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.

As an alternative to conventional methods of vegetative propagation, micropropagation attracts much attention, because the levels of multiplication are increased, somaclonal variation is limited and disease-free material can be obtained. The technique is invaluable to the conservation of Crinum species belonging to the Amaryllidaceae which, as a group, possesses several biological features that make them particularly vulnerable. This is in addition to other problems relating to their value as horticultural material, traditional medicines and sources of phytochemicals of interest to medical science. Two in vitro systems are widely used for the propagation of amaryllidaceous species; regeneration from young floral stem explants and from twin-scales excised from bulbs. Although plantlet regeneration could be obtained from peduncle explants of Crinum moorei, a complex of factors including: the age of the floral stem; explant position and; hormonal factors, limited growth. Callus production was poor and indirect organogenesis could not be achieved. Twin-scales were used for the induction of somatic embryos. Morphologically these were different depending on the concentrations of 2,4-D and BA used in the induction medium. Although some of them went on to germinate, the use of somatic embryos for large-scale culture is not an efficient micropropagation route, owing to the low frequency of both embryo production and germination and to the long culture times. Regeneration of shoots and bulblets could, however, be readily induced from twin-scales using a series of modified MS media, and this despite the fact that explants from the bulb were more difficult to decontaminate than the above ground parts. Shoots arose in the axes of the twin-scales close to the basal plate. Initiation was greatest on a basic Murashige and Skoog medium, containing 4 g ℓ ¯¹ sucrose, and in the dark. No hormones were required. At high concentrations, the hormones stimulated abnormal organogenesis. Bulbing of the shoots was further enhanced using higher than normal levels of sucrose i.e. 6% and 5 g ℓ ¯¹ activated charcoal. The response was also influenced by the size of the twin-scale and its position in the parent bulb. Greater numbers of bulblets with larger diameters developed in large twin-scales from an intermediate position between the inner and outer scales. Furthermore, light, and a temperature of 25°C were required for normal bulblet development. Bulblets grown in this manner were used as a source of secondary explants by splitting them vertically in half. The addition of 10 mg ℓ ¯¹ BA resulted in multiple shoot development. In a liquid-shake culture system, this same multiplication medium induced the formation of meristemoid clusters whose rates of proliferation were higher than that achieved for shoot multiplication on either solid or static liquid media. The advantage of using meristematic clusters is that shoot hyperhydricity is avoided. Furthermore, the clusters can be mechanically separated; making the system ideal for automated plant production. Shoot morphogenesis, followed by the formation of bulblets occurred on solid MS media containing activated charcoal or high concentrations (6%) of sucrose. The induction of bulblets by sucrose was, however, slower, which may be beneficial for long-term storage and conservation ex situ. Compared to smaller bulblets, bulblets with a diameter of approximately 9 mm, acclimatized readily and grew rapidly after transferring them to the soil in greenhouse conditions. Biotechnological processes such as cell and tissue culture provide an ideal system for producing secondary metabolites, especially where their production in situ is hampered by poor resource availability or when chemical synthesis is difficult. In vitro produced Crinum moorei bulblets were found to contain nine alkaloids of the Amaryllidaceae type; three of which were released into the culture medium. Light was essential for alkaloid biosynthesis while the inclusion of BA and activated charcoal stimulated the production of specific alkaloids. / Thesis (Ph.D.)-University of KwaZulu- Natal, Pietermaritzburg, 2002.

Thesis (Ph. D.)--University of Hawaii at Manoa, 1996. / Includes bibliographical references (leaves 145-153). / Microfiche. / viii, 153 leaves, bound ill. 29 cm

The propagation of wild-type virus and a transgenic viral vector was examined in cultured plant cells to identify factors affecting viral infection of and accumulation in cultured cells and to determine if viral vectors could be used to facilitate the expression of heterologous proteins in vitro. Tobacco mosaic virus (TMV) accumulation was examined in Nicotiana tabacum and N. benthamiana suspension and hairy root cultures. TMV accumulation was superior in N. benthamiana hairy roots. Hairy roots were infected by adding TMV to the liquid culture medium at the same time as root inoculation. Hairy root growth was unaffected by virus infection. The distribution of virus within root mats from shake-flask grown cultures was non-uniform and the concentration of virus accumulated in replicate cultures was variable. When N. benthamiana hairy roots were infected using 1.5 μg mL-1 TMV, the average maximum concentration of virus accumulated in the biomass was 1.6 ?? 0.25 mg g-1 dry weight, or 15-fold lower than in TMV infected N. tabacum leaves. Virus coat protein accumulated to a level of (26 ?? 10)% total soluble protein in the hairy roots. Inoculum virus concentration and the medium in which infection was performed affected the virus yield and the percentage of inoculated cultures that accumulated virus. When cultures were inoculated using 9.0 μg mL-1 TMV, virus accumulated in the biomass to 4.2 ?? 0.60 mg g-1 dry weight. Proportional scale-up of hairy root infection in shake flasks did not result in constant virus concentrations in the scaled cultures. TMV accumulation in bioreactor-infected and -grown hairy roots was poor. N. benthamiana hairy roots were infected with a TMV-based vector (30B-GFPC3) that encoded Cycle 3 green fluorescent protein (GFP). TMV-GFPC3 was (260 ?? 140)-fold less infectious than TMV as measured by local lesion assays. Propagation of TMV-GFPC3 could not be confirmed using mass balance. GFP was not detected in the infected hairy roots or the culture medium. Hairy roots represent a potentially viable culture-based system for the in vitro production of virus and virus products when field-grown agricultural systems do not adequately address issues of containment or product safety.

Thesis (M.S.)--University of Nevada, Reno, 2004. / "December 2004." Includes bibliographical references (leaves 29-42). Online version available on the World Wide Web.

Thesis (M.S.)--California Polytechnic State University, 2009. / Title from PDF title page; viewed on June 5, 2009. "May 2009." "In partial fulfillment of the requirements for the degree [of] Master of Science in Forestry Sciences." "Presented to the faculty of California Polytechnic State University, San Luis Obispo." Major professor: Walter R. Mark, Ph.D. Includes bibliographical references (p. 39-43). Also available on microfiche.

A growth chamber experiment was carried out for ten weeks to reduce post-rooting dormancy in juneberry micropropagation. An RCBD with a split plot arrangement and three replicates were used. Plantlets subjected to 750 mg/L GA, 100 mg/L BA, and 250 mg/L GA + 100 mg/L BA recorded the greatest leaf number. Pre-rooted ‘Thiessen’ plantlets recorded the greatest biomass (fresh and dry weight) and root volume. In a second study, a cultivar evaluation was conducted in Absaraka, ND, where ten juneberry cultivars and a native biotype planted were evaluated for plant and fruit characteristics. An RCBD with four replicates was used. The high yielding cultivars for total yield were ‘Thiessen’, ‘Martin’, ‘Parkhill’, ‘Pembina’, ‘Regent’ and Native. ‘Thiessen’, ‘Martin’, and ‘Parkhill’ maintained a significant higher marketable yield. ‘Thiessen’, ‘Regent’, ‘Martin’, ‘Parkhill’ and ‘Northline’ had the largest fruits, while ‘Thiessen’ and ‘Martin’ fruit had the greatest mass.

No description available.

Dormant bud and actively growing shoot tip explants of B. uber were cultured on Murashige & Skoog (MS) and modified woody plant (WPM) basal salts supplemented with organic nutrients and benzylaminopurine (BAP). Shoot tip explants were morphogenically more responsive than dormant bud explants. The modified WPM proved to be better than MS medium for the establishment and proliferation of B. uber cultures. Shoot proliferation was greatest at 8.9 μM BAP. However, BAP at 4.4 μM produced the greatest number of shoots > 10 mm. Indolebutyric acid (IBA) was more effective than naphthaleneacetic acid (NAA) for rooting of microcuttings in vitro. Rooting was best with a low level of IBA (2.5 μM) in the culture medium. Ex vitro rooting of microcuttings using a conventional potting mix of sand-peat-perlite (1:2:1 v/v/v) was superior to all in vitro rooting treatments. Plantlets were acclimated successfully to greenhouse conditions. Genotype differences with regard to both rooting and shooting abilities were observed. / Master of Science

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.