Masters Degrees (Botany)

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The effect of elevated atmospheric CO2 on the growth and physiology of Chromolaena odorata.
(2008) Lalla, Reshnee.
Rising atmospheric CO2 (Ca) concentrations have generated concern among scientists, mainly because of CO2’s role as a greenhouse gas and its influence on plant growth and development. Previous research has suggested that future CO2 enriched atmospheres may enhance the success of invasive aliens. Chromolaena odorata is an example of an invasive alien proving to be a serious threat to indigenous vegetation in South Africa, and effective control measures are desperately needed to curb infestations in the future. The current study aimed at assessing the response of C. odorata to elevated Ca and interactive factors, and was divided into two trials. During PART A, C. odorata was grown in competition with 2 grass species: Eragrostis curvula and Themeda triandra (selected for their differential preferences to nutrient availability). All three species were potted in a greenhouse at the University of KwaZulu-Natal (Howard College). There were 16 pots in total, and each pot contained four C. odorata plants, four T. triandra seedlings, and four E. curvula seedlings. Eight pots were exposed to elevated Ca (~700ppm), and eight pots were exposed to ambient Ca (~370ppm). The pots at each Ca treatment were further divided: four received high nutrient treatments (3L per addition), while the other four received low nutrient treatments (300 ml per addition). Studies on growth (e.g. plant height, dry weight, etc.), as well as physiology (e.g. Jmax), were undertaken. Results showed that generally, plants responded positively to high nutrient treatments. In contrast, elevated Ca did not affect growth or any of photosynthetic parameters of C. odorata significantly, but did reduce stomatal limitations. During PART B, C. odorata plants were grown monospecifically to assess whether there was a “chamber effect” associated with planting density. Pots at both Ca treatments contained either four C. odorata or two C. odorata seedlings. Growth and physiology were assessed. The fact that elevated Ca did not affect any of the photosynthetic parameters studied, suggests that photosynthetic down-regulation did not occur. This, together with the fact that no increase in stomatal limitations were observed in elevated Ca, implies that enhancement of photosynthetic assimilation could have occurred in C. odorata plants exposed to CO2 enrichment. Results from this study (PART A and PART B), when compared to previous research on this species, suggests that CO2 enrichment may enhance the success of monoculture populations of C. odorata. However, other species may gain competitive advantages over C. odorata occurring in mixed communites, under CO2 enriched environments. In addition, results of this study support the prediction that increasing Ca will reduce the importance of carbon as an external limiting resource, and that the extent of a plant’s response to Ca enrichment will depend on resources other than CO2. If increases in temperature caused by elevated Ca increases nutrient availability in the soil, then Ca could indirectly enhance the success of C. odorata occurring in mixed communities.
The performance and rooting of eucalyptus grandis x nitens cuttings.
(2007) Murugan, Nelisha.; Watt, Maria Paula Mousaco Deoliveira.; Mycock, David John.; Mokotedi, Mompe Edward Oscar.
Hybrid clones of Eucalyptus grandis and E. nitens (GN) have consistently been shown to be suitable for planting in cold, dry, marginal plantation sites, where they exhibit high yields and superior pulp properties. However, their clonal propagation is hindered by the very poor rooting success of cuttings. The present study aimed at assessing the effect of cutting type, time of year of setting cuttings and Seradix 2 application on rooting and development of cuttings of a commercially important Eucalyptus grandis x Eucalyptus nitens clone (GN107). Cuttings were prepared from clonal hedge coppice at the Mondi Business Paper, Trahar Technology Centre, Hilton. Three cutting types were used (cut at different distances from the node) for each terminal (situated below the apical bud) and non-terminal cuttings. The leaves were trimmed and, for half the cuttings, the base of the stem of cuttings were dipped in Seradix 2 rooting powder (3 g kg-1 4-(indole-3-yl)-butyric acid (IBA). They were then placed into rooting trays (128 inserts/ tray arranged as 8 rows x 16 columns). Seradix 2-treated and Seradix 2-untreated terminal and non-terminal cuttings, cut at, above and below the node (twelve treatments in total) were set in trays with one treatment per column of eight replicates, per tray. There were nineteen trays overall. The trays were filled with peat, perlite and vermiculite (3:3:1) and were maintained in a Mondi greenhouse, with air temperature at 25°C to 27°C (thermostatically activated fans), root zone temperature at 28°C (bed heaters) and 20 second misting at 10 minute intervals (automatic misters). The study was carried out in November 2005, April 2006 and June 2006. In the first experiment, both terminal and non-terminal cuttings were used; thereafter only non-terminal cuttings were used. The plantlet yield was very low, regardless of cutting type, Seradix 2 treatment and the time of year the cuttings were set. The highest plantlet production (12.5%) and rooting frequencies (13.8%) were achieved with non-terminal cuttings treated with Seradix 2. Although not statistically significant, Seradix 2 inhibited shoot production (31.4% for Seradix 2-untreated and 24.2% for treated cuttings). The position at which inserts were cut in relation to the node did not significantly affect the number of plantlets produced and non-terminal cuttings appeared hardier and performed better than terminal cuttings. The time of year of setting cuttings did not have any significant effect on plantlet yield, nonetheless, plantlet yield was highest in cuttings set in November (9.2%) and lowest in April (0.4%). In addition, cuttings set in November (spring), had superior shoot development in terms of the number of cuttings that produced shoots (regardless of root production), shoot length and the mass of shoots relative to root mass. The highest percentages of cuttings that produced roots (regardless of shoot growth) (10%) and the highest number of roots per cutting (2) were part of the June trial. Therefore, cuttings set in June (winter) had superior root development as compared with cuttings set in November (spring) or April (autumn). In all of the studies, three rooting patterns were observed in cuttings: roots produced only from the cut area only (type 1), only from the sides of the stem (type 2) and from both sites (type 3). Non-terminal cuttings treated with Seradix 2 showed a higher incidence of types 2 and 3 rooting patterns than the terminal cuttings. Seradix 2 application increased the prevalence of types 2 and 3 rooting patterns. Although not statistically different, cuttings dipped 2.5 cm into Seradix 2 produced more types 2 and 3 rooting patterns than cuttings dipped at the abaxial end only. Light microscopy of stem sections of cuttings indicated that roots appeared to originate from the xylem archs as well as from the cambium. The collected data indicate that it is necessary to continue research towards improving the efficiency of plantlet production of GN107 via cuttings. It appears that cuttings of this clone may be set throughout the year and that terminal cuttings should be avoided. In addition, the present practice at the Mondi Hilton nursery of treating cuttings with Seradix 2 needs to be reconsidered as although it increases rooting, it does not increase plantlet production due to its apparent inhibitory effect on shoot development.
Seed germination and medicinal properties of Alepidea species.
(2009) Mulaudzi, Rofhiwa Bridgeht.; Van Staden, Johannes.; Finnie, Jeffrey Franklin.
The rhizomes of Alepidea amatymbica and Alepidea natalensis are used for medicinal purposes. Because of the increase in demand for these plants the species is becoming scarce. As the seed biology of neither species is well defined, conditions as well as treatments required for optimum germination and vigour were studied. Seeds were exposed to various physical factors such as varying light and temperature conditions and cold stratification, sowing depth and seed storage. The effects of smoke-water, butenolide (3-methyl-2H-furo [2, 3-c] pyran-2-one) a novel smoke compound and chemical substances (gibberellins, kinetin and KNO3) were also tested in order to improve seed germination. Alepidea amatymbica and A. natalensis achieved the highest seed germination (72.5% and 80%, respectively) at 25 °C under a 16 h photoperiod with a mean germination time (MGT) of 18 and 12 days, respectively. Phytochrome studies showed that A. natalensis requires light for germination. Cold stratification (5 °C) for 14-28 days significantly improved the percentage germination of both species (> 90%) compared to non-stratified seeds (control) at 25 °C under a 16 h photoperiod. Sowing A. amatymbica and A. natalensis seeds at a depth of 0.5 cm resulted in higher percentage germination compared to 2.5 cm. The highest emergence rate for A. amatymbica was 40% at a sowing depth of 0.5 cm and the lowest emergence rate was 3% at 2.5 cm. Six months storage of A. natalensis seeds at room temperature (25 ± 2 °C) showed maximum germination (99%) with a MGT of 9 days. Smoke-water treatment of A. amatymbica seeds significantly enhanced germination from 72% to 91%. Smoke and butenolide at 10 °C and 25 °C promoted germination of A. natalensis seeds in a 16 h photoperiod. Smokewater application significantly improved both germination and seedling vigour of A. natalensis. GA3 (10-8 M) was the best treatment for achieving maximum percentage germination of A. natalensis seeds. Antibacterial (two Gram-positive bacteria: Bacillus subtilis, Staphylococcus aureus and two Gram-negative bacteria: Escherichia coli, Klebsiella pneumoniae), antifungal (Candida albicans), anti-inflammatory (COX-1 and -2) and genotoxicity tests (Ames test) were carried out on petroleum ether (PE), dichloromethane (DCM), 80% ethanol (EtOH) and water extracts of the two Alepidea species. Water extracts of A. natalensis rhizomes exhibited high activity (MIC values of 0.78 mg/ml) against the four bacterial strains. High activity was also observed in the PE and DCM leaf extracts of the same plant against the Gram-positive bacteria. The PE and DCM extracts of A. amatymbica rhizomes exhibited the best activity (MIC values of 0.39 mg/ml) against Bacillus subtilis. The rest of the extracts showed low activity (MIC values >1 mg/ml). All the extracts showed activity against Candida albicans, with A. natalensis leaf extracts exhibiting the highest antifungal activity with MIC values of 0.88, 0.20 and 0.78 mg/ml for PE, DCM and EtOH, respectively. EtOH extracts had inhibition less than 40% for both A. natalensis and A. amatymbica. All the PE extracts showed higher inhibitory activity for COX-2 than for COX-1. PE and DCM extracts had percentage inhibitions above 70% in both COX-1 and COX-2 assays. The Ames test for genotoxicity revealed that none of the plant extracts were genotoxic to the Salmonella TA98 tester strain.
Pharmacology and phytochemistry of South African plants used as anthelmintics.
(2009) Aremu, Adeyemi Oladapo.
Traditional medicine in South Africa is part of the culture of the people and has been in existence for a long-time. Although animal components form part of the ingredients used, plant material constitutes the major component. South Africa is endowed with vast resources of medicinal and aromatic plants which have been employed for treatment against various diseases for decades. A large number of South Africans still depend on traditional medicine for their healthcare needs due to its affordability, accessibility and cultural importance. Helminth infections are among the variety of diseases treated by traditional healers. These infections are regarded as neglected tropical diseases (NTDs) due to their high prevalence among the economically disadvantaged living in rural areas in different regions of the world.
In vitro bulb induction in Eucomis zambesiaca Baker.
(2009) Cheesman, Lee.; Finnie, Jeffrey Franklin.; Van Staden, Johannes.
Eucomis L’ Hér. is a genus of 10 species that fall within the Hyacinthaceae family. Eucomis zambesiaca Baker is a summer-blooming bulbous geophyte occurring from northern South Africa to Malawi. Eucomis species are used in southern African traditional medicine for the treatment of various ailments, in particular, pain and inflammation. As a result, the bulbs are heavily harvested for trade in South Africa’s traditional ‘muthi’ markets. Over-collection of Eucomis species has seriously depleted natural populations and now Eucomis plants are among the 15 scarcest medicinal species to be traded. Micropropagation is a useful technique for rapid clonal multiplication of plant material which could potentially yield useful secondary metabolites as well as alleviate the pressure on the wild plant populations. The in vitro induction of storage organs is especially beneficial as it can limit the loss of plants during acclimatization as bulblets are hardier than shoots or plantlets. The aim of this research was to determine optimal growth conditions for bulblet induction of Eucomis zambesiaca. The effect of environmental and physiological parameters on the initiation and growth of bulblets was investigated. These included the effect of temperature, photoperiod, various carbohydrates at different concentrations and combinations as well as various plant growth regulators. Maximum number of bulblets per explant was obtained at 20 °C, with an average of three bulbs p er leaf explant. The average bulblet mass was 57 mg, which was significantly higher than bulblets formed at other tested temperatures. An 8 h light regime was the optimum photoperiod. The highest mean number of bulblets (1.4 per leaf explant) developed under the 8 h photoperiod and the bulblets that formed were large in size. They had a mean bulb diameter of 3.4 mm and a mean bulb weight of 42 mg. Different carbohydrates such as fructose, sucrose and glucose were tested at concentrations of; 1, 3, 6, 9 and 12%. Fructose at a concentration of 3% was found to produce the best results. An average of 1.2 bulbs formed per explant. The mean bulb diameter was 3.4 mm and mean bulb weight was 56.6 mg. Plant growth regulators (GA3, IAA, IBA, NAA, BA, zeatin, iP and others) were tested at concentrations of 1, 2 and 5 mg/L. 1 mg/L IBA was found to be the optimum hormone treatment for bulblet induction. Bulblets were large, had good leaves and well established roots. Medium supplemented with 1 mg/L IBA produced bulblets that had an average bulb diameter of 4.36 mm and a mean bulblet weight of 79.1 mg. Bulblets grown in vitro were transferred to vermiculite and placed in a misthouse to acclimatize. After 2 months the plantlets were transferred to pots containing a sand:soil mixture of 1:1 and placed in a greenhouse. There was a 80 to 90% survival rate.
Some aspects of megagametophyte development and post-shedding seed behaviour of Encephalartos natalensis (Zamiaceae)
(2009) Woodenberg, Wynston R.; Pammenter, Norman William.; Berjak, Patricia.
Very little is known about the post-shedding seed behaviour and megagametophyte development of the cycads, the most primitive extant seed-bearing plants, which pre-date the dinosaurs. In the present investigation, seeds of Encephalartos natalensis Dyer and Verdoorn were shed with relatively high mean embryo (3.33 g g-1) and megagametophyte (1.25 } 0.16 g g-1) WCs, when the developing embryo consisted primarily of the coiled, elongated suspensor bearing a rudimentary sporophyte at its tip. It was not surprising that these seeds were revealed as desiccation sensitive in the present investigation, as the embryos continued to develop after seed-shed, reaching a germinable size (.15 mm) only 4 . 6 months after seed abscission from the strobilus. Maintenance of the seeds in hydrated storage conditions was precluded by the proliferation of fungi, despite the application of the fungicide: BenlateR. Some seeds were also found to germinate in hydrated storage, despite the hard physical barrier to germination imposed by the enclosing sclerotesta. Seeds dusted with BenlateR and placed in eopen f storage in loosely closed paper bags had a longer life-span than those placed in hydrated storage; however, seeds stored in open storage were also overcome by fungi, but only around 18 months after seed-shed. Therefore, while the vigour and viability of the seeds appeared to decline slowly in the months after the embryos reached a germinable size, the life-span of stored E. natalensis seeds devoid of fungi is yet to be determined and will be the subject of further research. The current investigation also combined ultrastructural and viability retention studies to observe the post-shedding behaviour of the storage tissue, the megagametophyte. The cells of the megagametophyte became progressively packed with starch and protein as the two main storage reserves, a limited number of discrete lipid bodies, and occasional mitochondria all of which appeared to be embedded in an homogeneous matrix. When the development of the megagametophyte cells was analysed ultrastructurally, it was found that the unusual matrix was present from the inception of megagametophyte cellularisation, and contained microtubules and numerous very faintly-visible vesicles. Newly-formed megagametophyte cells were thus not highly vacuolated as previously thought, but dominated by an homogeneous matrix. Enzyme-gold localisation was employed in an attempt to determine the organelles responsible for the deposition of cell wall components during cellularisation of the megagametophyte. It appeared that ER-derived vesicles (and not Golgi-derived vesicles) were the principal contributors of the primary cell wall components, pectin and xylan. While cellularisation took place over approximately 1 - 2 weeks, subsequent development of the megagametophyte cells involved the accumulation of storage reserves, this phase lasting approximately 8 months -when the seeds were shed whether pollination/fertilisation had recently occurred, or not. At seed-shed, the cells of the megagametophyte were nucleated and contained a few mitochondria of a metabolically-active appearance. The occurrence of aerobic metabolism in these cells was confirmed by the tetrazolium (TTZ) test. Judging from the TTZ reactivity, the viability of the megagametophyte cells of fertilised seed appeared to decline slowly in the months after seed-shed, in parallel with extension growth of the embryo. The cell layer comprising the external surface of the megagametophyte showed marked ultrastructural differences from the inner cells, and may emerge as having an ‘aleurone-like’ function. It is, however, possible that the cells of the body of the gametophyte participate actively – at least in the earlier stages of post-shedding seed development – in mobilisation of stored reserves, which must support the development of the embryonic sporophyte.
The influence of root chilling on the hydraulic characteristics of selected Eucalyptus taxa.
(2008) Cheek, Michael.
The hydraulic conductance of a plant is a significant factor in determining the
Genetic analysis of Chaerephon pumilus (Chiroptera: Molossidae) from southern Africa.
(2008) Reddy, Devendran.
Chaerephon pumilus, the little free-tailed bat, (family: Molossidae) has a distribution throughout most of sub-Saharan Africa and the eastern region of Madagascar. The vast geographical distribution of this species is accompanied by considerable phenotypic variation, which may conceal cryptic species. The cytochrome b (845 nucleotides) and D-loop (314 nucleotides) regions of the mitochondrial DNA were sequenced to assess phylogenetic relationships within C. pumilus (southern Africa) and in relation to Chaerephon species from Madagascar (C. pumilus, C. leucogaster). Samples were obtained from KwaZulu-Natal, South Africa, and localities in Swaziland. The cytochrome b sample (n = 11) comprised four haplotypes, with a haplotype diversity of 0.6727, whilst the D-loop (n = 34) dataset comprised 13 haplotypes with a haplotype diversity of 0.8342. Neighbour joining, maximum parsimony and Bayesian analyses revealed congruent tree structures for both mtDNA regions. All Chaerephon taxa in this study formed a monophyletic clade with respect to the outgroup Mops midas. Chaerephon pumilus from the eastern side of Madagascar formed a well-supported monophyletic group, sister to a clade comprising C. pumilus (southern Africa) and C. leucogaster, and is suggested to comprise a separate species. Southern African C. pumilus formed two paraphyletic clades, A and B, separated by a genetic distance of 0.9 %. Chaerephon leucogaster formed a monophyletic group nested within southern African C. pumilus, suggesting conspecificity. However, the well-characterized morphology of C. leucogaster lends support to its specific status, and suggests the possible existence of cryptic species among southern African C. pumilus. Population genetic analysis suggests that two C. pumilus (southern African) clades have been expanding, one for between 2432 and 4639 years, and the other for the 11156 to 21280 years. A combined cytochrome b analysis, trimmed to 343 nucleotides, was carried out on the data from this study and that of Jacobs et al. (2004), also on southern African C. pumilus. Haplotypes from the Jacobs et al. (2004) study, which also identified two 0.9 % divergent clades (light- and dark-winged) were found to be identical or very similar to haplotypes from this study and were interspersed among southern African C. pumilus haplotypes in phylogenetic analyses. Chaerephon pumilus haplotypes from Zambia and Tanzania were found to be more closely related to those from southern Africa and to C. leucogaster than to C. pumilus (Madagascar), further indicating that this may be a separate species. Haplotypes from the light-winged clade of Jacobs et al. (2004) were identical to those of dark-winged samples from this study, suggesting that wing shade may not be diagnostic of the two clades.
Some investigations towards the cryopreservation of sugarcane germplasm.
(2009) Jaimangal, Ashika.; Pammenter, Norman William.; Berjak, Patricia.
Sugarcane has become an increasingly important crop in recent years, with South Africa featuring as one of the prominent producers. This has led to a significant growth in the South African sugarcane industry, translating into an increased demand for planting material. Although this demand is now satisfied by recent biotechnological advancements such as protocols for somatic embryogenesis to increase the production of planting material, such techniques are limited as a result of the progressive loss of the embryogenic potential of calli over time. In order to facilitate management of this material, it is desirable to develop a protocol for the long-term storage of the germplasm. This study reports on investigations of the different parameters that influenced the cryoprocess in attempts to develop a protocol for the successful cryopreservation of sugarcane somatic embryos of the 88H0019 variety. Experiments were carried out to determine in vitro culture conditions for successful induction of somatic embryos via both the direct and indirect routes of micropropagation. A suitable regeneration medium for plantlet establishment pre- and post-cooling was established (Chapter 2). Investigations were also carried out to ascertain the responses of somatic embryos to both rapid and slow dehydration techniques (Chapter 3). Finally, several cooling techniques (both slow and rapid), were applied, on partially dehydrated somatic embryos, either without, or after cryoprotection, in an attempt to achieve survival after cryopreservation of the somatic embryos (Chapter 4). Both directly- and indirectly-derived somatic embryos were converted, most successfully, on full strength Murashige and Skoog medium without addition of plant growth regulators. The initial mean water contents of directly- and indirectly-derived somatic embryos were not significantly different from each other (8.38±0.19 g g-1 and 8.45±0.33 g g-1 [dry mass basis], respectively). The percentage conversion at these water contents was also not significantly different; 97% for directly- and 98% for indirectlyinduced embryos. Slow dehydration by culture on a series of media with increasing concentrations of sucrose (from 0.2 M to 1.2 M) for a period of 48 h each was the most effective technique, with water content being reduced to 0.94±0.03 g g-1 and 0.95±0.02 g g-1 after dehydration on media containing 1.0 M sucrose, while maintaining between 98% and 100% conversion, respectively. Of the various cryoprotectants tested, proline and casamino acid had the least adverse effects on the somatic embryos. The encapsulation-vitrification cooling technique was the most efficient of all techniques employed. The best conditions involved encapsulation of embryo clumps in a solution of MS medium with 3% (w/v) Na-alginate and loading solution containing 2 M glycerol plus 0.4 M sucrose, followed by infiltration and dehydration at 0°C for various time intervals (0, 5, 10, 15, 20, 25, 30 min) with 1 ml PVS2 solution and thereafter, rapid immersion in liquid nitrogen. Under such conditions, 30% of the cryopreserved somatic embryos retained viability, going on to form callus from which shoots and roots were produced. Although somatic embryos of sugarcane of the local variety 88H0019 have proved to be recalcitrant to cryopreservation, the results obtained with explants that had been processed by encapsulation-vitrification suggest that this approach may be worth pursuing and refining.
Leaf ultrastructural studies of Avicennia marina in response to salinity under natural conditions.
(2007) Hiralal, Omitha.; Naidoo, Gonasageran.; Naidoo, Yougasphree.
In Richards Bay Harbour, the mangrove Avicennia marina exhibits a distinct natural productivity gradient. The fringe site, which is regularly inundated twice daily by tides, supports luxuriant adult A. marina trees that are 6-10 m tall and which form a dense, well-developed canopy. The landward site which is only inundated during high spring tides, supports diminutive or dwarf A. marina that are less than 1.5 m in height. In this study we compared leaves from fringe and dwarf sites with respect to morphology, ultrastructure and ecophysiology. Alterations in leaf morphology, ultrastructure and physiology of A. marina were compared at the fringe site (35 ‰) and dwarf site (60 ‰) using morphometric measurements, light (LM), transmission (TEM) and scanning microscopy (SEM). SEM and light microscopy revealed that multicellular salt glands were located on the thick, cutinised adaxial surface from leaves of both sites. The glands appeared to be scattered and protruding from individual crypts in fringe mangrove leaves whilst they appeared sunken and occluded by cuticular material in dwarf mangrove leaves. The salt glands on the abaxial surface were not sunken but obscured by the indumentum of peltate trichomes. Ultrastructural changes observed in dwarf mangrove leaves were associated with cuticle, cell walls, chloroplasts, mitochondria of mesophyll tissue and salt glands. Fringe mangrove leaves had chloroplasts with typical well-developed grana and stroma. Ultrastructural changes of chloroplasts were evident in dwarf mangrove leaves and included swelling and separation of thylakoids, disintegration of granal stacking and integranal lamellae, as well as loss of the integrity of the chloroplast envelope. Multivesicular structures were commonly found in vacuoles and associated with chloroplasts and mitochondria in both leaf types. In fringe mangrove leaves, mitochondria appeared spherical to tubular with a relatively smooth outer membrane and a highly convoluted inner membrane. Swelling and vacuolation of mitochondrial membranes, cristae and mitochondrial clustering in the cytoplasm around the chloroplasts were evident in dwarf mangrove leaves. Extensive lipid accumulation in the form of large, dense plastoglobuli occurred in the chloroplasts of dwarf mangrove leaves. There were characteristic differences in salt gland morphology of fringe and dwarf mangrove leaves, namely in the cell walls, vacuoles, and vesicle formation. In salt glands of dwarf mangrove leaves, a distinct withdrawal of the cytoplasm from the cell wall was observed. This feature was not observed in salt glands of fringe mangrove leaves. Numerous large vacuoles were observed in the secretory cells of glands of dwarf mangrove leaves compared to those of fringe plants. Multivesicular structures, vesicles and mitochondria were common features in both leaf types. Physiological studies involved a comparison of osmotic and ionic relations as well as whole plant responses in fringe and dwarf mangrove leaves. Relative leaf water content decreased by 7.8 % and specific leaf area by 17 % in dwarf compared to those of fringe mangroves. Dwarf mangrove leaves were 27.6 % thicker and leaf cuticle thickness 37.4 % higher than those from fringe mangroves. Fringe mangrove leaves displayed higher total chlorophyll contents by 27 %, with chlorophylls a and b being 22 % and 39.6 % higher, respectively than those of dwarf mangroves. Salt gland frequencies were higher in the apex, mid-lamina and base of fringe than dwarf mangrove leaves by 36 %, 45 % and 51 %, respectively. The concentration of glycinebetaine, a compatible, N-containing osmolyte was significantly higher by 40 % in dwarf than in fringe mangrove leaves. Concentrations of proline were 27 % lower in dwarf than in fringe mangrove leaves. The predominant inorganic ion detected in mature leaves was Na+, which was 19 % higher in dwarf than fringe mangrove leaves. Phosphorus was an element that appeared deficient in dwarf mangrove leaves, being 50 % lower compared to fringe mangrove leaves. The results of this investigation indicated that there were cytomorphological alterations as well as differences in physiological responses in leaves of A. marina at fringe and dwarf sites.
The effect of developmental status and excision injury on the success of cryopreservation of germplasm from non-orthodox seeds.
(2007) Goveia, Meagan Jayne Theresa.; Kioko, Joseph Ivala.; Berjak, Patricia.; Pammenter, Norman William.
The zygotic germplasm of plant species producing desiccation-sensitive seeds can be conserved in the long-term only by cryopreservation. Usually the embryonic axis is excised from the cotyledons and is used as the explant for cryopreservation as it is small and provides a large surface area:volume ratio. However the shoot of the axis of most species studied does not develop after excision, with the result that survival after cryopreservation is often recorded as callus production or simply explant enlargement and/or greening. Thus, besides explant size, factors such as in vitro regeneration techniques, physical injury induced upon excision and developmental status of the seed could compromise the success of cryopreservation. This study investigated the effect of the factors mentioned above, with particular attention to the developmental status of the seeds on explant in vitro development (section 3.1), response to dehydration (section 3.2) and cryopreservation of the desiccation-sensitive embryonic axes (section 3.3) of two species: Trichilia dregeana, T. emetica and embryos of a third, Strychnos gerrardii. For all three species, investigations were conducted on the embryonic axes/embryos excised from mature seeds immediately after fruit harvesting and from mature seeds stored under hydrated conditions for different periods (in order to achieve different degrees of development). In addition, preliminary studies were carried out on axes of T. dregeana to assess whether generation of reactive oxygen species (ROS) occurs in response to wounding upon axis excision (section 3.4). Excised embryonic axes of T. dregeana and T. emetica did not develop shoots in vitro unless the explants included attached cotyledonary segments. Following the development associated with short-term storage, however, the excised axes could develop shoots after complete cotyledon excision. The embryos from the (endospermous) seeds of S. gerrardii which included the paper-thin cotyledons, developed normally in vitro, with percentage germination increasing with seed storage time. For all three species, in vitro axis germination was promoted when activated charcoal was included in the germination medium, regardless of the developmental stage of the seeds. When dehydrated to approximately 0.3 g H2O g-1 dry mass (g g-1), embryonic axes from all three species failed to develop shoots even though a minimum of 50% produced roots in all cases. Hence, shoot production was shown to be more sensitive to desiccation than was root production. Furthermore, the sensitivity of the shoot apical meristem to desiccation was not ameliorated with seed storage for T. dregeana and T. emetica, but did decrease for S. gerrardii when seeds were stored for 6 – 8 weeks. The application of certain cryoprotectants did facilitate production of shoots after dehydration by a few axes of both Trichilia spp. For T. dregeana explants, combination of glycerol and sucrose allowed for 10% of the axes to retain the ability for shoot production after dehydration while for T. emetica explants, the combination of DMSO and glycerol (10 - 20% shoot production after dehydration) was best. The efficacy of the cryoprotectants was not influenced by storage period. The provision of cryoprotectants still needs to be tested for S. gerrardii. Survival of subsequent cryopreservation of T. dregeana and S. gerrardii explants was best achieved with rapid cooling in nitrogen slush, with the cooling procedure for T. emetica explants still to be optimized. The highest post-cryopreservation survival of T. dregeana axes was achieved when seeds had been stored for three months, while the seed storage period did not affect post-thaw survival of the axes of T. emetica or S. gerrardii. A small proportion of S. gerrardii explants only, produced shoots after cryopreservation, whereas the surviving embryonic axes of T. dregeana and T. emetica regenerated only as non-embryogenic callus. Although callus production is less desirable than successful seedling establishment, it has the potential for micropropagation if embryogenicity can be induced. Ultrastructural examination of the shoot apical meristem of T. dregeana after a 3-d recovery period, following excision, revealed considerable cellular derangement, although damage of individual organelles could not be resolved microscopically. Preliminary studies on T. dregeana involving a colorimetric assay using epinephrine, confirmed the generation of ROS in response to wounding associated with axis excision. Reactive oxygen species generated appeared to persist over prolonged periods rather than occurring only as a single oxidative burst. Hence, ROS production at the wound site could be the primary factor contributing to lack of shoot development. Axes immersed in the anti-oxidant, ascorbic acid (AsA) immediately after excision, showed lower ROS production and 10% shoot development when cultured in vitro, indicating that the oxidative burst coincident with, and after excision might be counteracted if immediate ROS production can be adequately quenched. Future investigations should aim to identify the specific ROS associated with wounding and optimize an anti-oxidant treatment(s) that will facilitate shoot development. Thus, the successful cryopreservation of the germplasm of the species tested, and others producing recalcitrant seeds, depends on a spectrum of species-specific factors, some still to be elucidated.
Pharmacology and phytochemistry of South African traditional medicinal plants used as antimicrobials.
(2009) Fawole, Olaniyi Amos.; Finnie, Jeffrey Franklin.; Van Staden, Johannes.
Among all the major infectious human diseases, gastro-intestinal infections caused by microbial pathogens are a major cause of morbidity and infant death in developing countries, largely due to inadequate sewage disposal and contaminated water. Traditional health practitioners in South Africa play a crucial role in providing health care to the majority of the population. Many plants are locally used by South African traditional healers to treat microbial infections related to gastro-intestinal tracts. Ethnopharmacological and ethnobotanical studies using traditional knowledge as a selection strategy has given priority to certain plants for isolation and identification of plant novel bioactive compounds. Pharmacological and phytochemical studies of the investigated twelve medicinal plant species (from 10 families) extensively used as antimicrobials against gastro-intestinal infections was necessary to validate the use of the plants. Furthermore, to provide sufficient preliminary information for the isolation and identification of active compounds that are present in the investigated plants. Plant parts were sequentially extracted using petroleum ether (PE), dichloromethane (DCM) and 70% ethanol (EtOH). Cold water and boiled (decoction) extracts of the plant materials were prepared non- sequentially. Among the extracts, EtOH yielded the highest amount of plant substances. A total number of 85 extracts were evaluated for antibacterial activity, 80 for antifungal activity, 64 for anti-inflammatory activity, and 27 biologically active extracts were tested for genotoxicity. The microdilution method was used to determine the minimum inhibitory concentration values in the antibacterial assay against two Gram-negative bacteria (Escherichia coli ATCC 11775 and Klebsiella pneumoniae ATCC 13883) and two Gram-positive bacteria (Bacillus subtilis ATCC 6051 and Staphylococcus aureus ATCC 12600). A modified microdilution method was used to determine the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) values in the antifungal assay against Candida albicans. Cyclooxygenase assay was used to evaluate the anti-inflammatory activity of the extracts against cyclooxygenase-1 and -2 (COX-1 and COX-2) enzymes. The plant extracts were screened first at a concentration of 250 ƒÊg/ml per test sample, and then further screened at concentrations of 125 and 62.5 ƒÊg/ml for extracts that inhibited the COX-2 enzyme. The Ames test was used to test for genotoxicity in extracts that showed interesting pharmacological activities using Salmonella typhimurium strain TA98. Among the screened extracts, 25 extracts showed good antibacterial activity with MIC values . 1.0 mg/ml. Dichloromethane extracts exhibited the greatest antibacterial activity, and Gram-positive bacteria were most susceptible. The best antibacterial activity was exhibited by Becium obovatum leaf EtOH extracts with an MIC value of 0.074 mg/ml. A broad spectrum antibacterial activity was observed by leaf extracts of Cucumis hirsutus (PE), Haworthia limifolia (PE), Protea simplex (PE and DCM) and Dissotis princeps (EtOH) against both Gram-negative and Gram-positive bacteria. No interesting antibacterial activity was exhibited by water extracts with the exception of Dissotis princeps water extract with a good antibacterial activity against Gram-positive and Gram-negative bacteria. In the antifungal assay, 6 extracts showed interesting antifungal activity. Protea simplex leaf PE extract showed the best fungicidal activity with an MFC value of 0.014 mg/ml. The best overall antifungal activity was observed in plant EtOH extracts. Some extracts from Agapanthus campanulatus (leaves and roots), Dissotis princeps (leaves), Gladiolus dalenii (corms) and Protea simplex (leaves) showed good activity against Candida albicans. Twenty one extracts inhibited the COX-1 enzyme, while fifteen extracts inhibited the COX-2 enzyme at the lowest screening concentration of 62.5 ƒÊg/ml. The highest COX-1 inhibition at a concentration of 62.5 ƒÊg/ml was exhibited by Diospyros lycioides leaf PE extract (89.1%) while Agapanthus campanulatus root DCM extract showed the highest COX-2 inhibitory activity (83.7%) at the same concentration. In the Ames test, no genotoxicity was observed in any of the extracts, however more tests need to be done to confirm these results. Thin layer chromatograms of the organic solvent plant extracts were developed. The fingerprints of the plant extracts showed colours of bands at different Rf values when viewed under UV254 and UV366 suggesting that the investigated plant species contained different compounds in the extracts. In the quest to understand the source of the plants pharmacological activities, total phenolic compounds including condensed tannins, gallotannins and flavonoids were quantitatively investigated in terms of their amounts in the aqueous methanol extracts of the plants materials using spectrophotometric methods. Alkaloids and saponins were qualitatively determined. The amounts of total phenolics were determined by the Folin Ciocalteu assay, condensed tannins were determined by the butanol-HCl assay, while rhodanine and vanillin assays were used to determine the amounts of gallotannins and flavonoids respectively. Dragendorff reagent was used to detect alkaloids in the plant extracts on thin layer chromatographic plates, while the froth test was employed to detect saponins. Secondary metabolites varied with plant parts and species with Cyperus textilis (leaf) having the highest amounts of total phenolics, condensed tannins and flavonoids. The highest amount of gallotannins was detected in Protea simplex leaf extracts. All the investigated plant materials with the exception of Haworthia limifolia leaf, Protea simplex leaf, Antidesma venosum leaf and Dissotis princeps leaf tested positively to saponins. Alkaloids were detected in Haworthia limifolia leaf (PE and EtOH), Cucumis hirsutus leaf (EtOH), Becium obovatum root (DCM), Protea simplex root and bark (EtOH), Agapanthus campanulatus root (DCM) and leaf (EtOH), Cyperus textilis root (DCM), Vernonia natalensis leaf (PE), Antidesma venosum leaf (PE), Diospyros lycioides leaf (PE) and Dissotis princeps leaf (DCM) extracts. The results obtained from the investigation of the pharmacology and phytochemistry of the plant species used to treat microbial infections related to gastro-intestinal tracts, provide sufficient preliminary information to validate the use of some of the plants in traditional medicine. The information provided might be considered sufficient for further studies aimed at isolating and identifying the active compounds in the plant species, and evaluating possible synergism amongst the isolated compounds.
Establishment of an indirect organogenesis protocol for Eucalyptus grandis species and hybrids.
(2004) Hajari, Elliosha.; Watt, Maria Paula Mousaco Deoliveira.
The prospect of integrating transgenic eucalypts with conventional breeding programmes is of value to the Plantation Forestry and Forest Products Industries. However, significant progress in this regard has still to be reported, one constraint is the lack of appropriate high yielding regeneration culture methods for clonal material. Such was the main aim of the present study. The strategy was to develop a suitable protocol using in vitro shoots of an E. grandis x E. urophy/la clone (GU185) and thereafter to test its applicability to other clones. Explants from greenhouseestablished cuttings provided the in vitro shoots, which were multiplied via axillary bud proliferation either on semi-solid medium or using a RIT A system. To determine the best conditions for callus and shoot regeneration, parameters such as vessels (petri dishes and tubes) and types and levels of plant growth regulators were tested. The best callus production (100%) and shoot regeneration (78.9 - 100% callus with shoots) for GU185 occurred on MS, 30 g rl sucrose, 4 g rl Gelrite, 5 mg rl IAA and 0.25 mg rl BAP. Parameters tested to identify the most suitable explants for indirect organogenesis were the age of parent plants, different systems to generate in vitro shoots, elongation status of explants, 1 sI and 2nd generation in vitro shoots and the use of hyperhydric shoots. Of these, the most suitable explants for indirect organogenesis were shoots from axillary bud multiplication of 3-month-old parent plants using the semi-solid system (33 shoots/dish). Up to 90% rooting was achieved on 1f4 MS (Murashige and Skoog, 1962), 15 g rl sucrose, 0.1 mg rl biotin, 0.1 mg rl calcium pantothenate, 4 g rl Gelrite and mA. The highest rooting was obtained when regenerated shoots were first multiplied and then placed on medium without plant growth regulators for one week, before transfer to root induction medium containing 0.1 - 0.5 mg rl mA. Acclimatization success was 95% when rooted shoots were placed in pots with a rooting mix (2 perlite: 1 coir) enclosed in plastic bags and the humidity was gradually reduced over four weeks. The developed indirect organogenesis protocol appeared to have a broad general application, although the tested clones exhibited a genotype-dependent response, with GU180, GUI77 and TAG31 producing fewer shoots (9, 6 and 7 shoots/dish) than ZG14 and GU185 (24 and 18 shoots/dish). Similarly high levels of rooting were obtained for TAG3l (93.8%) and ZG14 (90%) and for hardening-off (90.7% for TAG31 and 91.4% for ZG14).
A revision of the genus Ledebouria Roth (Hyacinthaceae) in South Africa.
(1993) Venter, Stephanus.; Edwards, Trevor John.
Members of the genus Ledebouria Roth (Hyacinthaceae), which occur in South Africa, are revised. This genus occurs throughout Africa, India and Madagascar. 33 Species are recognized and placed into nine provisional infrageneric groups. A multidisciplinary approach was used in an attempt to provide natural groupings. The following characters were analysed; morphology, micromorphology, palynology and caryology. Aspects of ovary structure and leaf micromorphology proved especially useful in the synthesis of infrageneric and specific concepts. Keys, descriptions, illustrations, distributional, ecological, medicinal and toxicological data are provided. This study is based on plants in their natural habitat, cultivated specimens and representative herbarium specimens from herbaria in South Africa and in Europe.
Above-ground allometry, biomass and nutrient content of acacia mearnsii across four ages and three sites in the Kwazulu-Natal Midlands.
(2005) Dovey, Steven B.; Pammenter, Norman William.
Acacia mearnsii (black wattle) is one of the few tree crops of which both timber and bark are utilised, with branches sometimes being collected for fuel wood. There is a great potential for nutrient loss from plantations with intense harvesting practices. Allometric relationships were developed to estimate above-ground biomass across four ages and three site qualities of A. meamsii stands. The three sites were based on high, medium and low site quality classes of productivity. Differences in biomass and the distribution of biomass between the stem, bark, live branches, dead branches and foliage components are described in relation to site and age. Relationships between biomass and light interception and plant area index are investigated and show some merit. Nutrient concentrations were used with the above-ground biomass data to estimate quantities of nutrients held in the various biomass components in each of the stands. Nutrient distributions in the above-ground biomass (AGB) were examined and compared to other studies. Foliar phosphorus (P) and potassium (K) concentrations were shown to be sub optimal, agreeing with results and recommendations of South African fertilisation stupies. Some concentration differences were observed between site and age classes for certain nutrients, although these differences may have been due to seasonal effects. Biomass and nutrient quantities were adjusted to yield a wood production of 100 t ha01 and compared with adjusted calculations for similar studies on other plantation crops. Total nutrients contained in the AGB of the adjusted calculations were 540.8 kg ha01 nitrogen (N), 20.4 kg ha-I P, 200.6 kg ha01 K, 241 kg ha-I calcium (Ca), 55.7 kg ha-I magnesium (Mg), with a half to two thirds of the nutrients held in the stem and bark alone. Nutrients losses vary with harvesting intensity as bark and branches may be harvested with the stem wood. Levels of nutrient removal with harvesting intensity are discussed with reference to estimated losses and gains from natural processes and management practices. An incomplete nutrient budget calculation indicated that P, K, Ca and Mg might potentially be removed in quantities greater than replaced by natural processes under stem and bark harvesting. The budget calculations lack processes such as leaching and N-fixation. It is highly probable that these processes, once quantified, may yield more negative budget results, especially for the base cations K, Ca and Mg.
The use of a virtual world to address misconceptions held by students regarding photosynthesis and respiration.
(1998) Adams, Jillian Claire.; Amory, Alan M.; Criticos, C.
In an effort to contribute to the improvement of Matric Biology education, a survey was conducted in 1996 and 1997 of Matric pupils and first year students at several tertiary institutions, in order to identify those topics which learners found most difficult. Photosynthesis and respiration were among the topics with which Matric Biology learners experienced many conceptual difficulties. The aim of this project was twofold: firstly to identify specific misconceptions students had regarding these topics, and secondly, to develop and evaluate a learning tool that would address these misconceptions. In order to identify the most common specific misconceptions, a quantitative research approach was taken. A three-tiered multiple choice questionnaire was developed, and administered to first year students in the 1998 intakes at MLSultan Technikon and the Biology Department of the University of Natal, Durban. It was also administered to students at the end of their first, second and third years of Cell Biology (University of Natal), Analysis of the questionnaires revealed that students did not understand the complementary relationship between photosynthesis and respiration. Computer-based virtual worlds provide constructivist learning environments, in which visualisation and problem solving in a complex system is possible. It was proposed that use of a virtual world would be an effective means of addressing the misconceptions we identified. A game was developed that presented students with authentic tasks of filling an oxygen cylinder (as an air supply) and a carbon dioxide cylinder (which would later be used to extinguish a fire). In order to do this students were required to solve a series of three puzzles, all of which related to the processes of photosynthesis and respiration. To account for different learning styles, the puzzles were based on three of Gardner's multiple forms of intelligence. Evaluation of the virtual world made use of a combination of quantitative and qualitative research methods. Students' understanding of the processes was measured with the use of the questionnaire. A deeper evaluation of their understanding and affective response to the game was obtained through interviews. It was found that students who had played the game had a clearer understanding of the complementary relationship between photosynthesis and respiration, and understood that respiration in plant cells is a continuous process. Students also showed greater confidence in their grasp of the processes, and reported that playing the game had been an enjoyable way of complementing their traditional lecture material in order to master these concepts. The virtual world was an effective learning tool for addressing the misconceptions students held regarding photosynthesis and respiration.
Comparision of two promoters driving transgene expression in water-stressed sugarcane.
(1999) Cassim, Tasmien Nadine.; Huckett, Barbara Isobel.; Botha, Frikkie Coenraad.; Watt, Maria Paula Mousaco Deoliveira.
For the expression of transgenes in plant cells, appropriate promoter sequences have to be introduced upstream of the gene to ensure efficient transcription. Tissue- or signal-responsive promoters are in high demand in practical plant biotechnology. The present study sought to characterise the activities of two promoters in sugarcane, namely the UBI (ubiquitin) promoter and the SUC-1 promoter (UBI linked in tandem to the cauliflower mosaic virus 35S promoter). It was hypothesised that the activity of UBI would be maintained or even increased under conditions of environmental stress, since it is well documented that ubiquitin is a stress-related protein. A further hypothesis was that SUC-1 might enhance overall gene expression since the CaMV 35S component is a constitutive promoter widely and successfully used in plant transformation. Plants of the sugarcane variety NC0310, containing the cry1A(c) (Bt) gene from Bacillus thuringiensis, were used as models in a system in which the plants were stressed by withholding water supply in a controlled manner. Since large numbers of clones of both transgenic and wild-type plants were needed for the water stress and expression experiments, three micropropagation techniques, namely, shoot tip-, callus- and node culture, were optimised and compared. The objective was to propagate genetically stable plants rapidly. Compared to shoot tip culture, node and callus culture proved slow and inefficient. Shoot tip culture was thus chosen as the most suitable for the regeneration of experimental material. Relative Water Content (RWC) determination, leaf elongation measurements and Infra Red Gas Analysis (IRGA) were compared in order to find the most appropriate method of measuring plant water status. In addition to being destructive, no observable differences were evident between the control (non-stressed) and water-stressed plants when using RWC as a measure. Results obtained from leaf elongation measurements compared favourably to the more sophisticated IRGA readings, showing that leaf elongation is as sensitive a measure of water stress. On the basis of preliminary studies with untransformed plants using the latter two techniques, water regimes for stress-induction in the final experiments were designed. Leaf elongation measurements, which are simple and non-destructive, were ultimately chosen to measure plant water status. In the final water stress experiment non-transgenic NCo310 and clonal populations of six transformants were used (three containing the UBI promoter; three the SUC-1 promoter). Exactly half of the plants of each type were stressed by withholding water supply, while the other half (controls) were watered manually twice a day. Leaf elongation measurements were made at the same time daily on the third youngest leaf of 6 plants from each population per treatment. At the same time, leaf samples were taken daily for molecular analysis. The stress regime led to marked differences in leaf elongation between control and water-stressed plants. In terms of physiological response (leaf rolling and senescing), plants containing the SUC-1 promoter appeared least affected. The reverse transcription-polymerase chain reaction (RT-PCR) and Northern hybridisation were used to assay UBI and SUC-1 activity. RT-PCR revealed that both promoters drove Bt gene expression in controls and experimentals throughout the stress period, although differences in signal intensity were not observed. The extent of expression occurring in each type of plant was revealed in Northern blots probed with two genic sequences (1) the transgene and (2) sugarcane EST ME42, homologous to heat shock protein 82 in rice. Individual transformants showed overall levels of transgene expression that were variable, possibly due to insert position in the plant genome, as well as variations in relation to the application of stress. SUC-1 seemed superior to UBI in terms of driving transgene expression under stressful environmental conditions, since UBI promoter activity appeared to decrease under stress, while SUC-1 promoter activity remained constant. In addition to the expected 2.0 kb Bt transcript, transcripts of smaller than expected size were also obtained, leading to the suggestion of premature polyadenylation signals in the coding region of the wild-type Bt234 gene. Upon inspection of the transgene sequence, a number of motifs rarely present in plant genes were observed, namely A/T rich sequences, ATTTA motifs and numerous potential polyadenylation sites.
Recirculating hydroponic systems : evaluating cuttings yield and rooting ability of cold tolerant eucalyptus hybrids.
(2004) Wallis, Jacqueline Tanya.; Watt, Maria Paula Mousaco Deoliveira.; Pammenter, Norman William.
In South Africa, clonal forestry of Eucalyptus and its hybrids has been implemented to increase the productivity on existing forestry lands and marginal sites and to facilitate the production of desired fibre types for timber processing operations. The cold-tolerant Eucalyptus grandis x E. nitens hybrids have produced consistently high yields, and are propagated clonally with limited success via a macro-cutting system currently in use for other hybrid species. The heart of vegetative propagation operations is the clonal hedge and its management, and nutrition in particular, is an important element of any vegetative propagation programme. However, achieving and sustaining an optimum nutritional balance in macrohedges is difficult in practice and, in order to accurately predetermine the optimum plant nutrition required all year round and to ensure optimal levels of rooting, a more controllable nutrient environment is essential. Hydroponics may facilitate this control of nutrition. At the same time it may be possible to manipulate the system to determine accurately what levels of each nutrient may contribute to the highest rooting and more importantly allow forest nursery managers to maintain those levels in a practical manner. The main aims of the present work were to obtain and compare cuttings and rooting yields from hydro-ramets in different hydroponic substrates and systems and to investigate the possible roles of essential nutrients on those parameters. Modified Nutrient Film Technique (NFT), ebb-and-flow and aeroponic tables were used in this study. The former consisted of eight individual gutters, allowing for eight different substrates to be tested simultaneously. One gutter was set up as an unmodified NFT table and the other seven gutters had gravel, Leca, peat, perlite, perlite: vermiculite mix, Rockwool® and sand as substrates; all were supplied with the same nutrient solution. Three commercial clones were used throughout these trials: GN107, GN156 and NHOO. Rooting results and data from plant elemental analyses indicated that certain elements (Ca, Cu, Zn, Mn and B) appeared to play a more important role in rooting than others (N, P, K, Mg, Na and Fe). It was also found that when comparing the hydroponic systems, the substrate and / or method of irrigation affected the availability and uptake of different nutrients, which in turn affected the rooting of coppice collected from those ramets. The rooting performance of coppice from the eight different substrates tested in the NFT system was compared. Within each of the four harvests undertaken, both clone and substrate had a significant effect on the rooting performance. However, when the four harvests were compared, only harvest number/time had a significant effect on the rooting performance of the cuttings derived from the hydro-hedges . For both the ebb-and-flow and aeroponics systems (where there was no substrate), only the clone had a significant effect on the rooting performance. In addition to this, the plants from the ebb-and-flow system produced the highest number of cuttings to be placed overall (7.9 cuttings per mother plant per harvest) while those from the gravel substrate had the highest rooting percentage overall (26.9 %). When combining these two factors into a success rate, the perlite substrate rated highest (1.7 rooted cuttings per mother plant per harvest). From a cost efficiency perspective, perlite was the most cost effective substrate, as it required the least initial capital outlay to produce one million rooted clones per year from a hydroponics system (R6 533 655). The plants in the perlite substrate also produced the highest number (6 700) of rooted cuttings per year from 1 000 mother plants with a low cost per plant (R2.33 per rooted plant).
The development of in vitro rooting systems for cold-tolerant Eucalyptus grandis x nitens clones and the assessment of the hydraulic efficiency of roots produced by in vitro vs. cutting propagation.
(1999) Mokotedi, Mompe Edward Oscar.; Watt, Maria Paula Mousaco Deoliveira.; Pammenter, Norman William.
Hybrid clones of the fast-growing Eucalyptus grandis and cold-tolerant E. nitens (GN clones) have been identified by the South African Forestry Industry as being highly suitable for plantations in cold-dry marginal areas. However, one of the main problems regarding their propagation is the difficulty in rooting of cuttings, both in vitro and ex vitro. The aims of this investigation, therefore, were (1) to develop widely applicable and efficient in vitro rooting system(s) for these commercially important clones, and (2) to assess some physiological characteristics of the roots produced. Adventitious shoots (15-20 mm in length) were obtained (l0 shoots/explant) from axillary buds on Murashige and Skoog's (MS) medium containing 0.01 mg.l-1 NAA, 0.01 mg.l-1 IBA and 0.2 g.l-1 FAP. The effect of various medium components, as well as modification of culture environment on in vitro rooting, were investigated. The highest rooting frequencies in clones GN121 (75%) and GN107 (65%) were achieved on l/4 MS with additional 0.22 g.l-1 CaCl2..2H2O and 0.18 g.1-1 MgS04.7H2O, 0.1 mg.l-1 IBA, 0.1 mg.l-1 biotin, 0.1 mg.l-1 calcium pantothenate, 15 g.1-1 sucrose and 4 g.l-1 Gelrite. Best culture conditions were an initial 72-hours dark incubation followed by a 16-hours day/8-hours night photoperiod at a PPFD of 37 µmol.m-2.s-1 and 23°C day/21°C night for seven days, after which the PPFD was increased to 66 µmol.m-2.s-1 at 27°C day/21°C night for 18 days. Towards the development of a more widely applicable in vitro rooting protocol for GN clones, the use of Agrobacterium rhizogenes strains was investigated. Production of transgenic roots was observed on carrot discs and shoots from seedlings of Eucalyptus grandis and E. nitens, but not on shoots of GN clones. Therefore, a method needs to be established for the successful transfer and integration of the Ri plasmid of Agrobacterium into the hybrid plant genome for induction of transgenic roots. The quality of roots produced in vitro and from cuttings was assessed by examination of root anatomy and hydraulic characteristics. Adventitious roots were prepared for measurement of hydraulic conductivity by detopping explants, then filtered, acidified distilled water was drawn through undisturbed potted root systems under partial vacuum, causing no damage to the roots. Initial studies showed that tissue culture-derived roots exhibited a higher specific root mass hydraulic conductivity than those derived from cuttings (6.46 x 10-6 vs. 3.06 X 10-6 g.kPa-1.s-1.g-1 dry root), probably due to root architecture. Curves relating vulnerability to water potential were constructed and both types of roots showed vulnerability to cavitation at high water potentials. Differences were also observed in staining reactions (safranin and fastgreen) which might suggest differences in presence and level of secondary metabolites in these roots at the juvenile stage. Applications of the developed protocols and future research strategies are discussed.
Development of novel antibacterial and antiviral transgene vectors and techniques for their application and analysis in sugarcane.
(2002) Pepper, Timothy Bryan.; Watt, Maria Paula Mousaco Deoliveira.; Rutherford, Richard Stuart.; Huckett, Barbara Isobel.
Sugarcane is challenged by a number of phytopathogenic bacteria and viruses that are best managed by the development of resistant varieties. Genetic engineering is a promising strategy in such breeding efforts, as it allows novel mechanisms of resistance not available in any parent germplasm to be introduced into the crop. DNA sequences encoding cystatin from papaya (Carica papaya), and pleurocidin from the winter flounder (Pleuronectes americanus) were envisaged as transgenes in this work due to their theoretical potential to increase sugarcane resistance to viruses and pathogenic or opportunistic bacteria, respectively. Cystatin is a cysteine proteinase inhibitor. Cysteine proteinases are used by potyviruses to cleave the polyprotein gene product, an essential step in the viral life cycle. Constitutive expression of cystatin may therefore lend the host plant resistance to a range of potyviruses, including the economically important pathogen sugarcane mosaic virus (SCMV). Pleurocidin is an amphipathic, α-helical, cationic peptide, with broadspectrum anti-bacterial activity at physiological pH. By binding to the cell membranes of both Gram positive and Gram negative bacteria, pleurocidin disrupts the membrane potential, causing it to become more permeable, especially to cations, leading to death of the bacterial cell. Initial microbiological bioassays showed that pleurocidin has inhibitory and bactericidal effects on the organisms which cause leaf scald (Xanthomonas albilineans), gumming disease (Xanthomonas campestris pv. vasculorum) and post-harvest sucrose conversion in sugarcane, as well as inhibitory effects against Leifsonia xyli ssp. xyli, which causes ratoon stunting disease (RSD). For transformation vector construction, the cystatin and pleurocidin coding sequences were altered so that their start codons were in the most favourable consensus context for expression in monocotyledonous plants. In the case of pleurocidin, an extracellular peroxidase signal sequence was attached. The prepared sequences were spliced into the vector pUBI510 in which the gene of interest is driven by the CaMV 35S promoter linked in tandem to a derivative of the maize ubiquitin promoter. The constructs generated were named pUBI510-cys3 and pUBI510-pleur08 respectively. The plasmid structures were confirmed using restriction endonuclease analysis and DNA sequencing. Since the transformation of sugarcane is known to be inefficient, two routes of morphogenesis for the production of somatic embryos were compared in the transformation procedure. These were (1) indirect embryo production via callus and (2) the direct and indirect production of embryos from transverse sections of leaf roll. Field grown sugarcane varieties N12 and NCo376 were the source of explant material. Plasmids pUBI510-cys3 and pUBI510-pleuro8 were respectively co-delivered by microprojectile bombardment with the antibiotic resistance selection plasmid pUBIKN containing the neomycin phosphotransferase gene (npt-II). Cultures were maintained in the dark on selection medium containing various concentrations of the antibiotic geneticin (G418) for several weeks before being allowed to regenerate in the light. Plantlets coming through selection were hardened off in the glasshouse when approximately 100mm high. Primer pairs for amplification of the cystatin insert were designed in various ways. The primer pair which ultimately proved most useful was designed to be complementary to the 5' and 3' ends of the papaya cystatin nucleotide sequence. Primer Premier analysis of a sorghum cystatin sequence provided additional possible primers. A further pair for potential future use was devised based on complementarity to conserved regions on maize cystatins 1 and 2, sorghum, rice, and papaya cystatins. The nucleotide sequence was constructed using the most common monocotyledon codon permutations for each amino acid. Pleurocidin primers were designed to be complementary to 5' and 3' regions of the nucleotide sequence encoding the pleurocidin pre-pro-protein. PCR and RT-PCR protocols for the detection of transgenes and transcript production in putative transgenic plants were developed using these primers. No plants survived selection via the callus route, although some were regenerated via direct embryogenesis. Putative transformed plants were analysed using PCR to test for the presence of integrated transgenes and Southern hybridization to determine transgene copy number. Both types of transgene were reproducibly detectable by PCR in DNA from some immature plants, but results were negative in DNA from those same plants when mature. Southern hybridization analysis detected the cystatin transgene in DNA from immature plants but no transgenes were detected in up to 20 µg DNA from mature plants. Single copy constructions of the transgenes in backgrounds of non-transformed DNA were detectable by both PCR and Southern hybridization analysis. Overall, PCR, RT-PCR and Southern hybridization results indicated that the plants regenerated fell into two categories: non-transformed plants that had survived selection (escapes) and chimaeric individuals with a component of both transformed and non-transformed cells, in which the transgene had probably become diluted during plant development under non-selective conditions. A method for extracting leaf exudates was tested, in conjunction with a cysteine proteinase assay to detect the presence of cystatin transgenes in the intracellular spaces of sugarcane leaves of confirmed transformants. Although it could not be applied within the scope of this project, this assay will prove useful in future work.

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