Enhanced hairy root induction in garlic (Allium sativum L.) via Agrobacterium rhizogenes: Targeting the basal plate of intact sprout

Authors

  • Phuong B.T. VU 1) University of Science, Department of Plant Biotechnology and Biotransformation, Laboratory of Plant Biotechnology, Ho Chi Minh, 700000; 2) Vietnam National University, Ho Chi Minh, 700000 (VN) https://orcid.org/0000-0002-6083-9604
  • Tan Q. TRAN 1) University of Science, Department of Plant Biotechnology and Biotransformation, Laboratory of Plant Biotechnology, Ho Chi Minh, 700000; 2) Vietnam National University, Ho Chi Minh, 700000 (VN) https://orcid.org/0000-0003-4225-5952
  • Khoi D. NGUYEN 1) University of Science, Department of Plant Biotechnology and Biotransformation, Laboratory of Plant Biotechnology, Ho Chi Minh, 700000; 2) Vietnam National University, Ho Chi Minh, 700000 (VN)
  • Huy TRAN 1) University of Science, Department of Plant Biotechnology and Biotransformation, Laboratory of Plant Biotechnology, Ho Chi Minh, 700000; 2) Vietnam National University, Ho Chi Minh, 700000 (VN)
  • CAO Minh Dai 1) University of Science, Department of Plant Biotechnology and Biotransformation, Laboratory of Plant Biotechnology, Ho Chi Minh, 700000; 2) Vietnam National University, Ho Chi Minh, 700000 (VN) https://orcid.org/0000-0001-6411-315X
  • Phuong N.D. QUACH 1) University of Science, Department of Plant Biotechnology and Biotransformation, Laboratory of Plant Biotechnology, Ho Chi Minh, 700000; 2) Vietnam National University, Ho Chi Minh, 700000 (VN) https://orcid.org/0000-0001-7259-1960

DOI:

https://doi.org/10.15835/nbha53414670

Keywords:

Agrobacterium-mediated transformation, Allium sativum, in vitro garlic hairy roots, monocotyledonous species, stem disc

Abstract

Hairy root induction in garlic represents a promising approach to actively exploit biomass from a familiar plant species rich in antibacterial compounds. Previously, the stem disc region of garlic was identified as a potential site for morphogenesis; however, few studies have focused on utilizing this tissue for hairy root induction and optimizing the conditions to enhance transformation efficiency. Histological analysis revealed that the basal plate contains soft parenchyma tissue conducive to root and vascular development, making it a suitable target for transformation in monocot species. In this study, we investigated the factors influencing hairy root induction from this recalcitrant tissue. Our results indicated that maintaining the integrity of the basal plate of stem disc in 10-day-old garlic seedlings significantly enhanced induction efficiency compared to sectioning methods. Through preservation of the primary root system and inner clove tissue and addition of acetosyringone, roots exhibited further improved stability in genetic, morphological, and growth traits, with fresh biomass accumulation reaching 1 g per induced explant and the hairy-root induction rate achieved 100%. PCR analysis confirmed the presence of rolB and rolC genes in induced roots, verifying successful gene transfer by Agrobacterium rhizogenes ATCC 15834. Overall, this study provides important insights into improving hairy root induction in garlic-a monocotyledonous species typically considered recalcitrant-and highlights the stem disc as a reliable and efficient target tissue for generating bioactive root biomass. These findings not only advance transformation strategies in monocots but also open broader opportunities for metabolic engineering, enhanced production of valuable secondary metabolites, and the development of sustainable biotechnological platforms for pharmaceutical and agricultural applications.

References

Ahsan N, Lee SH, Lee DG, Anisuzzaman M, Alam MF, Yoon HS, Choi MS, Yang JE, Lee BH (2007). The effects of wounding type, preculture, infection method and cocultivation temperature on the Agrobacterium-mediated gene transfer in tomatoes. Annals of Applied Biology 151(3):363-372. https://doi.org/10.1111/j.1744-7348.2007.00181.x

Ankri S, Mirelman D (1999). Antimicrobial properties of allicin from garlic. Microbes and Infection 1(2):125-129. https://doi.org/10.1016/S1286-4579(99)80003-3

Atif MJ, Amin B, Ghani MI, Ali M, Liu X, Zhang Y, Cheng Z (2021). Allium sativum L. (Garlic) bulb enlargement as influenced by differential combinations of photoperiod and temperature. Food Chemistry 338:127991. https://doi.org/10.1016/j.foodchem.2020.127991

Atif MJ, Amin B, Liu X, Zhao Y, Ali M, Ghani MI, Cheng Z (2020). Effect of photoperiod and temperature on garlic (Allium sativum L.) bulbing and selected endogenous chemical factors. Environmental and Experimental Botany 180:104250. https://doi.org/10.1016/j.envexpbot.2020.104250

Bellini C, Pacurar DI, Perrone I (2014). Adventitious roots and lateral roots: Similarities and differences. Annual Review of Plant Biology 65(1):639-666. https://doi.org/10.1146/annurev-arplant-050213-035645

Brencic A, Angert ER, Winans SC (2005). Unwounded plants elicit Agrobacterium vir gene induction and T-DNA transfer: Transformed plant cells produce opines yet are tumour free. Molecular Microbiology 57(6):1522-1531. https://doi.org/10.1111/j.1365-2958.2005.04763.x

Chen K, Otten L (2017). Natural Agrobacterium transformants: Recent results and some theoretical considerations. Frontiers in Plant Science 8:1600. https://doi.org/10.3389/fpls.2017.01600

Christey MC (2001). Use of Ri-mediated transformation for production of transgenic plants. In Vitro Cellular & Developmental Biology - Plant 37(6):687-700. https://doi.org/10.1007/s11627-001-0120-0

Christey MC, Braun RH (2005). Production of hairy root cultures and transgenic plants by Agrobacterium rhizogenes-mediated transformation. In L Peña (Ed). Transgenic plants: Methods and protocols. Humana Press pp 47-60. https://doi.org/10.1385/1-59259-827-7:047

Colby SM, Juncosa AM, Meredith CP (1991). Cellular differences in Agrobacterium susceptibility and regenerative capacity restrict the development of transgenic grapevines. Journal of the American Society for Horticultural Science 116:356-361. https://doi.org/10.21273/JASHS.116.2.356

Currier TC, Nester EW (1976). Isolation of covalently closed circular DNA of high molecular weight from bacteria. Analytical Biochemistry 76:431-441. https://doi.org/10.1016/0003-2697(76)90338-9

Dhiman N, Patial V, Bhattacharya A (2018). The current status and future applications of hairy root cultures. In: Kumar N (Eds). Biotechnological approaches for medicinal and aromatic plants. Springer, Singapore pp 87-155. https://doi.org/10.1007/978-981-13-0535-1_5

Escudero J, Hohn B (1997). Transfer and integration of T-DNA without cell injury in the host plant. The Plant Cell 9(12):2135-2142. https://doi.org/10.1105/tpc.9.12.2135

Evert RF, Eichhorn SE (2006). Esau’s plant anatomy: Meristems, cells, and tissues of the plant body: Their structure, function, and development (3rd ed). John Wiley & Sons, New York.

Faraz R, Gokhale M, Gothalwal R (2024). Enhancement in production of baicalein through transformation in Oroxylum indicum (L.) Vent by Rhizobium rhizogenes. Vegetos 37(1):305-320. https://doi.org/10.1007/s42535-023-00602-8

Gelvin SB (1990). Crown gall disease and hairy root disease: A sledgehammer and a tackhammer. Plant Physiology 92(2):281-285. https://doi.org/10.1104/pp.92.2.281

Gelvin SB (2003). Agrobacterium-mediated plant transformation: The biology behind the “gene-jockeying” tool. Microbiology and Molecular Biology Reviews 67(1):16-37. https://doi.org/10.1128/mmbr.67.1.16-37.2003

Gelvin SB (2017). Integration of Agrobacterium T-DNA into the plant genome. Annual Review of Genetics 51:195-217. https://doi.org/10.1146/annurev-genet-120215-035320

Giri A, Narasu ML (2000). Transgenic hairy roots: Recent trends and applications. Biotechnology Advances 18:1-22. https://doi.org/10.1016/S0734-9750(99)00016-6

Gonin M, Bergougnoux V, Nguyen TD, Gantet P, Champion A (2019). What makes adventitious roots? Plants 8(7):240. https://doi.org/10.3390/plants8070240

Guillon S, Trémouillaux-Guiller J, Pati PK, Rideau M, Gantet P (2006). Hairy root research: Recent scenario and exciting prospects. Current Opinion in Plant Biology 9:341-346. https://doi.org/10.1016/j.pbi.2006.03.008

Kumar V, Parvatam G, Ravishankar GA (2006). Agrobacterium rhizogenes-mediated genetic transformation resulting in hairy root formation is enhanced by ultrasonication and acetosyringone treatment. Electronic Journal of Biotechnology 9(4):349-357. https://doi.org/10.2225/vol9-issue4-fulltext-4

Lan X, Quan H (2010). Hairy root culture of Przewalskia tangutica for enhanced production of pharmaceutical tropane alkaloids. Journal of Medicinal Plants Research 4:1477-1481. https://doi.org/10.5897/JMPR09.289

Lanzotti V (2006). The analysis of onion and garlic. Journal of Chromatography A 1112:3-22. https://doi.org/10.1016/j.chroma.2005.12.016

Li M, Wen Y, Wang X, Li Y, Li H, Zhang Y (2015). Establishment of hairy root cultures by Agrobacterium rhizogenes-mediated transformation of Isatis tinctoria L. for the efficient production of flavonoids and evaluation of antioxidant activities. Molecules 20:19777-19793. https://doi.org/10.1371/journal.pone.0119022

Liu H, Deng R, Huang C, Cheng Z, Meng H (2019). Exogenous gibberellins alter morphology and nutritional traits of garlic (Allium sativum L.) bulb. Scientia Horticulturae 246:298-306. https://doi.org/10.1016/j.scienta.2018.11.003

Mangena P (2021). Effect of Agrobacterium co cultivation stage on explant response for subsequent genetic transformation in soybean (Glycine max (L.) Merr.). Plant Science Today 8(4):905-911. https://doi.org/10.14719/PST.2021.8.4.1363

Moradi F, Mehrjerdi MZ, Vahdati K (2018). Agrobacterium rhizogenes -mediated hairy root induction in garlic. Bulgarian Journal of Agricultural Science 23(4):572-577.

Nester EW (2015). Agrobacterium: Nature’s genetic engineer. Frontiers in Plant Science 5:730. https://doi.org/10.3389/fpls.2014.00730

Phuong VTB, Anh NP, Thang NPQ, Phi T, Phuong QND (2023). Study on induction of Allium sativum L. hairy roots with antibacterial activity against Vibrio. Research Journal of Biotechnology 18:8. https://doi.org/10.25303/1808rjbt1530162

Porebski S, Bailey LG, Baum BR (1997). Modification of a CTAB DNA extraction protocol for plants containing high polysaccharide and polyphenol components. Plant Molecular Biology Reporter 15:8-15. https://doi.org/10.1007/BF02772108

Rana MM, Wei S, Abdullah M, Shamalla FL (2017). Wild-type Agrobacterium rhizogenes-mediated gene transfer in plants: Agrobacterium virulence and selection of transformants. Journal of Plant Science and Phytopathology 1(1):44-51. https://doi.org/10.29328/journal.jpsp.1001005

Schlappi M, Hohn B (1992). Competence of immature maize embryos for Agrobacterium-mediated gene transfer. The Plant Cell 4(1):7-16. https://doi.org/10.1105/tpc.4.1.7

Spanò L, Wullems GJ, Schilperoort RA, Costantino P (1981). Hairy root: In vitro growth properties of tissues induced by Agrobacterium rhizogenes on tobacco. Plant Science Letters 23:299-305. https://doi.org/10.1016/0304-4211(81)90040-7

Tepfer D (1984). Transformation of several species of higher plants by Agrobacterium rhizogenes: Sexual transmission of the transformed genotype and phenotype. Cell 37(3):959-967. https://doi.org/10.1016/0092-8674(84)90430-6

Veena S, Taylor CG (2007). Agrobacterium rhizogenes-mediated transformation of plants: Biology and applications. In Vitro Cellular & Developmental Biology - Plant 43:383-403. https://doi.org/10.1007/s11627-007-9096-8

Verstraeten I, Schotte S, Geelen D (2014). Hypocotyl adventitious root organogenesis differs from lateral root formation. Frontiers in Plant Science 5:495. https://doi.org/10.3389/fpls.2014.00495

Yang QQ, Fan JD, Liu CY, Zhao YQ, Xu ZS, Lu XJ, Ge J, Zhang BW, Li MQ, Yang Y, Yang F (2025). Physiological and transcriptome analysis of changes in endogenous hormone contents and related synthesis and signaling genes during the heat stress in garlic (Allium sativum L.). BMC Plant Biology 25:464. https://doi.org/10.1186/s12870-025-06346-8

Downloads

Published

2025-12-21

How to Cite

VU, P. B., TRAN, T. Q., NGUYEN, K. D., TRAN, H., CAO, D. M., & QUACH, P. N. (2025). Enhanced hairy root induction in garlic (Allium sativum L.) via Agrobacterium rhizogenes: Targeting the basal plate of intact sprout. Notulae Botanicae Horti Agrobotanici Cluj-Napoca, 53(4), 14670. https://doi.org/10.15835/nbha53414670

Issue

Section

Research Articles
CITATION
DOI: 10.15835/nbha53414670