The aim of this project was to investigate the feasibility of the development of in vitro methods for assisting in the plant breeding of the important culinary and medicinal plant, Wasabia japonica Matsumura, through rapid plantlet production and improved plant quality, by the application of micro-propagation and induced mutation techniques. The synthesis and accumulation of a key valuable chemical component was also examined, through callus culture, tissue culture, and induced mutation.
In vitro propagation systems by means of gelled and liquid culture were developed for W. japonica. In vivo dormant buds were used as a primary source of explants, following disinfection in alcohol, NaOCl and detergent. Explants were successfully initiated in culture followed by a rapid shoot multiplication on gelled basal media containing nutrient salts of Murashige and Skoog (MS), vitamins, sucrose and growth regulators. Amongst various cytokinins used (BA, TDZ, kinetin and zeatin) BA proved to be the most effective, with the best concentration at 5 µM. Combinations among growth regulators enhanced shoot proliferation. In order to establish a simple and efficient in vitro propagation protocol, ½MS liquid culture medium with 5°μM BA was used for effective shoot multiplication of W. japonica with reduced labour and cost. The best rooting occurred in shoots cultured on gelled ½MS basal medium supplemented with 10°μM IBA. Over ninety per cent of plantlets were successfully acclimatised to in vivo conditions, exhibiting normal development.
The effects of in vitro mutation induction by physical and chemical mutagens on wasabi shoot tips were also investigated. Gamma rays and X-rays as physical mutagens in the range of 10 to 40 Gy, reduced the allyl isothiocyanate content of in vitro explants, to a greater extent as doses increased. X-ionising irradiation had a more detrimental influence on survival and growth of wasabi tissues than gamma-ionising irradiation. Colchicine and oryzalin as chemical mutagens, however, did not reduce the content of this compound. Oryzalin appeared to be more phytotoxic than colchicine, with a concentration of 15 μM affecting explant growth. Conversely, a low concentration of colchicine at 25 μM appeared to be favourable for the growth and development in vitro. Leaf morphology appeared to be altered in several greenhouse-grown plants treated with chemical mutagens, exhibiting typical characteristics of polyploids. Further studies of morphological effects and assessments of the alteration of allyl isothiocyanate need to be undertaken on in vivo-grown plants.
The yield of allyl isothiocyanate was investigated in calluses, in vitro mutated- and non-mutated explants, and in vivo plants. Light conditions and leaf tissues proved to be more effective than dark conditions and petiole tissues, respectively, for the accumulation of this component in callus cultures. Shoot bases accumulated higher yield than leaves and petioles in the in vitro explants over three months in culture. Similarly, roots and rhizomes produced higher yields than leaves and petioles of in vivo plants over a period of 12 months. Leaves accumulated greater amounts of allyl isothiocyanate than petioles in vitro, but lower amounts than petioles in vivo. Thus it appears that he harvest of allyl isothiocyanate in plant leaves and petioles in vivo could be most effectively commenced after growth for nine months.
These results lay a scientific foundation on which a more elaborated and effective system of in vitro propagation and improvement for W. japonica can be achieved. Further studies on the analysis of chemical components need to be conducted for the pharmacautical uses of this plant.