NAA; b – hemmogenesis on the calluses obtained from hairy roots; c – regenerant plant acclimated to the given soil conditions (on the left) as compared with the wild type (on the right).

Fig. 8. Electropherogram of the PCR results with the following primers: a – RolAB1, PCR product size 1112 bps; b – RolAB2, PCR product size 1127 bps; c – RolC1, PCR product size 267 bps; M – the DNA size marker 1kb DNA Ladder; 1–10 – the shoots that regenerated on the roots under the influence of hormones; K – negative PCR control; +K – positive PCR control.

References

  1. Young J.M., Kuykendall L.D., Martínez-Romero E., Kerr A., Sawada H. A revision of Rhizobium Frank 1889, with an emended description of the genus, and the inclusion of all species of Agrobacterium Conn 1942 and Allorhizobium undicola de Lajudie et al. 1998 as new combinations: Rhizobium radiobacter, R. rhizogenes, R. rubi, R. undicola and R. vitis. Int. J. Syst. Evol. Microbiol., 2001, vol. 51, no. 1, pp. 89–103. doi: 10.1099/00207713-51-1-89.
  2. Flores-Félix J.D., Menéndez E., Peix A., García-Fraile P., Velázquez E. History and current taxonomic status of genus Agrobacterium. Syst. Appl. Microbiol., 2020, vol. 43, no. 1, pp. 126046–126062. doi: 10.1016/j.syapm.2019.126046.
  3. Smith E.F., Townsend C.O. A plant-tumor of bacterial origin. Science, 1907, vol. 25, no. 643, pp. 671–673. doi: 10.1126/science.25.643.671.
  4. Costantino P., Mauro M.L., Micheli G., Risuleo G., Hooykaas P.J.J., Schilperoort R. Fingerprinting and sequence homology of plasmids from different virulent strains of Agrobacterium rhizogenes. Plasmid, 1981, vol. 5, no. 2, pp. 170–182. doi: 10.1016/0147-619x(81)90018-4.
  5. Levesque H., Delepelaire P., Rouzé P., Slightom J., Tepfer D. Common evolutionary origin of the central portions of the Ri TL-DNA of Agrobacterium rhizogenes and the Ti T‑DNAs of Agrobacterium tumefaciens. Plant Mol. Biol., 2009, vol. 11, no. 6, pp. 731–744. doi: 10.1007/BF00019514.
  6. Srivastava S., Srivastava A.K. Hairy root culture for mass-production of high-value secondary metabolites. Crit. Rev. Biotechnol., 2007, vol. 27, no. 1, pp. 29–43. doi: 10.1080/07388550601173918.
  7. Stoger E., Fischer R., Moloney M., Ma J.K.-C. Plant molecular pharming for the treatment of chronic and infectious diseases. Annu. Rev. Plant Biol., 2014, vol. 65, pp. 743–768. doi: 10.1146/annurev-arplant-050213-035850.
  8. Kuluev B.R., Vershinina Z.R., Knyazev A.V., Chemeris D.A., Baimiev An.Kh., Chumakov M.I., Baimiev Al.Kh., Chemeris A.V. “Hairy” plant roots – an important tool for researchers and a powerful phytochemical biofactory for industrial workers. Biomika, 2015, no. 2, pp. 70–120. (In Russian)
  9. Aggarwal P.R., Nag P., Choudhary P., Chakraborty N., Chakraborty S. Genotype-independent Agrobacterium rhizogenes-mediated root transformation of chickpea: A rapid and efficient method for reverse genetics studies. Plant Methods, 2018, vol. 14, art. 55, pp. 1–13. doi: 10.1186/s13007-018-0315-6.
  10. Foti C., Pavli O.I. High-efficiency Agrobacterium rhizogenes-mediated transgenic hairy root induction of Lens culinaris. Agronomy, 2020, vol. 10, no. 8. doi: 10.3390/agronomy10081170.
  11. Gumerova G.R., Chemeris A.V., Nikonorov Yu.M., Kuluev B.R. Morphological and molecular analysis of isolated cultures of tobacco adventitious roots obtained by the methods of biolistic bombardment and Agrobacterium-mediated transformation. Plant Physiol., 2018, vol. 65, no. 5, pp. 740–749. doi: 10.1134/S1021443718050072.
  12. Knyazev A.V., Kuluev B.R., Fateryga A.V., Yasybaeva G.R., Chemeris A.V. Aseptic germination and Agrobacterium rhizogenes-mediated transformation of Taraxacum hybernum Steven. Plant Tissue Cult. Biotechnol., 2017, vol. 27, no. 2, pp. 141–151. doi: 10.3329/ptcb.v27i2.35019.
  13. Murashige T., Skoog F. A revised medium for rapid growth and bio-assays with tobacco tissue cultures. Plant Physiol., 1962, vol. 15, no. 3, pp. 473–497. doi: 10.1111/j.1399-3054.1962.tb08052.x.
  14. Knyazev A.V., Kuluev B.R., Vershinina Z.R., Yasybaeva G.R., Chemeris A.V. Agrobacterium rhizogenes mediated hairy root induction in Parasponia andersonii Planch. Asian J. Plant Sci., 2017, vol. 16, no. 4, pp. 227–234. doi: 10.3923/ajps.2017.227.234.
  15. Kuluev B.R., Knyazev A.V., Mikhaylova E.V., Ermoshin A.A., Nikonorov Y.M., Chemeris A.V. The poplar ARGOS-LIKE gene promotes leaf initiation and cell expansion, and controls organ size. Biol. Plant., 2016, vol. 60, no. 3, pp. 513–522. doi: 10.1007/s10535-016-0610-x.

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