The regeneration of Hypoxis rooperi S. Moore and production of hypoxoside in vitro.
Against the background of the increasing pharmaceutical importance of members of the genus Hypoxis L., methods for propagating these plants and for producing hypoxoside (the believed active compound found within Hypoxis species) using in vitro techniques, were investigated. These investigations were accompanied by anatomical observations. Hypoxis rooperi S. Moore was selected as experimental material because of its availability and common usage among researchers studying the genus Hypoxis. Two aseptic procedures were developed for propagating H. rooperi. These being the only procedures as yet to be established and documented, using a member of the family Hypoxidaceae. The first procedure involved the induction of callus and adventitious shoots from flower bud explants of H. rooperi . For this response to be initiated, the buds selected for culture had to be of a specific morphological and physiological age. The best medium determined for inducing a callusing and shooting response from these explants, was a MURASHIGE and SKOOG (1962) medium supplemented with low levels of I-naphthalene acetic acid and high levels of 6-benzylaminopurine. The rate of this response was enhanced by the wounding of flower bud explants (i.e. by the excision of the perianth segments, stamens and style from the buds). Investigations indicated that callus and adventitious shoot formation was inhibited by the acropetal positioning of damaged flower buds on the culture medium. This inhibition was not manifest when buds were placed basipetally or horizontally on the culture medium. Flower bud harvest time was not found to have a marked effect upon the numbers of explants responding in culture. On average 37,5 per cent of the buds formed callus and adventitious shoots throughout the flowering season. The subculturing of callus tissue established from H. rooperi flower buds, onto a MURASHIGE and SKOOG (1962) medium supplemented with the same hormone levels as were initially used to induce callus and shoot formation, resulted in the production of multiple adventitious shoots. Serial subculturing of this tissue indicated that the shoot producing capacity of the callus, was maintained for at least a year. Shoots produced via this method, when inoculated onto a hormone-free culture medium, formed roots. Seventy-five per cent of the plantlets regeneratro in vitro were successfully "hardened-off". Theoretically it was calculated that using the micropropagation procedure developed, almost 81000 H. rooperi plantlets could be established from 100 flower bud explants, within a year. The second aseptic procedure developed, involved the culturing of explants excised from the primary thickening meristem region of H. rooperi corms. The best medium determined for inducing the formation of adventitious shoots from these explants, was a MURASHIGE and SKOOG ( 1962) nutrient solution supplemented with: equivalent low concentrations of I-naphthalene acetic acid and 6-benzylaminopurine; 30 rather than 20 or 40 gl¯¹ sucrose; and 1,0 gl¯¹ casein hydrolysate. Random as opposed to a basal or side positioning of corm explants upon the culture medium, resulted in higher numbers of adventitious shoots being produced. The location of explant excision from within the donor plant was also found to influence shoot productivity. No significant difference was detected in the total number of shoots produced from corm explants harvested at various times of the year. The rooting of shoots differentiated from corm explants posed few problems, as most shooted explants eventually formed roots without being subcultured. Those which did not form roots could be induced to do so, by the inoculation of the shooted explants onto a culture medium either devoid of hormones or containing low I-naphthalene acetic acid levels. Following a rather simple procedure developed, ninety per cent of the plantlets were "hardened-off". From 100 corm explants it was therefore possible to regenerate 104 to 112 plantlets within a 3 to 4,5 month period. Prior to the assessment of the usefulness of in vitro cultures for producing hypoxoside, qualitative and quantitative techniques for detecting hypoxoside, were developed. Using these techniques it was established that only the root-like types of cultured tissue, contained hypoxoside. The levels of hypoxoside detected within these tissues were much lower than those found within mature in vivo grown plants. Using the cultured tissue containing the highest levels of hypoxoside, it was shown that the subculturing of this tissue resulted in a decrease in hypoxoside content. This effect could be overcome by lowering the levels of nitrogen in the medium or by culturing the tissue in the dark. These results showed that the cultured tissue was able to synthesize hypoxoside. To what extent this synthetic rate can be increased remains very much an academic problem and one which deserves more attention.