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Plant Science Today (PST)

Research Articles

Vol. 13 No. 2 (2026)

Agrobacterium-mediated transformation and enhanced regeneration system for genome editing in prominent cultivars of tomato (Solanum lycopersicum L.)

DOI
https://doi.org/10.14719/pst.12544
Submitted
1 November 2025
Published
04-06-2026 — Updated on 12-06-2026
Versions

Abstract

Agrobacterium-mediated transformation remains a keystone of functional genomics and crop improvement, enabling the effective application of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 and related genome editing technologies. This study reports a highly optimised regeneration and transformation protocol for three tomato (Solanum lycopersicum L.) cultivars: Punjab Chhuhara (PC), Utkal Pragyan (UP) and Arka Meghali (AM). A single transcript unit based CRISPR vector, pTX171 was employed to optimise the transformation process using EHA105 Agrobacterium strain. Two explant types, hypocotyl and cotyledon, were evaluated under different media compositions and hormone combinations to establish efficient regeneration. The optimal shoot induction medium was achieved on Murashige and Skoog (MS) medium supplemented with 1.5 mg L-1 zeatin and 0.1 mg L-1 indole-3-acetic acid (IAA), followed by 1.5 mg L-1 6-benzylaminopurine (BAP) and 0.1 mg L-1 IAA, both producing comparable regeneration response, while cotyledons produced a higher number of shoots than hypocotyls. Shoot elongation was most effective on half-strength MS medium containing 20 g L-1 glucose and 10 g L-1 maltose supplemented with 1 mg L-1 zeatin and 0.1 mg L-1 IAA, while efficient rooting was obtained on media supplemented with 0.1 mg L-1 indole-3-butyric acid (IBA). For Agrobacterium-mediated transformation, cefotaxime and timentin at 200 mg L-1 each effectively supressed bacterial overgrowth, with optimum results obtained following a 48 hr co-cultivation period at optical density (OD₆₀₀) = 0.4. The presence of the transgene in the plant genome was validated using polymerase chain reaction (PCR) and Southern blotting. The highest transformation efficiency was obtained in PC (54.33 %), followed by UP (53 %) respectively on cotyledon. Overall, the standardised regeneration and transformation systems established in this study provide a robust platform for functional genomics research and development of transgenic or genome-edited tomato cultivars with enhanced disease resistance and improved agronomic traits.

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