Crinum moorei : propagation and secondary metabolite production in vitro.
As an alternative to conventional methods of vegetative propagation, micropropagation attracts much attention, because the levels of multiplication are increased, somaclonal variation is limited and disease-free material can be obtained. The technique is invaluable to the conservation of Crinum species belonging to the Amaryllidaceae which, as a group, possesses several biological features that make them particularly vulnerable. This is in addition to other problems relating to their value as horticultural material, traditional medicines and sources of phytochemicals of interest to medical science. Two in vitro systems are widely used for the propagation of amaryllidaceous species; regeneration from young floral stem explants and from twin-scales excised from bulbs. Although plantlet regeneration could be obtained from peduncle explants of Crinum moorei, a complex of factors including: the age of the floral stem; explant position and; hormonal factors, limited growth. Callus production was poor and indirect organogenesis could not be achieved. Twin-scales were used for the induction of somatic embryos. Morphologically these were different depending on the concentrations of 2,4-D and BA used in the induction medium. Although some of them went on to germinate, the use of somatic embryos for large-scale culture is not an efficient micropropagation route, owing to the low frequency of both embryo production and germination and to the long culture times. Regeneration of shoots and bulblets could, however, be readily induced from twin-scales using a series of modified MS media, and this despite the fact that explants from the bulb were more difficult to decontaminate than the above ground parts. Shoots arose in the axes of the twin-scales close to the basal plate. Initiation was greatest on a basic Murashige and Skoog medium, containing 4 g ℓ ¯¹ sucrose, and in the dark. No hormones were required. At high concentrations, the hormones stimulated abnormal organogenesis. Bulbing of the shoots was further enhanced using higher than normal levels of sucrose i.e. 6% and 5 g ℓ ¯¹ activated charcoal. The response was also influenced by the size of the twin-scale and its position in the parent bulb. Greater numbers of bulblets with larger diameters developed in large twin-scales from an intermediate position between the inner and outer scales. Furthermore, light, and a temperature of 25°C were required for normal bulblet development. Bulblets grown in this manner were used as a source of secondary explants by splitting them vertically in half. The addition of 10 mg ℓ ¯¹ BA resulted in multiple shoot development. In a liquid-shake culture system, this same multiplication medium induced the formation of meristemoid clusters whose rates of proliferation were higher than that achieved for shoot multiplication on either solid or static liquid media. The advantage of using meristematic clusters is that shoot hyperhydricity is avoided. Furthermore, the clusters can be mechanically separated; making the system ideal for automated plant production. Shoot morphogenesis, followed by the formation of bulblets occurred on solid MS media containing activated charcoal or high concentrations (6%) of sucrose. The induction of bulblets by sucrose was, however, slower, which may be beneficial for long-term storage and conservation ex situ. Compared to smaller bulblets, bulblets with a diameter of approximately 9 mm, acclimatized readily and grew rapidly after transferring them to the soil in greenhouse conditions. Biotechnological processes such as cell and tissue culture provide an ideal system for producing secondary metabolites, especially where their production in situ is hampered by poor resource availability or when chemical synthesis is difficult. In vitro produced Crinum moorei bulblets were found to contain nine alkaloids of the Amaryllidaceae type; three of which were released into the culture medium. Light was essential for alkaloid biosynthesis while the inclusion of BA and activated charcoal stimulated the production of specific alkaloids.