Solanum lycopersicum L. is one of the most studied crops, with numerous genetic improvement techniques employed to enhance its fruit quality. The recalcitrance of tomatoes to certain regeneration mediums remains to be a limiting factor for a successful in vitro culture protocol. This study was conducted to establish an efficient regeneration system for the target tomato genotype Vb-15 for its use in gene editing using Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 vector system delivered by Agrobacterium tumafaciens. Cotyledons excised from 10 to 12-day-old in vitro germinated seedlings were cultured onto MS medium supplemented with BAP, TZD, and NAA. Explants cultured on TZD medium showed enlargement of the explant followed by callusing with multiple shoot development, while explants on BAP-medium showed early signs of callus formation and early development of shoot structures. The highest number of regenerants was obtained in MS with 0.5 mg-1 or 2.5 mg-1 TZD yielding a mean of 4.45–4.64 shoots per explant. When transferred onto MS basal medium, regenerants from 0.5 mg/L TZD or BAP showed maximum shoot elongation compared with other treatments. Rooted plantlets transferred to the greenhouse after acclimatization showed 78% survival. The optimum TZD concentration gave a significantly higher regeneration response in seven genotypes compared with Vb 15. Cluster analysis showed that at 0.6 average distances, the response of Vb-15 was unique showing its recalcitrance to in vitro culture.
Key words: in vitro, benzylaminopurine, naphthalene acetic acid, recalcitrance, callus