Development of a callus regeneration protocol for “Masan” (Ziziphus mauritiana) using shoot tip culture

Abstract

Plant tissue culture is a widely used technique for the commercial-scale production of uniform, disease-free, and high-quality plants under controlled laboratory conditions. Lack of an efficient regeneration protocol for Ziziphus mauritiana “Local Masan”, limits commercial cultivation in Sri Lanka’s dry zones despite its adaptability and economic value. This study aimed to develop a tissue culture protocol by optimizing sterilization conditions and cytokinin concentrations using shoot tip explants, to enable the large-scale propagation of “Local Masan”. The study was carried out in a completely randomized design (CRD), with ten replicates per treatment using Murashige and Skoog (MS) culture medium. Four 6-benzylaminopurine (BAP) levels (1.5 mg/L, 1.0 mg/L, 0.5 mg/L, and 0 mg/L) and two sterilization protocols (S1, S2) were tested. Protocol S1 consisted of 15% Clorox, 0.1% mercuric chloride and 70% alcohol. Protocol S2 had 8% Sodium hypochlorite, 0.1% mercuric chloride and 70% alcohol. Tested parameters included viability, contamination, callus number, callus width, and callus length. Protocol S1 resulted in a higher percentage of viable, green explants (90% ± 1.14; p= 0.0788) and significantly lower contamination rates (45% ± 3.37; p= 0.0461) compared to S2. Among the cytokinin treatments, 1.5 mg/L BAP induced the highest number of callus regeneration (1.8 ± 0.13; p=0.0006) followed by 1.0 mg/L (1.5 ± 0.22). In terms of callus morphology, the 1.5 mg/L BAP treatment resulted in the highest callus width (4.78 mm ± 0.62; p=0.008) and the highest callus length (3.47 mm ± 0.55; p=0.0107), significantly outperforming the lower cytokinin concentrations. The results indicate that higher cytokinin concentration of 1.5 mg/L boosts morphogenic responses consistently yielding the most robust callus regeneration in Z. mauritiana ‘Local Masan’. These findings highlight the importance of optimizing hormonal levels and sterilization conditions in improving in vitro culture success for Z. mauritiana.