The wide utilization and popularity of medicinal plants in African Traditional Medicine (ATM) has been recognized and attributed to the effectiveness, affordability and accessibility of these medicinal plants. However, the extensive exploitation of medicinal plants has exacerbated the strain on the wild populations. In vitro propagation/micropropagation is an effective method which allows for mass production or multiplication of pathogen-free plants that are morphologically and genetically identical to the parent plant. In addition, the technique is contributing to the understanding of metabolic pathways and regulating the production of plant secondary products. Eucomis autumnalis (Mill.) Chitt. subspecies autumnalis (Hyacinthaceae) is a valuable medicinal species in ATM and commonly traded in the urban street markets of South Africa. Currently, the conservation status of this species has not been evaluated. However, as with most bulbous plants, the wild population is continuously under threat due to over-harvesting and habitat loss via various anthropogenic factors. Thus, in vitro propagation is a viable means of ensuring conservation of the plant species. However, mass propagation of medicinal plants should be accompanied with increased secondary metabolite production to guarantee their therapeutic efficacy. Therefore, the current study was aimed at understanding the different factors that affect the growth and secondary metabolite production in micropropagated E. autumnalis subspecies autumnalis. The influence of the type of gelling agent (gelrite versus agar) and source of initial/primary explant source (LDL = leaf explant derived from primary leaf regenerants and LDB = leaf explant derived from primary bulb regenerants) were evaluated. Gelrite-solidified medium significantly improved shoot proliferation when compared to the use of agar as a solidifying medium. In contrast, quantified phytochemicals such as flavonoids and phenolics were more enhanced in agar-supplemented media. On the basis of the explant source, shoot proliferation and secondary metabolites in regenerants from LDB were similar to those from LDL in most cases. Overall, the type of gelling agents and primary explant source individually or/and interactively significantly influenced the growth parameters as well as the production of iridoid, condensed tannin, flavonoid and phenolic content. The influence of different types of plant growth regulators (PGRs) on growth, phytochemical and antioxidant properties were evaluated. The PGRs were BA (benzyladenine); mT (meta-topolin); mTTHP [meta-topolin tetrahydropyran-2-yl or 6-(3-hydroxybenzylamino)-9-tetrahydropyran-2-ylpurine]; MemT [meta-methoxytopolin or 6-(3-methoxybenzylamino)purine]; MemTTHP [meta-methoxy 9-tetrahydropyran-2-yl topolin or 2-[6-(3-Methoxybenzylamino)-9-(tetrahydropyran-2-yl)purine] and NAA (α-naphthalene acetic acid). Five cytokinins (CKs) at 2 μM in combination with varying (0, 2.5, 5, 10, 15 μM) concentrations of NAA were tested. After 10 weeks of in vitro growth, the regenerants were acclimatized in the greenhouse for four months. Growth, phytochemical content and antioxidant activity of in vitro regenerants and ex vitro-acclimatized plants were evaluated. The highest number of shoots (approximately 9 shoots/explant) were observed with 15 μM NAA alone or with BA treatment. Acclimatized plants derived from the 15 μM NAA treatment had the highest number of roots, largest leaf area and widest bulb diameter. While applied PGRs increased the iridoids and condensed tannins in the in vitro regenerants, total phenolics and flavonoids were higher in the PGR-free treatment. In contrast to the PGR-free regenerants, 5 μM NAA and 2 μM BA treatments produced the highest antioxidant activity in the DPPH (55%) and beta-carotene (87%) test systems, respectively. A remarkable carry-over effect of the PGRs was noticeable on the phytochemical levels and antioxidant activity of the 4-month-old plants. In addition to the development of an optimized micropropagation protocol, manipulating the type and concentration of applied PGRs may serve as an alternative approach to regulate phytochemical production in Eucomis autumnalis subspecies autumnalis. The influence of smoke-water (SW), karrikinolide (KAR1) and CK analogues (PI-55 = 6-(2-hydroxy-3-methylbenzylamino)purine and INCYDE= inhibitor of cytokinin dehydrogenase or 2-chloro-6-(3-methoxyphenyl)aminopurine) individually or in combination with some selected PGRs [BA (4 μM), NAA (5 μM) and both] for in vitro propagated E. autumnalis subspecies autumnalis was evaluated. While these compounds had no significant stimulatory effect on shoot proliferation, they influenced root length at varying concentrations and when interacted with applied PGRs. The longest roots were observed in SW (1:1500), PI-55 and INCYDE (0.01 μM) treatments. There was an increase in the concentration of quantified phytochemicals (especially condensed tannins, flavonoids and phenolics) with the use of these compounds alone or when combined with PGRs. In the presence of BA, an increase in the concentration of PI-55 significantly enhanced the condensed tannin, flavonoid and phenolic contents in the regenerants. Both phenolic and flavonoid content in E. autumnalis subspecies autumnalis were significantly enhanced with 0.01 μM INCYDE. Condensed tannins was about 8-fold higher in 10-7 M KAR1 with BA and NAA treatment when compared to the control. To some varying degree, the effect of the tested compounds on the antioxidant activity of the in vitro regenerants was also noticeable. In most cases, there was no direct relationship between the level of phytochemicals and antioxidant activity recorded. The current findings indicate the array of physiological processes influenced by SW and KAR1 during micropropagation. In addition, targeting or manipulation of phytohormone metabolic pathways using CK analogues demonstrated some noteworthy effects. Perhaps, it may offer other potential practical applications in plant biotechnology and agriculture. Thus, more studies such as quantification of endogenous hormones and identification of specific phytochemicals responsible for the bioactivity in this species will provide better insights on the mechanism of action for CK analogues as well as SW and KAR1. / M. Sc. University of KwaZulu-Natal, Durban 2014.

Made available in DSpace on 2014-06-11T19:31:00Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-11-16Bitstream added on 2014-06-13T19:19:53Z : No. of bitstreams: 1 salvaro_lms_dr_botib.pdf: 542129 bytes, checksum: 9f3ea451106084bdce151c44b55ad171 (MD5) / O 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... / 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)

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.

Graduation date: 1999

Graduation date: 1995

The responses of the tepary bean (Phaseolus acutifolius) to in vitro tissue culture systems were documented. Tests were conducted to identify the optimal auxin and cytokinin combinations required for optimal callus growth. Regeneration experiments were conducted to: (1) determine the effect of explant source and age on regeneration, (2) effect of callus age on regeneration, (3) the cultivation status of the explant source, and (4) the effects of nutritional additives on somatic embryogenesis. The callus was easily induced and maintained in all hormonal medias except those containing IAA and 6BA. The results for regeneration were most promising from cultures derived from immature cotyledon tissue. Ammonium Chloride, glutamine and Absisic acid appeared to have little affect on embryogenesis, however the addition of kinetin enabled the embryos to develop to the torpedo stage. Callus age and cultivative status of explant source had no effects on plantlet regeneration.

No description available.

Nodal sections of the winter-hardy 'John Franklin' rose cultivar from field-grown plants were cultured on a modified Murashige and Skoog (MS) nutrient medium. Very high levels of contamination from the surface of the initial sections required that plants be grown under greenhouse conditions. Rose plantlets obtained from subsequent subcultures were used for the first time in a radiotracer experiment with $ sp{32}$P to study the kinetics of phosphorus (P) uptake as a function of temperature of the nutrient medium. P uptake increased with time for rooted and non-rooted plantlets in a linear fashion that did not reach an equilibrium value even after 96 hours of exposure. An analysis of variance revealed that the plantlets with roots absorbed significantly greater amounts of P at the 0.01 level compared with non-rooted plantlets at 22$ sp circ$C. P uptake was significantly higher at the 0.05 level, for rooted versus non-rooted plantlets at 33$ sp circ$C. There was no significant difference in P uptake by rooted and non-rooted plantlets at 3$ sp circ$C. Interaction between time of exposures and rooting was found to be significant at 22$ sp circ$C and 33$ sp circ$C at the 0.01 level. The results indicated that the root system, previously thought to be inefficient in the nutrient absorption, played a key role in absorbing P from the nutrient medium at optimum temperature.

La laitue (Lactuca saliva L.) est un légume-feuille de climat frais, ayant un cycle annuel. Son importance à la fois sociale et économique facilite la mise sur pied de programmes d'amélioration où les génotypes choisis sont préservés par clonage in vitro. Cette étude porte sur des facteurs d'ordres génétique, hormonal et cultural dans le but d'établir les conditions optimales pour la régénération des explantats, issus de bourgeons apicaux et axillaires, en plants de laitue producteurs de semences. La première expérience (chap. 1) cherchait à optimiser le processus d'excision des tissus sous-jacents de l'explantat. Quatre types de coupes (diamant « DNT », carré « CAR », diamétral « DAL » et transversal « TRA ») ont été effectués, à différents niveaux (3, 6 et 9 mm), sur l'explantat. Les résultats ont montré que les explantats issus de bourgeons apicaux régénèrent mieux que ceux provenant des axillaires et que la coupe DNT6 a induit le meilleur pourcentage de régénération en plantules viables. L'expérience suivante (chap. 2) étudie l'effet génétique du cultivar sur l'organogénèse. Pour ce faire, des explantats issus de bourgeons apicaux et axillaires ont été prélevés sur des cœurs provenant de 4 cultivars de laitue (Lactuca saliva L.) pommée (cvs. Estival, Ithaca, Eldorado) et romaine (cv. Sunbelt). Nous avons observé que les explantats issus de bourgeons apicaux sont encore ceux qui ont le mieux régénéré. De plus des différences génotypiques ont été décelées, bien que le pourcentage d'explantats régénérés en plantules viables n'ait pas été influencé par le cultivar. La troisième expérience surveille l'effet de différentes combinaisons d'acide 3-indoleacétique (AIA) et de 6-benzylaminopurine (BAP) sur le développement et la viabilité des plantules de laitue pommée issues de bourgeons apicaux. Les résultats ont montré que l'AIA n'a pas affecté la régénération des plantules de laitue tandis que la BAP a négativement influencé chacune des variables à l'étude en dehors du nombre de feuilles. Nous avons déduit que la régénération peut se faire sur un milieu de culture contenant uniquement de l'AIA à une faible concentration. Le problème lié à l'aseptisation en culture in vitro a été évoqué dans le chapitre 4. Une expérience exploratoire a permis de démontrer que l'aseptisation faite simplement avec de l'hypochlorite de sodium (NaOC1) donnait de meilleurs résultats qu'en combinaison avec du peroxyde d'hydrogène. Une seconde expérience a été effectuée en utilisant différentes concentrations de NaOC1 et durées d'exposition pour aseptiser des explantats issus de bourgeons apicaux et axillaires prélevés sur des cœurs de laitue pommée. Elle a révélé qu'une aseptisation faite avec du NaOC1 à 1.2% pendant 10 minutes donnait des résultats satisfaisants. En dernier lieu, le brunissement des tissus et du milieu de culture, a été abordé au chapitre 5. Trois types de traitements antioxydants ont été utilisés, pour la première fois, sur des explantats de laitue issus de bourgeons axillaires : l'application d'une période à l'obscurité en début de culture Gours); le trempage et la coupe dans une solution d'acide citrique et d'acide ascorbique; le trempage dans une solution et/ou la mise en culture dans un milieu contenant du polyvinylpyrrolidone (PVP). Bien qu'il n'y ait pas eu de différence au niveau de l'indice d'opacité du milieu entre ces traitements, nous avons constaté que le fait de tailler l'explantat dans une goutte d'acide ascorbique et d'acide citrique est à considérer puisque ce traitement est facile à appliquer en plus de favoriser le développement de feuilles (≥ 1 cm) et de racines (≥ 1 cm). En somme, pour régénérer des explantats en plantules de laitue saines et viables provenant de n'importe quel cultivar, il faut utiliser les bourgeons apicaux comme explantats et les aseptiser avec du NaOC1 1.2% pendant 10 minutes. Puis, les tissus sous-jacents aux bourgeons doivent être excisés selon un plan de coupe DNT6, dans une goutte de solution antioxydante avant que les bourgeons soient placés sur un milieu de culture contenant uniquement de l'AIA. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : Lactuca saliva L., culture in vitro, bourgeons apicaux et axillaires, taille des explantats, hormones de croissance, aseptisation, brunissement

Tissue-cultured shoots and plantlets usually have leaves with non-functional, open stomata and little epicuticular and cuticular wax, resulting in excess evapotranspiration after transplantation. Various strategies were evaluated to decrease ex vitro acclimatization difficulties for 'Silvan' blackberry, including transplanting unrooted shoots, increasing the medium agar concentration from 6 to 9 or 12 g/l and diluting the basal medium. Increased medium agar concentrations and medium dilution did not improve survival or growth. Stomatal function resumed sooner in new leaves of plantlets than shoots. High relative humidity ($>$95%) and low light intensity (90 $ mu$mol s$ sp{-1}$ m$ sp{-2}$) negatively affected stomatal closure both on acclimatizing transplants and greenhouse-grown plants. Guard cells developed on leaves in vitro were physiologically active but had apparent anatomical abnormalities that inhibited closure. A rapid clearing and staining method was developed for examination of foliar morphology using intact in vitro blackberry (Rubus sp. 'Silvan') and strawberry (Fragaria x ananassa Duch. 'Totem') plantlets and sections of greenhouse-grown 'Silvan' and 'Totem' leaves. This method involved three steps: (1) removing the chlorophyll by autoclaving in 80% ethanol; (2) dissolution of the protoplasm using 5% NaOH at 80$ sp circ$C; (3) post-alkali treatment with 75% bleach (4.5% NaClO) at room temperature for tissue-cultured plantlets and at 55$ sp circ$C for greenhouse-grown leaves. Aqueous safranin (10 mg/l) was used for staining.