Multiple shoots were produced from nodal explants of thirty-day-old in vitro grown seedlings and from pretreated three, five- and nine-month-old greenhouse grown Acacia mearnsii plants, respectively. Explants were sterilized for 15 minutes using 0.1 % HgCl₂ for the three-month-old explants and 0.2 % for the five and nine-month-old explants. Nodal explants were induced to form multiple shoots when placed on Murashige and Skoog (MS) medium supplemented with 2.0 mg l ¯¹ benzyladenine (BA). Rooting of these shoots was achieved on MS medium supplemented with 1.0 mg l ¯¹ indole-3-butyric acid (IBA). Plantlets were acclimatized in transparent plastic containers under greenhouse conditions with a 90 % success rate. These plantlets were successfully acclimatised under greenhouse conditions and planted in the field together with plants regenerated by cuttings. In an attempt to overcome maturation effects and loss of juvenile characteristics, when using adult plant material in vitro, investigations were undertaken into the use of coppice material, as an alternative explant source. A. mearnsii trees from five ages (two, four, six, eight and ten-years old, respectively) were decapitated to a height of 1.5 m. After three weeks, coppice was noted on the stumps of trees from all ages. A linear response to coppice production was noted, with the greatest coppice production being on the two-year-old tree stumps and the least on the ten-year old tree stumps. Decontamination of the coppice was successful and multiple shoot production was obtained from coppice taken from all age groups on MS medium supplemented with 2.0 mg l ¯¹ BA. The effect of various sucrose concentrations were investigated. Greater shoot production occurred with increased sucrose concentrations (20 and 30 g l ¯¹). It was evident that rejuvenation of mature tissue could be achieved through the use of coppice material. A second approach to rejuvenate adult material and to overcome the deleterious effects of maturation, was in the use of apical meristems. Meristems were taken from 30-day-old in vitro grown plants, from coppice (rejuvenated tissue) and adult material of five various tree ages (two, four, six, eight and ten-years-old, respectively). Plant material were taken over two seasons (1997 to 1999) and the use of agar and liquid support media were tested under both light and dark conditions. The coppice and adult material was successfully decontaminated in both seasons. In the first season (1997/1998), shoot production was obtained from meristems of in vitro grown plants, coppice and adult material from all trees on MS medium alone or MS medium supplemented with 2.0 mg l ¯¹ BA. In the following season (1998/1999), the use of a solidified agar medium was superior to the use of a liquid culture. There appeared to be no significant difference (p<0.05) between the use of light or dark culture conditions. Various media were tested and maximum shooting occurred on half-strength MS medium and Woody Plant Medium (WPM). However, once multiple shoot primordia were initiated, shoot elongation posed a problem. It was for this reason that the size of the meristems excised from the coppice material was increased from 0.5 mm to 1.0 mm in the 1997/1998 season, to 1.0 to 2.0 mm in the 1998/1999 season. The use of gibberellic acid and 100 ml jars were also investigated to see if this might enhance shoot elongation. Sufficient plant material was not available for a thorough investigation. Environmental conditions under which the plant material (adult or coppice) was harvested was similar in both seasons, with respect to temperature, but differed in rainfall. Rainfall was high (105.1 mm) in 1997/1998 season and low (ranging from 59.8 to 71.45 mm) in the 1998/1999 season. Shoot production from meristems taken from coppice material in the 1998/1999 season was significantly greater (p>0.05) than that in the 1997/1998 season, whereas shooting from the adult plant material remained unchanged. The disadvantage with using coppice material is that its production on decapitated tree stumps is dependant on rainfall, which is unpredictable. The differences in results from coppice material could be attributed to the fact that the trees felled in the two seasons were not related to each other in any way. In both seasons meristems, tree age was not a limiting factor, for meristems from adult and from coppice material. Meristems from the ten-year-old trees were as productive as those taken from the two-year-old trees. In the 1997/1998 season the results from the meristems from the adult material was equal if not greater than those obtained from the coppice material. In the 1998/1999 season, there was no significant difference (p<0.05) in percentage shoot production between the meristems from the adult and coppice material throughout the age groups. This suggests that the use of rejuvenated tissue in the form of coppice is not essential. This re-emphasized the advantage of using meristems taken from adult plant material. This study provided suitable protocols for the micropropagation of both in vitro and ex vitro grown nodal explants of A. mearnsii. However, as the plant material obtained from the field matures so the ease of obtaining sterile material decreased, thus reducing the chances of in vitro micropropagation. For this reason suitable pretreatments and rejuvenation methods are necessary if explants from mature field tissue are to be introduced into culture and successfully micropropagated. This study has shown that through the use of nodal material (taken from coppice produced on adult tree stumps) and apical meristems taken from both coppice and mature plant material, adult material can be successfully decontaminated, introduced into culture and stimulated to produce shoots. Analysis of tannin production was conducted to see if there was any indication that the presence of tannins in the plant material effected in vitro culture of nodal explants. However, no trends were obtained suggesting any influence of tannins on in vitro performance. In future years after further optimisation, these techniques could be incorporated in an A. mearnsii clonal programme, with the advantage of possibly eliminating maturation effects, commonly noted in vegetative practices. This will allow for easy manipulation and amplification of superior quality adult material. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1999.

Research into the micropropagation of black wattle in South Africa is important for two reasons. Firstly micropropagation technology allows breeders to select and propagate mature tissue, which in turn allows them to better capture selected traits. Secondly, tissue culture may control the highly invasive nature of black wattle. If triploid black wattle can be developed, foresters will then have to rely on clonal propagation to supply material for their growing operations. This research was part of the Institute for Commercial Forestry’s Acacia mearnsii vegetative propagation programme. The main focus of this research was to overcome various problems associated with direct organogenesis of ex vitro material. The shoot apex region was used as the explant in all studies because this region is thought to harbour relatively few internal microbial contaminants and is of sufficient size to withstand stresses associated with micropropagation. The initial research was focussed on the screening of sterilants, searching for a viable alternative to mercuric chloride. Surface sterilisation is integral to any micropropagation technique. This process should do the least amount of plant damage, whilst reducing microbial contamination to an acceptable level. Explants were cultured on Murashige and Skoog (MS) medium supplemented with 2.0 mg L-1 BA and monitored for signs of contamination and shooting. Household bleach proved an excellent alternative to mercuric chloride because it did significantly less damage to the explants than mercuric chloride and is handled easily. There was no significant effect of sterilant exposure time on explant decontamination levels, whilst the shortest exposure time resulted in significantly higher levels of shoot development than the other two times tested. The results of this initial research was developed into a protocol and utilised in subsequent investigations. Due to a considerable variation in the success of the developed surface sterilisation protocol according to different times of the year, a further investigation into the effects of season and mother plant material on shoot apex culture of Acacia mearnsii was undertaken. The success of any tissue culture technique depends on a large array of ex vitro and in vitro variables. The objective of this research was to determine the ii effect of two ex vitro variables, season and mother plant, on shoot apex culture of Acacia mearnsii. Explants from individual mother plants were cultured on MS medium supplemented with 2.0 mg L-1 BA during four separate seasons and monitored for signs of contamination and shooting. Spring was found to be the best harvesting season because spring explants showed significantly higher decontaminated explant levels and shooting levels than explants harvested in the other three seasons. The effect of mother plant selection on the performance of Acacia mearnsii explants during shoot apex culture was also found to be significant, especially with regard to shooting levels. Finally factors influencing shoot elongation of A. mearnsii during shoot apex culture were investigated. In the past, induction of shoot elongation during micropropagation of A. mearnsii was attained through the addition of plant growth regulators and other supplements to the basal culture medium. However, some micropropagation methods in other species have utilised red light as a means of promoting shoot elongation. The objective of this study was to test the effects of an alternative basal medium, red light and differing concentrations of chemical additions to the culture medium on shoot elongation of Acacia mearnsii during shoot apex culture. Four independent experiments were undertaken comparing: shoot elongation on Woody Plant Medium (WPM) to the MS basal medium control; shoot elongation under a red cellophane box compared to control culture light conditions; shoot elongation on media supplemented with various concentrations of GA3 to the un-supplemented control and shoot elongation on media supplemented with combinations of BA and IBA compared to a control. Although no significant effects were observed, many trends were noted. The results indicated that there was no advantage to using WPM instead of MS medium when attempting to elongate shoots, rejuvenated through shoot apex culture of A. mearnsii, whilst the effect of GA3 showed a negative trend. The effects of red light and some BA and IBA combinations showed positive trends on the elongation of initiated shoots. This research successfully addressed some of the problems associated with micropropagation of A. mearnsii. Shoot apex culture shows promise and further research into this technique should be considered. A viable surface sterilant alternative to mercuric chloride was successfully identified. This alternative is not only iii safer to use but shows a large reduction in phytotoxic effects. The effects of season and mother plant on shoot apex culture was successfully investigated, resulting in a better understanding of mother plant influences on tissue culture as well as the identification of an optimum season for explant selection. Finally two possible shoot elongation promoters were identified for further research and a more affordable alternative to red light sources and screens was identified. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2007.