In vitro propagation of eucalytpus clones using a temporary immersion bioreactor system (RITA)

Abstract

Breeding and clonal programs in South African Forestry industry are designed to provide genetically superior trees to supply the forest product industry. Applied biotechnology, and in particular tissue culture, has been used to increase productivity in Eucalyptus clones (genetically superior trees) for trials and clonal hedges for commercial production. Improved growth using bioreactors has been increasingly recognized and the traditional semi-solid culture system was evaluated against a temporary immersion bioreactor (RITA®) system. The temporary immersion bio reactor (RITA®) system was tested across different clones for: ease of initiation into the vessels; multiplication numbers required to achieve production targets; and rooting. In addition costs of the RITA® system were evaluated. Contaminant free shoots in the RITA® system were obtained by initiating shoots on a semi-solid medium and thereafter pre-treating with 0.1 g.1¯¹ Rifampicin in liquid MS medium with visual selection of contaminant free plants. Cultures with fungal contamination were discarded as fungicides used as preventives or curatives measures were found to be ineffective against fungal contamination. Bacterial contamination could be reduced or controlled with the use of 0.1 g.1¯¹ Rifampicin. This however sometimes led to a fungal flush or, if Rifampicin was removed, a flush of bacterial contamination then occurred. Factors such as vessel ventilation, times of immersion and rest, size of vessel, ability to have a liquid substrate rather than a semi-solid substrate, and the physical covering of the plants with the nutrients, led to increased multiplication. Number of ex plants at the start, medium composition and flush and interval times particularly influenced multiplication. Initiating 50 shoots in a vessel with a flush time of 30 seconds and a rest period of 10 minutes gave the highest multiplication (3.8x) after 14 days. Depending on the clone, various media tested had different effects on multiplication. However, MURASHIGE & SKOOG (1962) medium with the following added: 0.1 g.1¯¹ Biotin and Calcium pantothenate; 0.2 mg.1¯¹ BA; 0.01 mg.1¯¹ NAA; and 25 g.1¯¹ sucrose (M1 medium) for both cold-tolerant and sub-tropical clones gave the highest average multiplication after 14 days (5.63x). Maximum shoot multiplication was achieved over 14 to 21 days. After 21 days the nutrients were depleted and the plants began to senesce by day 28. The time period for multiplication in the RITA® system was shorter than for in vitro propagation on semi-solid medium, with improved multiplication in half the time using the RITA® system. Nutrients from the media were utilized at different rates in the two systems. Plants from the RITA® system were superior in quality and this had a positive effect on rooting. The size of the shoot was important for rooting and thus elongation media were tested prior to rooting, with MS and ½ MS giving the best elongation. For rooting in the RITA® system, 1 mg.1¯¹ IBA for two cold-tolerant and one sub-tropical clones gave an average of 66 % normal rooting in the vessels. The type of media used prior to rooting affected rooting and acclimatization percentages. M1 media for 14 days transferring to MS media for 14 days and then placement onto RM media for a further 14 days gave the highest rooting percentage (55 %) after 28 days in the greenhouse. The period of time that the plants were exposed to a particular media played a role in rooting, as did the size of the plants, with bigger shoots (three to seven centimeters) resulting in better rooting. Sub-tropical clones showed no differences in rooting percentages between the semi-solid and the RITA® system rooting environments. However with the cold tolerant clones rooting was substantially improved with the RITA® system. The plants produced in the RITA® system were of a superior quality and acclimatized more readily than those grown on the semi-solid system. The costs involved in producing plants in the RITA® system were lower, as more plants were produced from the medium in shorter time. Although the initial outlay of vessels for the RITA® system was high, it was offset by reduced labour and media cost, together with significantly higher rooting and survival percentages, thus making the RITA® system a very cost effective option for in vitro propagation of Eucalyptus clones.