Efficient plant regeneration from multiple shoots formed in the leaf-derived callus of Lavandula vera, using the "open culture system".
Abstract To develop an efficient procedure for plant regeneration from leaf-derived callus of Lavandula vera DC, the production of multiple shoots and the formation of roots from these shoots were studied. When calli were cultured in a medium with 4.0×10−7 M N-(2-chloro-4-pyridyl)-N′-phenylurea (CPPU), urea-type cytokinin, multiple shoots were obtained from the greenish surface of the callus efficiently at a rate of 52.2%. For multiple shoot development, the shoots were divided into 7–20 shoot clusters with 2–3 shoots and subcultured every 2 weeks. Two modified culture-systems were employed for subculture; a “closed culture system” and an “open culture system”. In the open-system, shoot clusters grew and elongated vigorously. In the closed-system, most of the clusters showed vitrification and did not elongate. In root induction culture, shoot clusters in the open-system were well rooted and grew vigorously, the highest rate of root formation being 74.0%, in the 1 2 MS medium supplemented with 1.0×10−6 M indoleacetic acid (IAA). On the other hand, shoot clusters in the closed-system showed vitrification and did not root well. About seven times more regenerants were obtained from the multiple shoots than from normal shoots treated with 4.4×10−6 M 6-benzylamino purine (BA), a purine-type cytokinin. These results indicate that efficient plant regeneration in L. vera from callus using multiple shoots is possible by means of an open-system.
Plant Regeneration from the Mass of Shoot Primordia in Rhododendron pentaphyllum Maxim. var. nikoense Komatsu
A mass of shoot primordia were induced effectively from the shoot apex of Rhododendron pentaphyllum Maxim. var. Komatsu on WP media containing 30μM 2iP+IAA (10, 30, 100μM). Of the shoot primordia obtained, those cultured on WP medium supplemented with 10μM IAA and 30μM 2iP showed the most active proliferation, being 2-4 times in size, after one month of subculture. When shoot primordia obtained were transferred onto the differentiation media, multiple shoots were formed on WP media containing zeatin (1, 10μM) or 0.1μM CPPU after one month of culture, irrespective of the presence of IAA. The multiple shoots induced on the medium containing 10μM IAA and 10μM zeatin showed active elongation growth. Rooting from the multiple shoots subcultured on the same fresh medium occurred two months after the shoot differentiation.
Plant Regeneration via Somatic Embryogenesis from Cotyledon Protoplasts of Tomato(Lycopersicon esculentum Mill.)
Plant regeneration from cotylendon protoplasts of tomato (Lycopersicon esculentum cv. Kyoryokutoko) was obtained by somatic embryogenesis. Protoplasts were isolated from cotyledons of 8-10 day old in vitro-grown seedlings raised in a controlled chamber (20 °C, 3, 000-4, 500 lux, 16h). During initial protoplast culture, the addition of TM-2 medium (Shahin 1985) containing 0.15M sucrose, at 4 day intervals (sucrose concentrations were progressively reduced, 0.2M-0.17M-0.16M), resulted in vigorous cell divisions and prevented the browning of the cells. Somatic embryogenesis was induced from up to 50% of selected greenish calli when cultured on TM-4 medium supplemented with 2.0 mg/l zeatin riboside. Multiple adventitious shoots were formed by clonal propagation of somatic embryos on TM-4 or Murashige and Skoog (1962) medium containing 1.0 mg/l zeatin and 0.1 mg/l indoleacetic acid. Ninety-eight regenerated plants obtained from rooting shoots on Murashige and Skoog medium (0.8% agar, 20 g/l sucrose) were grown to maturity.