| Summary: | 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. === Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1984.
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