Development of genetic transformation methods for Oxalis tuberosa using Rhizobium rhizogenes and Agrobacterium tumefaciens



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Abstract

BACKGROUND: Oca (Oxalis tuberosa M.) is an understudied Andean tuber crop with significant nutritional value. However, its genetic improvement and functional analysis are hindered by the lack of an established genetic transformation system.

AIM: This study aims to establish transformation protocols for O. tuberosa using Rhizobium rhizogenes and Agrobacterium tumefaciens.

METHODS: Composite plants with transgenic roots were generated by inoculating oca stem explants with R. rhizogenes strain ARqua1 carrying reporter constructs (eGFP, DsRED1, GUS, RUBY). Transformation was confirmed using fluorescence microscopy (GFP, DsRED1), histochemical staining (GUS), and visual assessment (RUBY). To obtain fully transgenic plants, oca internodes and petioles were transformed with A. tumefaciens strain AGL1 carrying a vector containing the GUS reporter gene. 

RESULTS: Expression of all four reporter genes was detected in transgenic oca roots. Furthermore, the regeneration potential of O. tuberosa explants was confirmed. Following A. tumefaciens transformation, regions with GUS activity were observed on calli, indicating successful transformation events.

CONCLUSION: This study established the first methods for genetic transformation of O. tuberosa using R. rhizogenes and A. tumefaciens. The effectiveness of four reporter systems in transgenic roots was demonstrated, and a regeneration protocol for shoot formation from callus was evaluated. The potential of stable transformation using A. tumefaciens was shown, opening perspectives for the genetic modification of this valuable yet understudied crop.

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About the authors

Anastasia V. Timoshicheva

ITMO University, St. Petersburg

Email: timoshicheva.nastya@yandex.ru
ORCID iD: 0009-0009-4903-5697
Russian Federation

Karina I. Petrova

Saint Petersburg State University, St. Petersburg

Email: karinkakriukova@gmail.com
ORCID iD: 0009-0003-5833-9939
Russian Federation

Alyona A. Gurina

Saint Petersburg State University, St. Petersburg

Email: a.gurina@vir.nw.ru
ORCID iD: 0000-0002-1791-3063
SPIN-code: 8398-1264
Russian Federation

Maria S. Gancheva

Saint Petersburg State University, St. Petersburg

Author for correspondence.
Email: m.gancheva@spbu.ru
ORCID iD: 0000-0002-9631-6143
SPIN-code: 3694-4470
Russian Federation

References

  1. Khan MRI, Heyes JK, Cohen D. Plant regeneration from oca (Oxalis tuberosa M.): the effect of explant type and culture media. Plant Cell, Tissue and Organ Culture. 1988;14:41–50. doi: 10.1007/BF00043384
  2. Llaja-Zuta E, Fernández-Poquioima DM, Añazco-Urbina B, Hernández-Amasifuen AD, Condori-Apfata JA. In vitro micropropagation of oca (Oxalis tuberosa Mol.): an important plant genetic resource from the high Andean region. Plants. 2026;15(1):62. doi: 10.3390/plants15010062
  3. de Ruijter NCA, Verhees J, van Leeuwen W, van der Krol AR. Evaluation and comparison of the GUS, LUC and GFP reporter system for gene expression studies in plants. Plant Biology. 2003;5:103–115. doi: 10.1055/s-2003-40722
  4. Murashige T, Skoog F. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiologia Plantarum. 1962;15(3):473–497. doi: 10.1111/j.1399-3054.1962.tb08052.x
  5. Ilina EL, Logachov AA, Laplaze L, Demchenko NP, Pawlowski K, Demchenko KN. Composite Cucurbita pepo plants with transgenic roots as a tool to study root development. Annals of Botany. 2012;110(2):479–489. doi: 10.1093/aob/mcs086
  6. Karimi M, Inzé D, Depicker A. GATEWAY vectors for Agrobacterium-mediated plant transformation. Trends in Plant Science. 2002;7(5):193–195. doi: 10.1016/s1360-1385(02)02251-3
  7. He Y, Zhang T, Sun H, Zhan H, Zhao Y. A reporter for noninvasively monitoring gene expression and plant transformation. Horticulture Research. 2020;7(1):152. doi: 10.1038/s41438-020-00390-1
  8. Jyothishwaran G, Kotresha D, Selvaraj T, Srideshikan SM, Rajvanshi PK, Jayabaskaran C. A modified freeze–thaw method for efficient transformation of Agrobacterium tumefaciens. Current Science. 2007;93:770–772.
  9. Wu H-Y, Liu K-H, Wang Y-C, Wu J-F, Chiu W-L, Chen C-Y, et al. AGROBEST: an efficient Agrobacterium-mediated transient expression method for versatile gene function analyses in Arabidopsis seedlings. Plant Methods. 2014;10:19. doi: 10.1186/1746-4811-10-19
  10. Berg RH, Beachy RN. Fluorescent protein applications in plants. Methods in Cell Biology. 2008;85:153–177. doi: 10.1016/S0091-679X(08)85008-X
  11. Shi L, Li X, Fu Y, Li C. Environmental stimuli and phytohormones in anthocyanin biosynthesis: a comprehensive review. International Journal of Molecular Sciences. 2023;24(22):16415. doi: 10.3390/ijms242216415
  12. Collier R, Fuchs B, Walter N, Kevin Lutke W, Taylor CG. Ex vitro composite plants: an inexpensive, rapid method for root biology. The Plant Journal. 2005;43(3):449–457. doi: 10.1111/j.1365-313X.2005.02454.x
  13. Kiryushkin AS, Ilina EL, Guseva ED, Pawlowski K, Demchenko KN. Hairy CRISPR: genome editing in plants using hairy root transformation. Plants. 2021;11(1):51. doi: 10.3390/plants11010051
  14. Gancheva M, Tkachenko A. Genome and transcriptome sequencing of oca (Oxalis tuberosa Molina) reveals photoperiod-induced FT homologs as candidate tuberigens. International Journal of Plant Biology. 2026;17(2):11. doi: 10.3390/ijpb17020011

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