Evaluation of the efficiency of the optimal combination of  hormonal, physical  and biological stimuli in the induction and growth of callus in Lavandula angustifolia Mill

<ns5:p> Background Lavender is a crucial source of secondary metabolites with medicinal and industrial importance. Its exploitation faces challenges due to the limited availability of raw materials and their sensitivity to climate. Tissue culture techniques, particularly callus culture, represent the most sustainable method for the continuous production of these active compounds. Methods This study focused on developing an effective protocol for inducing and stimulating callus cultures in lavender plants. Callus formation was stimulated from leaf tissue has cultured on MS medium supplemented with different concentrations of 2, 4-D (0, 1, 2 and 3) mg L <ns5:sup>−1</ns5:sup> . The cultures were incubated under light or dark conditions. In the multiplication: calli (Cultured in MS medium containing 1 mg, 2,4-D incubated in the dark) was transferred to MS medium containing different 2,4-D (0, 1, 2 and 3 mg.L <ns5:sup>−1</ns5:sup> ) and BA (0,1 and 2 mg.L <ns5:sup>−1</ns5:sup> ). The multiplied callus (Cultured in MS medium containing 1mg L <ns5:sup>−1</ns5:sup> 2, 4-D and 2 mg L <ns5:sup>−1</ns5:sup> BA) was transferred into two nutrient media, MS and B5, each supplemented with different levels of chitosan (0, 50, 100, and 200 mg.L <ns5:sup>−1</ns5:sup> ) along with combination of 1 mg.L <ns5:sup>−1</ns5:sup> 2, 4-D and 2 mg.L <ns5:sup>−1</ns5:sup> BA. Results The results indicated that the optimal concentration for callus induction was 1 mg L <ns5:sup>−1</ns5:sup> of 2,4-D, with the highest callus formation rate 95% recorded under dark conditions. Among all treatments, the combination of 2, 4-D 1 mg L <ns5:sup>−1</ns5:sup> and BA 2 mg L <ns5:sup>−1</ns5:sup> was the most effective for sustainable callus growth. Regarding the nutrient medium, B5 was more efficient than the MS medium. For the biostimulants, Chitosan at 100 mg L <ns5:sup>−1</ns5:sup> recorded the highest efficiency in both fresh and dry callus weights. Conclusions This study underscores the necessity of integrating hormonal, nutritional and environmental factors for optimal callus production. </ns5:p>