Micropropagation of Acacia mearnsii (de willd)
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.