A Study on the Optimization of Callus Culture and Shoot Organogenesis in Pinus sylvestris L.: Effects of Plant Growth Regulators and Antioxidants

Scots pine (Pinus sylvestris L.) is an economically important conifer; however, its in vitro application is limited by rapid callus browning and loss of morphogenic potential. This study aimed to overcome these limitations by optimizing the basal medium, plant growth regulators, and antioxidant and organic supplementation. Seeds were germinated on half-strength Murashige and Skoog (½ MS) medium supplemented with 50 mg·L−1 gibberellic acid (GA3). Callus formation was initiated from cotyledons and hypocotyls of 3-week-old seedlings. Gamborg (B5) medium was unsuitable, causing rapid browning and necrosis. The most effective medium for prolonged proliferation (up to four weeks without subculturing) consisted of ½ MS supplemented with 3 mg·L−1 2,4-dichlorophenoxyacetic acid (2,4-D), 0.8 mg·L−1 6-benzylaminopurine (BAP), 500 mg·L−1 casein hydrolysate, 500 mg·L−1 L-glutamine, 200 mg·L−1 ascorbic acid, 5 mg·L−1 sodium diethyldithiocarbamate (Na-DDC), and 20 g·L−1 sucrose. This medium extended callus viability to four weeks, markedly reduced browning, and induced morphogenesis. Upon transfer to shoot-inducing medium (½ MS supplemented with 3 mg·L−1 BAP, 0.1 mg·L−1 α-naphthaleneacetic acid (NAA), and the same additives), 57.8 ± 1.4% of calli with green foci produced shoots. These results provide a basis for prolonged callus cultivation and shoot organogenesis in Scots pine. Rooting and acclimatization were not evaluated and remain to be optimized.