A protocol is presented for the rapid (28 days) induction of microtubers on micropropagated layered potato plantlets of 'Kennebec', 'Russet Burbank' and 'Superior' in medium devoid of growth regulators. With this method the addition of coumarin, 6-(2-chloroethyl)-trimethylammonium chloride and 6-benzylamino-purine to the microtuberization medium either had no effect or significantly reduced microtuber weight per plantlet. Increasing the incubation period from 28 to 56 days significantly increased the weight of microtubers per plantlet and the proportion of microtubers heavier than 1 gram. Increasing the volume of microtuberization medium from 50 to 100 ml significantly increased the number of microtubers per plantlet. Microtuber dormancy periods were cultivar-specific and microtubers $ le$250 mg had longer dormancy periods as compared to microtubers $>$250 mg. A positive correlation was established between endogenous abscisic acid levels and microtuber dormancy periods. Microtubers $ le$250 mg had lower specific gravity, fewer eyes and produce fewer sprouts than microtubers $>$250 mg. Microtuber-derived plants were generally single-stemmed. Severe physiological ageing treatment ($>$2500 degree-days) had no effect on microtuber sprout development, stem number, tuber number and only minimally influenced tuber weight of microtuber-derived plants. Decreasing field in-row planting density from 30 to 10 cm reduced tuber weights and numbers per plant but increased them on a per hectare basis. Economic analysis indicated that optimum planting density varied depending on plantlet cost. The optimum planting density was 10 cm if the cost of the plantlet was $0.10 or less, 20 if plantlet cost were from $0.10 and $0.20 and 30 cm for plantlet cost greater than $0.20. A potato seed tuber certification program adapted to the needs and constraints of Egypt is presented.

Various aspects of Ca2+ uptake into greenhouse-grown potato plants and micropropagated potato plantlets were examined, including the movement of Ca2+ into potato plants and tubers, identification of Ca-packing genotypes and assessment of the effect of improved medium Ca2+ level on different parameters such as plantlet growth, microtuber induction, yield and tissue Ca concentration. The effect of exogenous vitamin Ds and UV-elicited endogenous vitamin D synthesis on regulation of 45Ca 2+ uptake into plantlet shoots was also examined. Safranin dye was not as suitable as the tracer 45Ca2+ for monitoring translocation of Ca2+ into tubers. Uptake of Ca2+ into tubers occurred indirectly through the main basal roots and stolon roots and directly through tuber periderm. Liquid scintillation counting and flame atomic absorption spectrophotometry were used to screen six potato cultivars and two wild species for ability to take up Ca2+ from treatment solutions containing high or low Ca2+ levels. In vitro potato micropropagation, microtuberization, and tissue calcium concentration were compared for six cultivars when Murashige-Skoog basal medium Ca2+ level was increased from 3, to 5 or 15 mM. All aspects of growth including shoot dry weight, early microtuber induction, microtuber yield and tissue Ca concentration were improved when medium Ca2+ level was 15 mM. Cultivar Bintje was the most efficient genotype at accumulating Ca 2+ from treatment solutions or growth media containing high or low Ca2+ levels. Vitamin Ds improved 45Ca 2+ uptake into shoots of micropropagated potato plantlets and D 3 was more efficient in this regard than D2 or a combination of D3 and D2. Plantlets had increased 45Ca 2+ uptake when exposed to UV irradiation compared with the untreated control plantlets. Plantlets given a 24 hour interval in the dark after UV exposure had even greater 45Ca2+ uptake, suggesting that vitamin D, metabolites, specifically calcitriol (1, 25(OH)2-v

Micropropagation was investigated in order to develop protocols for rapid mass production of shoots ofSorbus aucuparia and Alnus glutinosa. Removal of apical dominance either physically by pruning the plantlets or chemically by using anti-auxins TIB A (2,3,5-triiodobenzoic acid) and NPA (1-naphthylphthalamic acid) was investigated. There was a 6-fold increase in the number of rooting-ready shoots of S. aucuparia produced by the pruning of plantlets grown in vitro. A. glutinosa however, needed more drastic measures to remove apical dominance and block the endogenous auxin transport. Incorporation of TIBA (3 μm) in the medium produced an initial 8- fold increase in the number of shoots. However, repeated subculture of shoots of Alnus on TIBA containing medium proved detrimental to shoot multiplication. There was 100% rooting of shoots of S. aucuparia on agar solidified medium. The auxin: cytokinin ratio of the multiplication medium played an important role in the rooting ability of shoots. A. glutinosa also had 100% rooting on agar solidified medium. Plants were acclimatised in Baumgartner vessels before transferring to soil. There was 100% rooting and survival of the shoots of A. glutinosa both after transfer to Baumgartner vessels and subsequent transfer to soil. In S. aucuparia the survival rates in Baumgartner vessels was 70% and after transfer to soil was 65%. Direct somatic embryogenesis from zygotic tissue of both S. aucuparia and A. glutinosa was achieved. Embryos of S. aucuparia were produced on medium containing MS salts and vitamins supplemented with 1 μM BAP, 1 μM kinetin, 0.5 μM NAA, 250 mg/L L-glutamine and 500 mg/L casein hydrolysate. A. glutinosa embryos were obtained on medium containing salts and vitamins of Driver and Kuniyuki (1984) supplemented with 3 μM BAP. No auxin was required. Adventitous shoot regeneration from leaves of S.aucuparia was also achieved at a frequency of 40% on medium containing MS salts and vitamins supplemented with 10 μM TDZ and 1 μM NAA. A method for chromosome doubling of S. aucuparia using 15 uM pronamide to treat shoot tips immersed in a semi-solid medium was developed. After 14 days of treatment, 86.5% treated shoots survived and there was 44.5% chromosome doubling of the survivors. The tetraploid shoot had a higher rate of multiplication than the diploid shoots. The involvement of extracellular proteins in direct somatic embryogenesis of Prunus 'Colt' was studied. Changes in the expression of proteins were observed from the first day of transferring the tissue to embryo induction medium. Most changes were seen on the days 28 and 35 when the embryos became visible.

Micropropagated potato (Solanum tuberosum L.) plantlets are routinely used for producing specific pathogen tested minitubers in the Canadian seed tuber certification industry. In vitro hardening methods for micropropagated 'Norland' were investigated, involving full and half strength Murashige and Skoog (1962) based propagation medium supplemented with NaCl, KCl, CaCl2, polyethyleneglycol, or paclobutrazol. Ten different media treatments were compared for their effect on stomatal function and early transplant performance using porometry, microscopy, and direct ex vitro transplanting. PEG, NaCl and 1/2 MS + 3 mM Ca treatments did lead to decreased leaf water losses. However, these treatments did not improve ex vitro transplant performance compared to controls. Minituber production was investigated using ex vitro plantlets in a rockwool-based hydroponic system. Productivity was evaluated for treatments involving photoperiod pretreatment, planting orientation, planting density, and hilling. No difference in total yield was detected when plantlets were exposed to 12 compared with 16 hour photoperiod pretreatments. However, short photoperiod pretreatment increased the number of minitubers in the most desirable size range. Increased planting density reduced yield per plant. However, small increases in yield per m2 occurred with increased planting density. Hilling, pinching, and planting orientation had no effect upon minituber size, number, or overall fresh weight yield. Significant differences in minituber yield occurred in field experiments. Larger minitubers (10--40 g) had larger yields compared with smaller minitubers (1--5 g). These results will contribute to improved minituber production technology for the Canadian certified potato seed tuber industry.

M.Sc. / Ophiopogon planiscapus var. nigrescens belongs to the family Convallariaceae. This plant exhibits unique black pigmented leaves and is, therefore. used in growing arrangements. The natural rate of multiplication. however. is slow and a reversion to green leaves in some plants necessitates the replacement of these plants, which is a time consuming activity. In vitro techniques allow the establishment of black clones and also speed up the multiplication process. A cultivation medium for optimal growth was formulated. The influence of other parameters on growth. multiplication and rooting was investigated and final hardening was done to yield plants suitable for the greenhouse.

The work on Spiraea in vitro shoot cultures was done to determine the feasibility of using light quality to modify endogenous phytohormone balances to decrease apical dominance. Such an effect would enable a reduction in the high levels of exogenous cytokinin benzyladenine (BA) applied in culture and thus reduce potential side-effects. The Spiraea in vitro light quality response was characterized by examining the effects of different light wavelengths on growth. A mixture of red/FR induced rates of shoot proliferation with 0.25 mg/1 BA that were as high as rates obtained under white light with 0.5 mg/1 BA. Shoot quality, as determined by the proportion of shoots 1 cm or longer (useful shoots), was highest under red/FR light. The lowest shoot proliferation rate was observed under blue light. When light wavelengths intermediate between blue and red light (green, yellow, and orange) were applied to explants only minor growth modifications occurred. Green light did not inhibit shoot initiation but inhibited shoot elongation at the 0.5 mg/1 BA level. The efficacy of the light source-filter combinations in the first experiment was studied in two further experiments. With the three light sources (tungsten filament, fluorescent, and metal halide) together with a blue filter, results supported the putative blue light inhibitory effect suggested in the first light quality experiment. Under the red filter, the tungsten filament source induced the highest shoot number means at both BA levels used (0.25 and 0.5 mg/1). Two factors may have contributed to the red/FR effect observed in the first experiment; the time under an incubation light regime before transfer to the treatment regime, and the photon fluence rate of each regime. In the subsequent study to examine these factors, shoot initiation was optimized at the lower BA levels of 0.25 and 0.4 mg/1 when cultures under low fluence red/FR were transferred after four weeks to white light of a higher fluence for one more week. Glyphosate, a known promoter of IAA oxidation, was used to investigate the presumed effect of lowered IAA-cytokinin interactions. Two types of responses to glyphosate occurred, each one dependent on the glyphosate concentration. At the lower glyphosate level (0.087 mg/1), cultures under both light regimes with 0.25 mg/1 of BA, showed a strong inhibition of shoot initiation. This inhibitory effect was overcome in cultures with 0.5 mg/1 of BA and an overall stimulatory response occurred as shoot initiation rates were as much as four-fold higher than in the previous experiments. For both BA levels, changes in shoot number were greater under white light than under red/FR. At the higher glyphosate level (0.2 67 mg/1), the shoot initiation rates were greater than glyphosate-free controls for both BA levels under white light although under red/FR the rates were virtually unchanged from controls. The glyphosate effect investigated for Spiraea cultures appears to be influenced by the levels of the cytokinin BA resulting in pleiotropic effects which depend on the specific concentrations of each component. / Land and Food Systems, Faculty of / Graduate

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Shoot cultures of Aloe polyphylla were initiated from young shoot explants of in vitro grown plants. The basal medium was MS medium (MURASHIGE and SKOOG, 1962), supplemented with 100 mgl ¯¹ myo-inositol, and 30 gl ¯¹ sucrose. Agar (0.8 %) was used as the gelling agent. Different cytokinins, singly or in combination with auxins (IBA and NAA), were tested for shoot proliferation activity. All the cytokinins tested (kinetin, zeatin, iP, and BA) gave a good shoot proliferation response. The optimal concentrations for shoot proliferation of each of the cytokinins tested were: zeatin (0.5 mgl ¯¹), kinetin (1.5 mgl ¯¹), iP (1.0 mg ¯¹) and BA (1.5 mgl ¯¹). In combination with auxins, the optimal combinations were kinetin/NAA (2.0/0.1 mgl ¯¹), kinetin/lBA (1.5/1.0 mgl ¯¹), zeatin/lBA (1.0/0.5 mgl ¯¹), zeatin/NAA (1.0/1.0 mgl ¯¹), BA/IBA (1.0/1.0 mgl ¯¹), BA/NAA (1.5/0.1 mgl ¯¹). Although it gave the highest number of shoots per explant, BA was responsible for hyperhydricity. Temperature and sucrose also influenced shoot proliferation. The optimal temperature was 25°C, while 30 gl ¯¹ was the optimal concentration of sucrose for shoot proliferation. Plants rooted well in plant growth regulator-free MS medium. Amongst the potting mixtures tested, soil: sand: vermiculite (1:1:1 v/v) was the best with 98 % plantlet survival. In the second part of this project, Platycerium bifurcatum cultures were established using leaf explants. The basal medium was MS medium (MURASHIGE and SKOOG, 1962), supplemented with 100 mgl ¯¹ myo-inositol and 30 g l ¯¹ sucrose. For bud initiation, 1.0 mgl ¯¹ BA was used, while 0.8 % agar was used as the gelling agent. Three different strengths of MS medium (full, half, and one-quarter strength) without plant growth regulators were tested for further bud growth and development. Half-strength MS proved to be the best for further bud growth and development. Rooting was best achieved in one-quarter strength MS medium without plant growth regulators. In vitro grown plantlets were successfully acclimatized using peat as the potting medium. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2001.