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 aimed 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, and 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, offering prospects for the genetic modification of this valuable yet understudied crop.

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About the authors

Anastasia V. Timoshicheva

Saint Petersburg National Research University of Information Technologies, Mechanics and Optics

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

Karina I. Petrova

Saint Petersburg State University

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

Alyona A. Gurina

N.I. Vavilov All-Russian Institute of Plant Genetic Resources

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

Cand. Sci. (Biology)

Russian Federation, Saint Petersburg

Maria S. Gancheva

Saint Petersburg State University; All-Russia Research Institute for Agricultural Microbiology

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

Cand. Sci. (Biology)

Russian Federation, Saint Petersburg; Saint Petersburg

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2. Fig. 1. Reporter gene expression in Oxalis tuberosa roots: a, c, fluorescence of eGFP and DsRED1, respectively; b, d, bright-field view of the same roots; e, GUS histochemical staining; f, visual detection of RUBY.

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3. Fig. 2. Assessment of shoot regeneration in Oxalis tuberosa: a, b, shoot regeneration from petiole- (a) and stem-derived calli (b); c, red pigment accumulation in callus tissues; d, an oca plantlet on a medium with 5 mg/L hygromycin; e, control plantlet on MS10 medium without hygromycin; f, GUS activity in callus tissues; g, shoot regeneration after transformation.

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