Browsing by Author "Mycock, David John."

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The actions of, and interactions between, auxins and cytokinins and their effect on in vitro rooting of selected Eucalyptus clones.
(2011) Nakhooda, Muhammad.; Watt, Maria Paula Mousaco Deoliveira.; Mycock, David John.
Clonal propagation of Eucalyptus spp. and its hybrids allows for competitiveness in the commercial forestry industry through the propagation and preservation of superior/elite genotypes. Vegetative propagation through rooted cuttings is the industry‟s standard and the choice of clones selected for plantations are determined by their rooting ability. However, as many potentially valuable genotypes are recalcitrant to adventitious rooting, micropropagation is the only effective means of propagating them. Micropropagation results in high plantlet yields, achieved primarily through the empirical use of the key plant growth regulators (PGRs) cytokinins and auxins, for shoot and root production, respectively. Their selection for use in vitro is driven by their effects on percent rooting rather than root quality. Little is known regarding the quality of the roots of the plantlets ex vitro, but there is some evidence that they are different from those of seedlings and cuttings. It was therefore hypothesized that the properties of exogenous PGRs and their interaction with other exogenous and endogenous PGRs, influenced root development and subsequent root quality. This was tested in vitro using a good-rooting E. grandis (TAG31) and two poor-rooting E. grandis x nitens hybrid clones (GN155 and NH58). In the former, the auxins supplied during the pre-rooting culture stages (multiplication and elongation) were sufficient for 100% rooting in an auxin-free rooting medium. Different combinations of PGRs in the two pre-rooting stages, followed by rooting without auxins, revealed a direct relationship between the stability of the supplied auxin and the rooting ability of TAG31. Gas chromatographymass spectrometry (GC-MS) analyses indicated that endogenous shoot levels of indole- 3-acetic acid (IAA) influenced graviperception. Also, low IAA content was associated with atypical starch grain accumulation or its absence from root tips (53.1 nmol IAA gˉ¹ DW compared with 325.7 nmol IAA g-¹ DW in gravisensing roots). The specific roles of the natural auxins IAA and IBA on root morphogenesis were then investigated using 2,3,5-triiodobenzoic acid (TIBA; inhibits IAA transport), ρ-chlorophenoxyisobutyric acid (PCIB; inhibits auxin signal transduction), and the auxin antagonist kinetin in the rooting medium, following root induction. After 3 weeks, the mean root diameter was significantly reduced from 552.8μm (control) to 129.2μm (with PCIB) and 278.6μm (with kinetin). TIBA increased root diameter to 833.4μm, decreased Δ root length, increased root vasculature and resulted in agravitropism. Hence, whereas rooting could be induced by IBA, IAA was necessary for the maintenance of vascular integrity and graviperception. This critical role of IAA in root development is of importance as IBA, owing to its higher stability, has been traditionally relied upon for root induction in the majority of micropropagation protocols. The potential of incorporating IAA into the media formulations of in vitro protocols for poor-rooters that do not respond well to IAA was then investigated, using GN155 and NH58. While PCIB in the rooting medium of GN155 completely inhibited rooting, the addition of dihydroxyacetophenone (DHAP), an inhibitor of auxin conjugation, to the rooting medium, did not significantly increase % rooting in the presence of 0.1 mg 1ˉ¹ IBA (i.e. 50% rooting with 2mM DHAP and IBA, compared with 45% with IBA alone). The results suggested that the inability of some eucalypts to induce roots easily in vitro was not due to a deficiency in auxin signal transduction or to auxin conjugation. Instead, rooting was inhibited by an accumulation of kinetin within shoots during the pre-rooting culture stages. The endogenous levels of PGRs in shoots of GN155 and NH58 showed a strong relationship (R² = 0.943) between the shoot kinetin:auxin and shoot rootability. Substituting kinetin with the relatively less stable natural cytokinin trans-zeatin in the elongation stage resulted in a significant increase in % rooting in both clones, from 19% to 45% (GN155) and from 31% to 52% (NH58), with 0.1 mg 1ˉ¹ IAA in the rooting medium. However, omitting all cytokinins from the elongation medium, resulted in over 95% and 75% rooting of shoots of GN155 and NH58, respectively, with 0.1 mg 1ˉ¹ IAA. These results suggest that IAA is a requirement for root development and cannot be substituted by its analogues in certain root developmental events. Hence, IAA should be the preferred auxin for eucalypt micropropagation. As fundamental research, the approach taken in this study circumvents the empirical method used in improving micropropagation protocols. The importance of the properties and the interactions between endogenous and exogenous PGRs in regulating root morphogenesis, and the practical implications of these findings is emphasised.
The development of short-to-medium and long-term germplasm storage protocols for Eucalyptus spp.
(1998) Thokoane, Novungayo Lucy.; Watt, Maria Paula Mousaco Deoliveira.; Mycock, David John.
Eucalyptus trees are a significant source of fuelwood, timber and raw material for the paper and pulp industry. In South Africa, Eucalyptus grandis and its hybrids are in high demand due to their fast growth and suitability of their timber for a wide range of products. Breeding and selection for superior quality eucalypts could sustain this high demand through selection and subsequent multiplication of superior genotypes and their use in controlled crosses. However, for this to be successful, a wide genepool must be available and maintained. Germplasm conservation of both vegetative and sexual material is therefore an integral part of such activities. However, in the case of trees, in vivo conservation practices are expensive and hazardous. The aim of this project was therefore to establish alternative conservation strategies for short-to-medium and long-term use of Eucalyptus spp. to facilitate on-going breeding and clonal programmes. For short-to-medium term storage, shoot cultures were subjected to various minimal growth conditions. Of the investigated treatments, reducing nutrients was the best storage method and shoots were maintained for 10 months with multiplication rates of 13.75 ± 7.05 shoots/explant. Encapsulated axillary buds were stored in jars at 10 °C or 28 °C. Of these two treatments, viability was sustained for longer (6 months) at 4 °C. Before establishing pollen storage regimes, viability assessment methods were evaluated and these consisted of in vitro germination on a BK (Brewbaker and Kwack, 1963) medium for 24 hours (26 ± 3.0%) and staining with two tetrazolium salts. Medium-term storage of pollen was best achieved by maintenance in the fridge (4 °C) without any desiccating substance (8 months at 6.73 ± 1.21%). Cryopreservation protocols were investigated for axillary buds and pollen. Buds that were 2 mm long were pretreated with chemical cryoprotectants, and a mixture of DMSO (dimethylsulphoxide) and glycerol was found to induce high survival rates (63%) after washing with MS (Murashige and Skoog, 1962) and 4 g.l-1 sucrose solution. Explants precultured in 1M sucrose showed increased tolerance (explant retained high survival rates of 80%) when desiccated to 20% moisture content fresh weight basis (fwb). Although pretreatments were successfully established, explants did not survive storage in liquid nitrogen indicating the need for further optimization of protocols. Pollen was successfully cryopreserved for 12 months with 23% survival rates. Applications and future research strategies of the developed protocols to Eucalyptus breeding programmes are discussed.
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.
A study of some of the inter-relationships between maize and the seed storage fungi as typified by Aspergillus flavus var. columnaris.
(1990) Mycock, David John.; Berjak, Patricia.
The seed storage fungi (xerotolerant) species of the genera Aspergillus and Penicillium} are renowned for their devastating effects on stored grain and grain products. In view of the fact that most of these fungi Iiberate toxins which can be harmful to both man and his livestock this problem is becoming increasingly relevant, particularly in developing countries. The seed storage fungi are said to be saprophytes and opportunistic invaders of dead or naturally dried organic matter, and as such no direct host-pathogen relationship has been ascribed to them. This dissertation reports aspects of an investigation into the modes/pathways utilised by these fungi in their infection of maize caryopses (seeds) and plants. The work involved studies on: the effects of protracted storage on maize seeds; the morphology of storage fungi; extra-cellular enzymes of storage fungi; the pathways utiIised by the storage fungi in invasion of seed tissues; and the effects of the storage fungi on the seeds. Correlations have been made on a species basis between the extent of seed deterioration and fungal aggressiveness. The results of these investigations indicated that apart from affecting seed vigour and viability, these fungi can also affect plant vigour. This latter aspect was further investigated to determine whether a seed storage fungus could infect germinating maize seeds, and remain an internal contaminant of the tissues during plant growth and development. These latter studies revealed that Aspergillus flavus var. columnaris is capable of systemic transmission from one seed generation to the next. This hitherto unrecognised phenomenon apart from indicating that the fungal species is in fact a biotroph as well as a saprophyte, also has implications In control measures.