Establishment of Micropropagation Protocol for Double Petunia “Pink Diamond” from Leaf Tissues

Petunias are ornamental plants of high economic interest. A micropropagation protocol from leaf tissues of Petunia hybrid cv. ‘Pink Diamond’ was established in this study. Murashige and Skoog (MS) medium supplemented with 1 mg L⁻¹ BA and 30 g L⁻¹ sucrose was the most suitable for callogenesis and shoot regeneration from leaf explants. “Pink Diamond” cultured on this medium showed the highest callus induction rate (94.44%), the maximum shoot regeneration percentage (83.33%), the highest multiplication rate (15.28 shoots/explant), and the best quality of shoots. For the growth of shoot tip-derived shoots, MS medium fortified with 40 g L⁻¹ sucrose and 10% coconut water was the optimal medium, achieving the highest shoot length (7.78cm) and the largest number of leaves (16.89 leaves/shoot). Meanwhile, MS medium containing 40 g L⁻¹ sucrose and 15% coconut water was optimal for the growth of node-derived shoots, resulting in the largest leaf size (1.88cm in length and 0.75 cm in width) and the highest number of leaves (10.83 leaves/shoot). MS medium supplemented with 30 g L⁻¹ sucrose and 0.1 g L⁻¹ AC was the most suitable for rooting, achieving the highest shoot length (3.22cm), the largest number of leaves (7.06 leaves/plantlet), and the highest number of roots (16 roots/plantlet). The in vitro plantlets transplanted to 100% peat moss were successfully acclimatized under poly-house conditions, with an 88.39% survival rate. These plantlets reached 8.16 cm in plant height and 15.35 leaves per plant after four weeks of being transferred.