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



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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.
MATERIALS AND 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 AND CONCLUSIONS: 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. 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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Об авторах

Вероника Юрьевна Симонова

Sirius University

Автор, ответственный за переписку.
Email: nikasimonova14@gmail.com
ORCID iD: 0000-0002-9037-4684

student

Россия, Sochi

Elina Potsenkovskaia

Sirius University

Email: potsenkovskaya.ea@talantiuspeh.ru
ORCID iD: 0000-0002-5045-2641

specialist in plant biology and biotechnology

Россия, Sochi

Alexandra Vanina

Санкт-Петербургский государственный университет

Email: alexandraspb15@gmail.com
ORCID iD: 0009-0006-9753-2004
Россия

Anna Kiseleva

Sirius University

Email: anykisely@gmail.com

student

Россия, Sochi

Андрей Георгиевич Матвеенко

Санкт-Петербургский государственный университет

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

кандидат биологических наук

Россия, Санкт-Петербург

Daria Pavlova

Санкт-Петербургский государственный университет

Email: db_pavlova@mail.ru
ORCID iD: 0009-0006-7828-4105
Россия

Kirill Smirnov

Санкт-Петербургский Государственный Университет; Всероссийский научно-исследовательский институт сельскохозяйственной микробиологии

Email: kirill.vad.smirnov@gmail.com
ORCID iD: 0000-0002-2875-3798
Россия

Elena Efremova

Saint Petersburg State University

Email: elena.efremova@spbu.ru
ORCID iD: 0000-0002-2565-1155

student in biology, department of genetics and biotechnology

Россия, Saint Petersburg

Anna Brynchikova

Научно-технологический университет «Сириус»

Email: annbv19@gmail.com
SPIN-код: 8700-4413
Россия

Varvara Tvorogova

Saint Petersburg State University

Email: krubaza@mail.ru
ORCID iD: 0000-0002-0549-1457

phd, senior researcher, department of genetics and biotechnology

Россия, Saint Petersburg

Список литературы

  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; 60:2593–2605. 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; 101:3551–3563. doi: 10.1002/jsfa.11059.
  3. Tao A, Afshar RK, Huang J, et al. Variation in yield, starch, and protein of dry pea grown across Montana. Agronomy Journal 2017; 109: 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. Agrosystems 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; 76:735–745. 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. Ecol Genet 2025; 23:81–98. doi: 10.17816/ecogen640891.
  7. Ludvíková M, Griga M. Pea transformation: history, current status and challenges. Czech J Genet Plant Breed 2022; 58:127–161. 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: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; 2:780–787. doi: 10.1021/acsagscitech.2c00084.
  10. Atif RM, Patat-Ochatt E, Svabova L, et al. Gene transfer in legumes. In Progress in botany 2013; 74:408.
  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 Agrobacterium protocols, Wang K (Ed). Humana Press: Totowa, NJ, 2006; 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; 153:161–170. 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.). Genetica 1988; 24:1632-1640.
  17. Stejskal J, Griga M. Somatic embryogenesis and plant regeneration in Pisum sativum L. Biol Plant 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. Methods Mol Biol 2021; 2238:37–61. 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; in press.
  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; 10:1–16. 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; 3:839–843. 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: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; 7:1–6. 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; 133:462–469. 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; 7:193–195. 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; 11:51. doi: 10.3390/plants11010051.
  30. Jyothishwaran G, Kotresha D, Selvaraj T, et al. A modified freeze–thaw method for efficient transformation of Agrobacterium tumefaciens. Curr Sci 2007; 93:770–772.
  31. Simonova V, Potsenkovskaia E, Kozlov N., et al. Peas in rouge: how tyrosine helps RUBY shine. 2025. Under review.
  32. Fåhraeus, G. The Infection of Clover Root Hairs by Nodule Bacteria Studied by a Simple Glass Slide Technique. J Gen Microbiol 1957; 16: 374–381. doi: 10.1099/00221287-16-2-374.
  33. Wickham H, François R, Henry L, et al. Dplyr: a grammar of data manipulation. 2025.
  34. Wickham H. Ggplot2: elegant graphics for data analysis. Springer-Verlag New York, 2016.
  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. Biol Plant 2005; 49:361–370. doi: 10.1007/s10535-005-0009-6.
  39. Soulard C, Monfort M, Pillot J, et al. Efficient and heritable gene editing through CRISPR‐Cas9 in Pisum sativum. Plant Biotechnol J 2025; 23:3398–3400. 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. 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; 101:751–757. 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; 11:132–139. 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; 13:102. doi: 10.3390/plants13010102.
  45. Kudriashov AA, Zlydneva NS, Efremova EP, et al. MtCLE08, MtCLE16, and MtCLE18 transcription patterns and their possible functions in the embryogenic calli of Medicago truncatula. Plants 2023; 12:435. 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; 127:329–340. 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. Magnifying transgenic tissue: development of transformation protocol for Lens culinaris Medik. 2025.
  50. Mookkan M, Nelson‐Vasilchik K, Hague J, et al. Morphogenic regulator-mediated transformation of maize inbred B73. Curr Protoc Plant Biol 2018; 3:e20075. doi: 10.1002/cppb.20075.

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