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.

The Asteraceae family is frequently used to describe several medicinal plants which contain various phytochemical compounds including phenols, flavonoids and terpenoids. Among the Asteraceae family German chamomile (Matricaria chamomilla L.) and yarrow (Achillea millefolium L.) plants are extant species used in contemporary medicine. These phytochemical compounds have been traditionally used since ancient times in health care systems worldwide as a source of medicines. The use of micropropagation is essential to improve and increase these active compounds via plant tissue culture within a short period of time using the application of key plant growth regulators (PGRs). Furthermore, quantitative and qualitative analysis using high performance liquid chromatography- ultraviolet detector (HPLC-UV) and gas chromatography- flame ionisation detector (GC-FID) of potential medicinal compounds expressed by both chamomile and yarrow are important points. The protocol of in vitro shoots, roots and callus formation of chamomile and yarrow seeds culture were investigated using Murashige and Skoog (MS) medium supplemented with different concentrations of plant growth regulators (PGRs). MS culture medium containing 0.5 mgL-1 IAA and 1.0 mgL-1 of GA3 were found to be the best culture medium for chamomile and yarrow seeds. In this project in vitro and in vivo growth rates of selected plant species were also investigated. In the earlier growth stages yarrow plants were found to grow much quicker than chamomile, while the yield of chamomile flowers was significantly (p ≤ 0.001) more than yarrow flowers. The phenolic, flavonoid and terpenoid compounds content of leaves and flowers of plants produced from both cultures were also studied. HPLC-UV analysis showed that chlorogenic acid, apigenin-7-O-glucoside and luteolin dominated as the main phenol and flavonoid compounds recovered in both in vitro and in vivo chamomile and yarrow cultures. However, GC-FID analysis indicated that farnesene and nerolidol were detected as the main terpenoid compounds present in the two culture conditions used to grow chamomile and yarrow plants. Moreover, this research examines how chamomile and yarrow plants can produce and improve their phytochemical compounds content not only under well-watered conditions but also under drought stress conditions. The main phenol and flavonoid compounds of chlorogenic acid, caffeic acid, apig-7-glucoside, umbelliferon and luteolin were found in chamomile and yarrow varieties grown under both well-watered and drought stress conditions using (HPLC-UV), however farnesene, nerolidol, chamazulene, α-(-)- bisabol and bisabolol oxide A were observed in the plant essential oils (EOs) using Soxhlet extraction and GC-FID analysis. The antibacterial activity of plant EOs was also investigated using disc diffusion and 96 well plates. In vivo chamomile EO showed the highest antibacterial activity against gram-positive and gram-negative bacteria strains. In addition, in vitro yarrow EO showed the greatest effect on the death of bacteria strains.

In vitro experiments were conducted using leaf explants from organically grown Petunia x hybrida &lsquo`Blue Picotee&rsquo' cultured on media prepared from organic hydroponic solutions, Murashigi and Skoog (MS) vitamins and benzyladenine. Explants cultured on MS regenerated the best. Explants cultured on Aqua Power produced shoots, had higher weights and lower necrosis than on other organic media. The Aqua Power results were not repeatable. In testing Aqua Power, Earth Juice and Peaceful Valley at ⅓, ⅔ and full concentrations, the explants on Aqua Power were 100% necrotic at all concentrations. Explants on Earth Juice and Peaceful Valley were chlorotic but had the best growth, measured by weight and necrosis, at the lowest concentration. Shoot organogenesis occurred on explants at all concentrations on Peaceful Valley. When using liquid petunia extract media, no shoots regenerated. On these media, the highest explant weights were with 7.5 g&middotL-1 agar and necrosis decreased as agar concentration increased.

Resumo: 0 objetivo deste trabalho foi verificar a eficiência da técnica de milcropropagação para a espécie Piper hispidinervium, utilizando reguladores vegetais e poliaminas e verificar a influência destes sobre o desenvolvimento dos explantes (número de raízes, número de brotações, formação de calo e altura), bem como verificar influência da época de avaliação das plântulas sobre os teores de flavonóides totais, atividade antioxidante, atividade da peroxidase e teores endógenos de poliaminas livres. Microestacas obtidas da germinação in vitro foram inoculadas nos devidos tratamentos, os quais foram divididos em dois experimentos, o primeiro, utilizando concentrações de benzilaminopurina (BAP) isoladas e combinadas com ácido indolilbutírico (IBA) e o segundo, utilizando poliaminas (espermina- Spm e espermidina-Spd) adicionadas ao meio de cultura, isoladas, combinadas entre si e com BAP. Desta forma, no primeiro experimento os tratamentos foram: BAP (0,25; 0,50; 0,75 e 1,0 mg L-1), isoladas ou combinadas com IBA (0,25 mg L-1 ), o qual também foi utilizado isolado, totalizando 10 tratamentos, 3 repetições com 6 microestacas por repetição. No segundo experimento os tratamentos foram: T1: MS (testemunha); T2: 1mg L-1 BAP; T3:1mM Spd; T4: 1mM pd + 1mg L-1 BAP; T5: 1mM Spm; T6: 1mM Spm + 1mg L-1 BAP e T7: 1mM Spd + 1mM Spm. Aos 30 e 60 dias após a inoculação (DAI) foram realizadas as análises biométricas (formação de calo, número de raízes, número de brotações e altura das plântulas). No momento da inoculação (tempo 0), aos 30 e 60 DAI foram coletadas amostras para a realização das análises bioquímicas do teor de flavonóides totais, atividade antioxidante, atividade da peroxidase e teor endógeno de poliaminas livres (experimento 2). Nas condições realizadas no primeiro experimento, o IBA propiciou o maior crescimento das plântulas e induziu maior... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The purpose of this paper was to evaluate the efficiency of the technique of micropropagation for the Piper hispidinervium species, using plant growth regulators and polyamines and also evaluate their influence on formation of callus, number of roots, number of shootings and height of explants and the influence of cutting time on the tenors of contents of totals flavonoids, antioxidant activity, activity of the peroxidase and endogenous contents of free polyamines. Microcuttings obtained of the germination in vitro were inoculated in the proper treatments, they were divided into two experiments, the first experiment, using benzilaminopurine (BAP) isolated and combined with indolilbutiric acid (IBA), and the second one, using polyamines (Espermine- Spm and Espermidine- Spd) added to the culture medium, isolated, combined among them and with BAP. Thus, in the first experiment, the treatments were: BAP (0.25; 0.50; 0.75 and 1.0 mg L-1), isolated or combined with IBA (0.25 mg L-1), and it was also used isolated, totalizing 10 treatments, 3 repetitions with 6 microcuttings for repetition. In the second experiment the treatments were: T1: MS (control); T2: 1 mg L-1 BAP; T3:1 mM Spd; T4: 1 m M Spd + 1 mg L-1 BAP; T5: 1 mM Spm; T6: 1 mM Spm + 1mg L-1 BAP and T7: 1 mM Spd + 1 mM Spm. After 30 and 60 days of the inoculation (DAI) analysis were carried out (formation of callus, number of roots, number of shootings and height of explants). At the moment of the inoculation (time 0), at 30 and 60 DAI, samples were collected for the realization of biochemical analyses of contents totals of flavonoids, antioxidant activity, activity of the peroxidase and endogenous contents of free polyamines (only in the experiment 2). In the realized conditions, in the first experiment, the IBA favored the highest growth of the seedlings and induced a higher number of roots, as well as a higher content... (Complete abstract click electronic access below) / Orientador: Elizabeth Orika Ono / Coorientador: Giuseppina Pace Pereira Lima / Banca: Carmem Silvia Fernandes Boaro / Banca: Maria de Lourdes Lúcio Ferrarese / Banca: Vandeir Francisco Gumarães / Banca: João Domingos Rodrigues / Doutor

The exotic blooms of the indigenous South African flowering plants and the biodiversity of the floral material available, have led to a high demand for these flowers on the international market. A wide variety of high-quality flowers, of which Protea cynaroides L. is an important component, are exported from South Africa. However, because of the high demand for the export market the producers are unable to supply enough plant material. Therefore, due to the limited supply, poorer quality flowers are exported which results in lower prices obtained. One of the reasons for the lack of flowers available for export is that being a woody perennial, P. cynaroides require a longer period of time to grow when conventional propagation methods are used. Micro propagation of P. cynaroides may become the most suitable alternative propagation method for rapid production and to ensure high standards of quality. In this study, Stage I (establishment) and Stage 2 (multiplication) of micro propagation were investigated. Establishment (Stage I) of the explants was achieved by using Murashige and Skoog medium with the addition of 30 mg.1-1 GA3. However, it was found that browning of the explant tissues caused by phenolic oxidation was a major problem during the establishment stage, as the browning of many explants led to the inability of the axillary buds to sprout which resulted in death. Browning of tissues occurs when phenolic compounds are oxidized, this usually happens when the plant tissues are stressed or injured during explant preparations. Therefore, methods to control oxidation were tested in an experiment which included the use of sterilants (mercuric chloride and sodium hypochlorite) and antioxidants (ascorbic and citric acid). The selection of a few treatments which showed potential in controlling oxidation, followed by further tests, led to positive results. Phenolic oxidation was reduced by stirring the explants in 100 mg.1-1 ascorbic acid and 1500 mg.1-1 citric acid for 1 hour. This was followed by growing the explants in a 16-hour photoperiod which was suitable for the axillary buds to sprout. Subsequently, Stage 2 of micropropagation (multiplication) was successfully done by subculturing the explants from the establishment stage onto the multiplication media. The effects of phosphorous on the growth of the explants were tested by using two media, where no ammonium phosphate was added into one medium, while 1400 mg.1-1 ammonia phosphate was added to the other. Surprising results were obtained when explants in both media grew well, illustrating that P. cynaroides may be tolerant to high levels of phosphorous. However, a possible reason for this is that because no roots were formed by the explants in the multiplication medium, the phosphorous in the medium were not taken up by the explants. These results also illustrated that two-budded explants achieved a higher survival rate and longer mean length than one-budded explants. Investigation into the rooting requirements of the explants, Stage 3 (rooting) of micropropagation must still be achieved. / Dissertation (M Inst Agrar (Horticultural Science))--University of Pretoria, 2006. / Plant Production and Soil Science / unrestricted

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.