In vitro organogenesis of jojoba has been attempted using a variety of explants, and highly concentrated nutrient media similar to Murashige and Skoog medium. This study found that, unlike many other woody plant species, jojoba tissue is very sensitive to high concentration of mineral salts. Modified versions of Woody plant medium, and Lyrene medium were very successful for growth and proliferation of shoot apices. A multiplication rate of 50 shoots per original explant in 3 months was achieved in the presence of high concentration of Ca (22 meg/L), and an auxin cytokinin ratio of 20 (2 mg/L of BAP, and 0.1 mg/L of NAA). An average of 5 roots/shoot were obtained when the base of the shoots were wounded prior to treatment with 100 ppm IBA solution for 5 sec, and then subsequently cultured on a MS medium containing 10 mg/L IBA, and 0.1 mg/L NAA. (Abstract shortened with permission of author.)

This thesis describes experiments designed to improve microtuberization efficiency, and to evaluate a range of dormancy-breaking agents for microtubers and minitubers. Provision of continuous darkness, agitation to cultures, mechanical resistance to stolons, or lower levels of medium nitrogen did not improve microtuberization. The 16/8 h d/n cycle at step 1 of microtuberization was significantly better than 12/12 or 8/16 h d/n cycles based on microtuber yield. Cultures exposed to prolonged step 2 or 2 successive harvests, rather than a single harvest at 30--35 d, had significantly improved microtuber yield. In a series of chemical and mechanical treatments applied to microtubers and minitubers, with or without variable periods of cold storage, 500 mg l -1 GA3 was the most efficient in breaking-dormancy and inducing precocious sprouting. GA3 was the only agent that was able to break dormancy of minitubers that had not been cold stored. After 2 weeks of cold storage, minitubers treated with GA3 also broke dormancy, while Signal was less effective in promoting sprouting. (Abstract shortened by UMI.)

No description available.

In South Africa, large quantities of Scilla natalensis are harvested from wild populations and sold as traditional medicine, which is reducing the density, distribution and genetic diversity of wild populations. The enforcement of existing legislation, however, has proved ineffective with plants being traded locally and internationally. It has therefore, been suggested that ex situ conservation through cultivation may alleviate pressures on natural resources. Conventional propagation of these plants, however, is usually fairly slow. In vitro propagation provides a rapid means of propagating selected chemotypes or cultivars, serving both conservation and commercial interests. In the first part of the study, continuous culture systems were established for the three forms of Scilla natalensis, S. natalensis sensu stricto (Form A), S. natalensis syn. S. kraussii (Form B) and S. natalensis syn. S. dracomontana (Form C). The efficiency of the systems was strongly influenced by genetic factors, viz the form and epigenetic factors, viz the explant type, carbohydrate source, plant growth regulators and gelling agents. The form, Form A, Form B or Form C respectively, influenced shoot initiation with the larger forms generally producing more shoots than the smaller forms (Form A > Form B > Form C). The data confirmed that the three forms are significantly different in terms of their physiological response to carbohydrates, plant growth regulators and gelling agents in vitro. Since the effect of form could not be compensated for by the addition of either carbohydrates, plant growth regulators or gelling agents, this may provide some support for the reinstatement of these forms as three species, Scilla natalensis Planch., S. kraussii Bak. and S. dracomontana Hilliard & Burtt. The explant type, that is bulb or leaf explants respectively, significantly influenced shoot initiation. Leaf explants generally produced more shoots than bulb explants. The carbohydrate source significantly influenced shoot initiation. The explants generally produced more shoots when cultured on media containing glucose or sucrose than on media containing fructose, lactose, maltose and particularly mannitol. The combination of cytokinins and auxins significantly influenced shoot initiation. Shoot initiation was higher for combinations of kinetin: IAA than for combinations of kinetin: NAA or TDZ: NAA. Optimal shoot initiation for Form A, Form B and Form C occurred on media containing 1 to 2 mg I-1 kinetin and 1 to 2 mg I-1 IAA. The gelling agent also influenced shoot initiation with media solidified with Gelrite® producing more shoots than media solidified with Oxoid or Unilab agar. Shoots were then rooted on media containing IAA, IBA or NAA and the plantlets were successfully acclimatised. These continuous culture systems can be used to produce large quantities of plantlets, which may alleviate pressures on natural resources and provide an alternative source of high quality plants for the burgeoning medicinal plant market. In the second part of the study, the effect of carbohydrate source and concentration on growth and development of shoots of S. natalensis syn. sensu stricto (Form A) were determined. This has applications for the acclimatisation and germplasm storage of bulbous plants. The carbohydrate source and concentration significantly influenced the growth and development of shoots. In the absence of carbohydrates, the shoots were short with spindly leaves and short roots. When media were supplemented with high concentrations of fructose, the shoots were long with broad leaves, small bulbs, and few short to medium length roots at low concentrations. At higher fructose concentrations, however, the shoots were robust and short with narrow, sometimes deformed leaves, large bulbs, and few stunted, brown roots. When sucrose was substituted for fructose, the shoots were robust and long with narrow and often red-pigmented leaves, large bulbs, and many long, thick roots. When AC was used in combination with sucrose, however, the shoots were robust and short with few, and occasionally red-pigmented leaves, small to medium bulbs, and few, severely stunted roots. Optimal shoot growth and development in terms of shoot weight (FW) and quality occurred on media containing glucose or sucrose (40 to 60 g I-1). The carbohydrate source and concentration also significantly influenced the physical properties of media particularly pH, electrical conductivity (EC) and gel-strength. The pH decreased slightly with increasing glucose concentration but decreased significantly with increasing fructose concentration when fructose was used alone or in combination with glucose. The pH also decreased significantly with increasing sucrose concentrations when sucrose was used in combination with Sigma AC. The EC decreased significantly with increasing fructose concentration when fructose was used alone but remained fairly constant irrespective of glucose concentration when glucose was used alone or in combination with fructose. The EC also remained fairly constant irrespective of the sucrose concentration but decreased with increasing sucrose concentration when used in combination with AC. The gel-strength remained fairly constant irrespective of glucose. The gel-strength decreased with increasing fructose concentration when used alone or in combination with glucose. The gel-strength of media increased with increasing sucrose concentration although the addition of Sigma AC significantly decreased the gel-strength of media, which decreased with increasing sucrose concentration. The brand and concentration of AC also influenced gel-strength. The matrix plot suggested that the effect of carbohydrate source and concentration on the growth of shoots may be largely due to the indirect effects of these physical properties such as hydrolysis of carbohydrates, the spectrum and quantity of the breakdown products and the availability of nutrients, plant growth regulators and water rather than the direct effects of pH, EC and gel-strength per se. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1999.

Cell division was obtained from cultured microspores of Douglas-fir on medium supplemented with auxin, cytokinin, and sucrose, but without medium salts. Embryogenic callus was initiated from excised mature and immature zygotic embryos of Douglas-fir on media supplemented with cytokinin and auxin. Precotyledonary embryos produced most of the embryogenic calli in the cultures. Secondary embryogenic callus production, and subsequent subculturing, were required for the establishment of stable embryogenic callus lines for both mature and immature zygotic embryos. Somatic embryos at the precotyledonary stage were obtained in high frequency when Douglas-fir embryogenic callus was transferred onto hormone-free medium supplemented with 1% activated charcoal, while some cotyledonary somatic embryos were obtained from hormone-free medium supplemented with low ABA levels (0-10 uM). The level of ABA in the maturation medium significantly affected the quality of the somatic embryos produced. Cell suspensions were established from embryogenic calli and have been maintained for over one year. Protoplasts were isolated from suspension, cell colonies and calli were regenerated from protoplasts. GUS and CAT genes were successfully introduced into protoplasts of Douglas-fir via electroporation, and their transient expression was obtained 2-4 days after electroporation. The results so far indicate that the production of somatic embryos via embryogenesis in vitro is obtainable, and the application of direct gene transfer via electroporation for genetic engineering of trees in this species is promising. / Forestry, Faculty of / Graduate

The objective of the present study was to develop a comprehensive and reproducible regeneration system for spinach (Spinacia oleracea L. ) from commercially important cultivars and to assess the potential use of spinach for Agrobacterium tumefaciensmediated transformation. Tissue cultures of spinach were initiated from seed material. Axenic shoot cultures of spinach were established on MS-based medium containing 1.0 VM NAA at a temperature of 15°C and under a 16 h photoperiod. These three parameters were found most suitable for the establishment of shoot cultures and the encouragement of axillary shoot growth. Attempts to enhance axillary shoot production of spinach were investigated by the use of a double phase culture system, employing semi-solid and liquid culture media. The application of liquid medium was feasable with a volume of 5 ml for a duration of 7 or 14 d or with a volume of 10 ml for a duration of 7 d, but the multiplication rate of spinach was not increased. Adventitious shoot production was initiated from cultured spinach root explants derived from axenic shoots or hypocotyl explants. Sections from root tips and middle segments exhibited the highest shoot regeneration capacity when cultured on Nitsch and Nitsch (1969) medium supplemented with 20 μM NAA and 5.0 QCM GA3. Histological analysis demonstrated that the regenerating shoots originated directly from the root explants. Adventitious shoots were rooted on MS-based medium containing 1.0 μM NAA and transferred to the glasshouse, where the plants were grown to maturity. Seeds collected from regenerated plants were 95 % viable, producing a homgenous, fertile Rl-generation. Flow cytometric analysis was used to determine ploidy levels of regenerated plants and their progenies and showed that spinach leaf tissue from all generations displayed an even proportion of Go/G1 cells and G2/M cells, which may be characteristic for this species. Transformation studies using in vitro derived spinach explants demonstrated a positive response using two strains of Agrobacterium tumefaciens. The highest transformation rate was achieved with 25 % of explants being GUS-positive, therefore confirming susceptibility of spinach to the binary vector containing both T-DNA border sequences. It was found that best results were obtained with root explants which had been incubated for 8 weeks prior to co-cultivation with Agrobacterium and in vitro material which had been maintained in culture for up to 2 years. This reproducible regeneration system for spinach and the demonstration that spinach is amenable to Agrobacterium-mediated transformation provides the basis for potential commercial application within spinach breeding, aiming to generate an improved crop plant.

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No description available.

No description available.

The development of the science of micropropagation from the very initial concept of totipotency to the modern day advancement and sophistication has been affected by a wide range of problems such as hyperhydricity, shoot-tip necrosis and somaclonal variation. These problems are largely the result of the obvious fact of trying to grow plants in an environment that is different from the one plants are used to naturally. The extent of these problems ranges from minor technical inconvenience to significant economic loss. Characterization and control of micropropagation problems has been one of the priorities of plant tissue culture research due to the enormous contribution of this discipline for plant production, improvement and conservation. The prevalence and severity of these tissue culture problems varies widely among plant species. The rationale of this research project was therefore, to identify plant species most affected by the problems studied, characterize the problem and find mechanism(s) to control or minimize the damage caused by the problem. The literatures reviewed provide sufficient background information for the experimental chapters. Due to the different nature of the problems and variation in the plant species they affect, the model plant, the methodologies used and parameters analysed were also different. The findings of these investigations, in their own different way, addressed certain problems that individually and collectively pose difficulties to the micropropagation industry. The difference in the content of the experimental chapters is therefore the result of the broader objective of the research project to tackle such difficulties. The success and failure of tissue culture system greatly depends on the choice of PGR’s. This choice can be made based on comparative study of their biological activity. Some promising reports on the role of topolins in micropropagation led to the idea of testing these cytokinins for their potential in tissue culture. As a prerequisite to subsequent investigations, the biological activity of some selected topolins and BA derivatives was tested using the soybean callus bioassay. The activity of the cytokinins tested varied significantly. The results demonstrated that the structure of a cytokinin dictates its activity. Modifications of side-chain improved the activity of oT but had no effect on pT. The presence of the methyl group had an enhancing effect on cytokinin activity of topolins or at least it did not reduce it. BA derivatives BA9THP (conjugated at N9 position), 3FBA and 2Cl6(3OHBA)R (halogenated derivatives) also showed good cytokinin activity and hold good promise for future research. In an attempt to alleviate hyperhydricity in Aloe polyphylla and optimize the micropropagation protocol, meta-topolin and its derivatives were tested at various concentrations together with BA and zeatin. Of all the cytokinins tested mT produced the best results in terms of shoot and root growth. Five μM was found to be the optimum concentration at which complete control of hyperhydricity was achieved without compromising shoot and root growth. Plantlets rooted in a multiplication media. BA generally had a negative effect on growth and development both in vitro and ex vitro. Acclimatization of plantlets was achieved easily by initially transferring plantlets to a mist house (for three weeks) followed by transfer to the greenhouse. The type of cytokinin also had an effect on ex vitro growth with BA-treated plants producing the lowest shoot and root biomass. Various experiments were conducted to characterize and control factors affecting STN in Harpagophytum procumbens. Media type and strength, PGR, carbon sources, sub-culturing, calcium and boron were tested. Results indicated that all of the tissue culture components tested affected STN. From the different media types tested, half strength was MS found to be the preferred medium. Increasing cytokinin concentration increased the incidence of STN and the problem was aggravated by the addition of auxin to the multiplication medium. Optimum shoot multiplication was achieved by omitting auxin and using the cytokinin mTR. Plantlets produced basal callus which interfered with rooting. The quantity of this basal callus was minimum when mTR was used. Sub-culturing plantlets onto fresh medium every two weeks helped minimize STN. Off all the sugars tested 3% sucrose was optimum. Other sugars either aggravated STN or inhibited growth when compared at equi-molar concentration. Increasing the concentration of either Ca or B prevented the development of necrotic shoots. When the concentration of both elements is increased simultaneously negative effects on both growth and STN were observed. Using 6 mM Ca in half strength MS medium was optimum. B was toxic at higher concentrations. Plantlets rooted readily in half strength cytokinin-free MS media supplemented with 2.5 μM IAA. Rooted plantlets produced using the optimized protocol were acclimatized successfully by transferring directly to a greenhouse in a 1:1 ratio of sand and soil mixture. The effect of meta-toplins on micropropagation and somaclonal variation of banana was investigated. Tissue cultured explants of cultivars ‘Williams’ and ‘Grand Naine’ were cultured in MS media containing the cytokinins BA, mT, MemT, MemTR and mTR at various concentrations. Results of the investigation revealed that superior multiplication and lower abnormality index was recorded from the mTR and mT treatments at 22.2 μM concentration. These treatments, however, had an inhibitory effect on rooting. The effect of these treatments (22.2 μM mT and mTR) in comparison with equi-molar concentration of BA on somaclonal variation of ‘Williams’ banana was tested using RAPD-PCR at the 7th multiplication cycle. No significant difference was found between the treatments. It should however be highlighted that cultures were initially maintained for three multiplication cycles in media containing BA. The inherent stability and initial effect of BA could have influenced the results. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2008.