Evaluation of selective agent requirements for pea callus culture expressing foreign DNA

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Abstract

BACKGROUND: Genetic modification of pea remains challenging, which can likely be attributed to its low regeneration efficiency. To obtain transgenic pea plants, shoot regeneration followed by rooting is usually applied. Another regeneration pathway, somatic embryogenesis, is not used for pea genome modification due to very low frequency of this process. If a gene stimulating somatic embryogenesis was identified, it could be used as a morphogenic regulator to enable regeneration of pea plants from genetically modified callus cells. The search for such genes relies on the development of a cultivation system, allowing production of a significant amount of callus tissue in which a potential morphogenic regulator is ectopically expressed.

AIM: The aim of study was to evaluate the possibility of obtaining pea callus tissue expressing foreign DNA with or without usage of selective agents, specifically, kanamycin and hygromycin B.

METHODS: In this study, we combined agrobacterial transformation protocol with a method of callus induction from pea shoot apex explants. To evaluate the effectiveness of this transformation system with different selective agents, we used two different reporters: RUBY and DsRed.

RESULTS: Our results demonstrate that transformation of pea shoot apices using the developed system yields a significant percentage of calli containing tissue regions which express foreign DNA. Addition of the antibiotics as selective agents doesn’t increase frequency of calli expressing introduced DNA.

CONCLUSION: Results obtained in this study suggest that searching for regeneration stimulators is feasible in Pisum sativum without usage of selective agents.

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About the authors

Veronika Yu. Simonova

Sirius University of Science and Technology

Author for correspondence.
Email: nikasimonova14@gmail.com
ORCID iD: 0000-0002-9037-4684
SPIN-code: 9475-2980
Russian Federation, SochiSirius

Elina A. Potsenkovskaia

Sirius University of Science and Technology; Saint Petersburg State University

Email: potsenkovskaya.ea@talantiuspeh.ru
ORCID iD: 0000-0002-5045-2641
SPIN-code: 4484-7595
Russian Federation, Sirius; Saint Petersburg

Alexandra A. Vanina

Saint Petersburg State University

Email: alexandraspb15@gmail.com
ORCID iD: 0009-0006-9753-2004
SPIN-code: 4763-4324
Russian Federation, Saint Petersburg

Anna S. Kiseleva

Sirius University of Science and Technology

Email: anykisely@gmail.com
ORCID iD: 0000-0002-1425-7013
SPIN-code: 1478-4870
Russian Federation, Sirius

Andrew G. Matveenko

Sirius University of Science and Technology; Saint Petersburg State University

Email: a.matveenko@spbu.ru
ORCID iD: 0000-0002-9458-0194
SPIN-code: 9877-5352

Cand. Sci. (Biology)

Russian Federation, Sirius; Saint Petersburg

Daria B. Pavlova

Saint Petersburg State University

Email: db_pavlova@mail.ru
ORCID iD: 0009-0006-7828-4105
SPIN-code: 8002-0762
Russian Federation, Saint Petersburg

Kirill V. Smirnov

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

Email: kirill.vad.smirnov@gmail.com
ORCID iD: 0000-0002-2875-3798
SPIN-code: 2440-2405
Russian Federation, Saint Petersburg; Saint Petersburg

Elena P. Efremova

Saint Petersburg State University

Email: elena.efremova@spbu.ru
ORCID iD: 0000-0002-2565-1155
SPIN-code: 4033-2739
Russian Federation, Saint Petersburg

Anna V. Brynchikova

Sirius University of Science and Technology

Email: annbv19@gmail.com
ORCID iD: 0009-0002-8358-8139
SPIN-code: 8700-4413
Russian Federation, Sirius

Varvara E. Tvorogova

Sirius University of Science and Technology; Saint Petersburg State University

Email: krubaza@mail.ru
ORCID iD: 0000-0002-0549-1457
SPIN-code: 6489-5206

Cand. Sci. (Biology)

Russian Federation, Sirius; Saint Petersburg

References

  1. Lu ZX, He JF, Zhang YC, Bing DJ. Composition, physicochemical properties of pea protein and its application in functional foods. Crit Rev Food Sci Nutr. 2020;60(15):2593–2605. doi: 10.1080/10408398.2019.1651248
  2. Guindon MF, Cazzola F, Palacios T, et al. Biofortification of pea (Pisum sativum L.): a review. J Sci Food Agric. 2021;101(9):3551–3563. doi: 10.1002/jsfa.11059
  3. Tao A, Afshar RK, Keshavarzafshar R, Mohammed YA. Variation in yield, starch, and protein of dry pea grown across Montana. Agron J. 2017;109(4):1491–1501. doi: 10.2134/agronj2016.07.0401
  4. Sainju UM, Lenssen AW, Allen BL, et al. Pea growth, yield, and quality in different crop rotations and cultural practices. Agrosyst Geosci Environ. 2019;2:1–9. doi: 10.2134/age2018.10.0041
  5. Stevenson FC, van Kessel C. The nitrogen and non-nitrogen rotation benefits of pea to succeeding crops. Can J Plant Sci. 1996;76(4):735–745. doi: 10.4141/cjps96-126
  6. Zhuravlev IY, Subkhanov LR, Sulima AS, et al. Genome editing of pea (Pisum sativum L.) using CRISPR/Cas9 technology: review. Ecological Genetics. 2025;23(1):81–98. doi: 10.17816/ecogen640891 EDN: XYWMFY
  7. Ludvíková M, Griga M. Pea transformation: history, current status and challenges. Czech J Genet Plant Breed. 2022;58(3):127–161. doi: 10.17221/24/2022-CJGPB
  8. Hodgins CL, Salama EM, Kumar R, et al. Creating saponin-free yellow pea seeds by CRISPR/Cas9-enabled mutagenesis on β-amyrin synthase. Plant Direct. 2024;8(1):e563. doi: 10.1002/pld3.563
  9. Kaur R, Donoso T, Scheske C, et al. Highly efficient and reproducible genetic transformation in pea for targeted trait improvement. ACS Agric Sci Technol. 2022;2(4):780–787. doi: 10.1021/acsagscitech.2c00084
  10. Atif RM, Patat-Ochatt E, Svabova L, et al. Gene transfer in legumes. In: Luttge U, Beyschlag W, Francis D, Cushman J, editors. Progress in botany. Vol. 74. Springer; 2013. P. 408. doi: 10.1007/978-3-642-30967-0_2
  11. Grant JE, Thomson LMJ, Pither-Joyce MD, et al. Influence of Agrobacterium tumefaciens strain on the production of transgenic peas (Pisum sativum L.). Plant Cell Rep. 2003;21:1207–1210. doi: 10.1007/s00299-003-0640-7
  12. Grant J, Cooper P. Peas (Pisum sativum L.). In: Wang K, editors. Agrobacterium protocols. Humana Press: 2006. P. 337–346.
  13. Krejčí P, Matušková P, Hanáček P, et al. The transformation of pea (Pisum sativum L.): applicable methods of Agrobacterium tumefaciens-mediated gene transfer. Acta Physiol Plant. 2007;29:157–163. doi: 10.1007/s11738-006-0020-3
  14. Polowick P, Quandt J, Mahon J. The ability of pea transformation technology to transfer genes into peas adapted to western Canadian growing conditions. Plant Sci. 2000;153(2):161–170. doi: 10.1016/s0168-9452(99)00267-8
  15. Rubluo A, Kartha KK, Mroginski LA, Dyck J. Plant regeneration from pea leaflets cultured in vitro and genetic stability of regenerants. J Plant Physiol. 1984;117:119–130. doi: 10.1016/S0176-1617(84)80024-3
  16. Lutova LA, Zabelina YK. Callus and shoot in vitro formation in different forms of peas (Pisum sativum L.). Russian Journal of Genetics. 1988;24(9):1632–1640. (In Russ.)
  17. Stejskal J, Griga M. Somatic embryogenesis and plant regeneration in Pisum sativum L. Biologia Plantarum. 1992;34:15–22. doi: 10.1007/BF02925784
  18. Kysely W, Jacobsen H-J. Somatic embryogenesis from pea embryos and shoot apices. Plant Cell Tissue Organ Cult. 1990;20:7–14. doi: 10.1007/BF00034751
  19. Nalapalli S, Tunc-Ozdemir M, Sun Y, et al. Morphogenic regulators and their application in improving plant transformation. In: Bandyopadhyay A, Thilmony R, editors. Rice genome engineering and gene editing. Methods in Molecular Biology. Vol. 2238. New York: Humana; 2021. P. 37–61. doi: 10.1007/978-1-0716-1068-8_3
  20. Simonova V, Tvorogova V, Potsenkovskaia E, et al. Analysis of intraspecific variability in somatic embryogenesis capacity in Pisum sativum. Biol Commun. 2025;70(3):223–230.
  21. Wu H-Y, Liu K-H, Wang Y-C, 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
  22. Engler C, Youles M, Gruetzner R, et al. A golden gate modular cloning toolbox for plants. ACS Synth Biol. 2014;3(11):839–843. doi: 10.1021/sb4001504
  23. Weber E, Engler C, Gruetzner R, et al. A modular cloning system for standardized assembly of multigene constructs. PLoS One. 2011;6(2): e16765. doi: 10.1371/journal.pone.0016765
  24. He Y, Zhang T, Sun H, et al. A reporter for noninvasively monitoring gene expression and plant transformation. Hortic Res. 2020;7(1):152. doi: 10.1038/s41438-020-00390-1
  25. Curtis MD, Grossniklaus U. A gateway cloning vector set for high-throughput functional analysis of genes in planta. Plant Physiol. 2003;133(2):462–469. doi: 10.1104/pp.103.027979
  26. Tvorogova VE, Fedorova YA, Potsenkovskaya EA, et al. The WUSCHEL-related homeobox transcription factor MtWOX9-1 stimulates somatic embryogenesis in Medicago truncatula. Plant Cell Tissue Organ Cult. 2019;138:517–527. doi: 10.1007/s11240-019-01648-w
  27. Xing H-L, Dong L, Wang Z-P, et al. A CRISPR/Cas9 toolkit for multiplex genome editing in plants. BMC Plant Biol. 2014;14:327. doi: 10.1186/s12870-014-0327-y
  28. Karimi M, Inzé D, Depicker A. GATEWAYTM vectors for Agrobacterium-mediated plant transformation. Trends Plant Sci. 2002;7(5):193–195. doi: 10.1016/S1360-1385(02)02251-3
  29. Kiryushkin AS, Ilina EL, Guseva ED, et al. Hairy CRISPR: genome editing in plants using hairy root transformation. Plants. 2022;11(1):51. doi: 10.3390/plants11010051
  30. Dupadahalli K. A modified freeze–thaw method for efficient transformation of Agrobacterium tumefaciens. Curr Sci. 2007;93(6):770–772.
  31. Simonova V, Potsenkovskaia E, Kozlov N., et al. Peas in Rouge: Tyrosine Supplementation Enhances RUBY Reporter Visibility in Pisum sativum. Plants. 2025;14(24):3719. doi: 10.3390/plants14243719
  32. Fåhraeus G. The infection of clover root hairs by nodule bacteria studied by a simple glass slide technique. J Gen Microbiol. 1957;16(2): 374–381. doi: 10.1099/00221287-16-2-374
  33. Wickham H, François R, Henry L, et al. Dplyr: a grammar of data manipulation. Am J Oper Res. 2023;13(6).
  34. Wickham H. Data analysis. In: ggplot2. Use R!. Springer, Cham; 2016. doi: 10.1007/978-3-319-24277-4_9
  35. Graves S, Piepho H-P, Dorai-Raj LS. multcompView: visualizations of paired comparisons. 2023.
  36. Wickham H. Stringr: simple, consistent wrappers for common string operations. 2022.
  37. De Kathen A, Jacobsen HJ. Agrobacterium tumefaciens-mediated transformation of Pisum sativum L. using binary and cointegrate vectors. Plant Cell Rep. 1990;9:276–279. doi: 10.1007/BF00232301
  38. Svabova L, Smykal P, Griga M, Ondrej V. Agrobacterium-mediated transformation of Pisum sativum in vitro and in vivo. Biologia Plantarum. 2005;49:361–370. doi: 10.1007/s10535-005-0009-6
  39. Soulard C, Monfort M, Pillot J-P, et al. Efficient and heritable gene editing through CRISPR-Cas9 in Pisum sativum. Plant Biotechnol J. 2025;23(8):3398–3400. doi: 10.1111/pbi.70091
  40. Bhowmik P, Yan W, Hodgins C, et al. CRISPR/Cas9-mediated lipoxygenase gene-editing in yellow pea leads to major changes in fatty acid and flavor profiles. Front Plant Sci. 2023;14:1246905. doi: 10.3389/fpls.2023.1246905
  41. Grant JE, Cooper PA, Gilpin BJ, et al. Kanamycin is effective for selecting transformed peas. Plant Sci. 1998;139:159–164. doi: 10.1016/S0168-9452(98)00184-8
  42. Schroeder HE, Schotz AH, Wardley-Richardson T, et al. Transformation and regeneration of two cultivars of pea (Pisum sativum L.). Plant Physiol. 1993;101(3):751–757. doi: 10.1104/pp.101.3.751
  43. Li G, Liu R, Xu R, et al. Development of an Agrobacterium-mediated CRISPR/Cas9 system in pea (Pisum sativum L.). Crop J. 2023;11(1):132–139. doi: 10.1016/j.cj.2022.04.011
  44. Krasnoperova EY, Tvorogova VE, Smirnov KV, et al. MtWOX2 and MtWOX9–1 effects on the embryogenic callus transcriptome in Medicago truncatula. Plants. 2024;13(1):102. doi: 10.3390/plants13010102
  45. Kudriashov AA, Zlydneva NS, Efremova EP, et al. MtCLEo8, MtCLE16, and MtCLE18 transcription patterns and their possible functions in the embryogenic calli of Medicago truncatula. Plants. 2023;12(3):435. doi: 10.3390/plants12030435
  46. Scholz-Starke J, Carpaneto A, Gambale F. On the interaction of neomycin with the slow vacuolar channel of Arabidopsis thaliana. J Gen Physiol. 2006;127(3):329–340. doi: 10.1085/jgp.200509402
  47. Padilla IMG, Burgos L. Aminoglycoside antibiotics: structure, functions and effects on in vitro plant culture and genetic transformation protocols. Plant Cell Rep. 2010;29:1203–1213. doi: 10.1007/s00299-010-0900-2
  48. Burgos L, Alburquerque N. Ethylene inhibitors and low kanamycin concentrations improve adventitious regeneration from apricot leaves. Plant Cell Rep. 2003;21:1167–1174. doi: 10.1007/s00299-003-0625-6
  49. Dubenko T, Smirnov K, Tvorogova V. The role of explant type and selective agent application in the initial transformation rate of Lens culinaris Medik. Vavilov Journal of Genetics and Breeding. 2026;30:233–240. doi: 10.18699/vjgb-26-26
  50. Mookkan M, Nelson-Vasilchik K, Hague J, et al. Morphogenic regulator-mediated transformation of maize inbred B73. Curr Protoc Plant Biol. 2018;3(4):e20075. doi: 10.1002/cppb.20075

Supplementary files

Supplementary Files
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1. JATS XML
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2. Supplement 1. Structure of plasmids used in the study.
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3. Supplement 2. Composition of media used in the study.
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4. Supplement 3. Representative images of calli, developed from explants transformed with pAGM4673_GUS_KmR_RUBY cultivated on the MSmod medium with different kanamycin concentrations at 7th and 35th day of cultivation (a) or calli, developed from explants transformed with pAGM4673_GUS_HygR_RUBY cultivated on the MSmod medium with different hygromycin B concentrations at 7th and 35th day of cultivation (b). (c) Boxplots demonstrating length of roots developed on media with different kanamycin concentrations from control seedlings treated with empty ARqua1 strain (beige) or from seedlings treated with ARqua1 strain with pAGM4673_KmR_EGFP_DsRed_HygR vector (grey). Statistical significance of length difference between roots obtained from control and vector-transformed seedlings was evaluated using pairwise Wilcoxon signed-rank test with Holm p-value adjustment. *p < 0.05.
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5. Fig. 1. Barplots demonstrating frequencies of explants developing calli with different numbers of tissue spots expressing reporter genes, RUBY (a) or DsRed (b), on media with different kanamycin concentrations. For RUBY, explants were divided into 3 categories: beige, no visible RUBY staining; pink, one or two spots of RUBY stained tissue; purple-red, several spots of RUBY stained tissue. For DsRed, explants were divided into 4 categories: black, no fluorescence; brown, single spot of DsRed fluorescence; dark red, two spots of DsRed fluorescence; bright red, three and more spots of DsRed fluorescence. Numbers at the top part of each bar show the amount of explants analyzed for each antibiotic concentration. Significance annotations represent results of pairwise Fisher tests evaluating differences between each treatment group and the control group without antibiotic (0 mg/L kanamycin). Fisher test was performed with Bonferroni adjustment for multiple comparisons, taking into account 5 comparisons; ns (non-significant)—p > 0.05, **p < 0.01.

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6. Fig. 2. Boxplots demonstrating weight of calli developed on media with different kanamycin concentrations from control explants, treated with empty AGL1 strain (a), or from explants, transformed with pAGM4673_KmR_EGFP_DsRed_HygR (b). Statistical significance of differences between weight of calli obtained on media with different antibiotic concentrations was evaluated with the Kruskall–Wallis test with post hoc Dunn test using Benjamini–Hochberg p-value adjustment. Different lowercase letters represent values with statistically significant differences (p < 0.05). (c) Combined boxplots demonstrating comparison between weight of calli developed from control and transformed explants on media with different kanamycin concentrations. Statistical significance of weight differences between calli obtained from control and transformed explants was evaluated using pairwise Wilcoxon signed-rank test with Bonferroni p-value adjustment; ns—non significant, p > 0.05.

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7. Fig. 3. Barplots demonstrating frequencies of explants developing calli with different numbers of tissue spots expressing reporter genes, RUBY (a) or DsRed (b, c), on media with different hygromycin concentrations, after transformation with pAGM4673_GUS_HygR_RUBY (a), pMDC32_DsRed (b), or pAGM4673_KmR_EGFP_DsRed_HygR (c) vectors. For RUBY reporter, explants were divided into 3 categories: beige, no visible RUBY staining; pink, one or two spots of RUBY stained tissue; purple-red, several spots of RUBY stained tissue. For DsRed, explants were divided into 4 categories: black, no fluorescence; brown, single spot of DsRed fluorescence; dark red, two spots of DsRed fluorescence; bright red, three and more spots of DsRed fluorescence. Numbers at the top part of each bar show the amount of explants analysed for each antibiotic concentration. Significance annotations represent results of pairwise Fisher tests evaluating differences between each treatment group and the control group without antibiotic (0 mg/L hygromycin). Fisher test was performed with Bonferroni adjustment for multiple comparisons, taking into account 5 comparisons; ns—non significant, p > 0.05.

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8. Fig. 4. Boxplots demonstrating weight of calli developed on media with different hygromycin concentrations from control explants, treated with empty AGL1 strain (a), or from explants, transformed with pAGM4673_KmR_EGFP_DsRed_HygR (b). Statistical significance of differences between weight of calli obtained on media with different antibiotic concentrations was evaluated with the Kruskall–Wallis test with post hoc Dunn test using Benjamini–Hochberg p-value adjustment. Different lowercase letters represent values with statistically significant differences (p < 0.05). (c) Combined boxplots demonstrating comparison between weight of calli developed from control and transformed explants on media with different hygromycin concentrations. Statistical significance of weight differences between calli obtained from control and transformed explants was evaluated using pairwise Wilcoxon signed-rank test with Bonferroni p-value adjustment; ns—non significant, p > 0.05.

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