The development of clone-unspecific micropropagation protocols for three commercially important Eucalyptus hybrids.
Micropropagation methods are often used to supplement existing clonal programmes for Eucalyptus species. However, genotypic differences among clones require the implementation of clone-specific protocols, an expensive and labour-intensive exercise. Hence, this study aimed at determining high-yielding hybrid-specific rather than clone-specific, micropropagation protocols for E. grandis x nitens (GN), E. grandis x nitens (NH), and E. grandis x urophylla (GU). Different conditions for surface sterilisation, bud-break (3 protocols, 2 media), multiplication (4 media), elongation (2 protocols) and rooting (4 media) were tested. A single successful surface sterilisation approach was possible for all clones of the tested hybrids (0.0-11.8% contamination, 0.0-22.9% necrosis). It involved rinsing nodal explants in a fungicide mixture (lg/l Benlate, 1g/1 boric acid, 0.5ml/1 Bravo, Tween 20) for 15 minutes followed by calcium hypochlorite (10g/l with Tween 20) for three minutes. Results at each culture stage were dependent on genotypes, and results indicated here represent ranges in values among the clones of each hybrid. The highest bud-break values for GN clones (87-90%) and NH clones (17-75%) were achieved on a medium containing MS, 0.1mg/1 biotin, 0.1mg/l calcium pantothenate, 0.04mg/1 NAA, 0.11mg/l BAP and 0.05mg/1 kinetin. In GU clones, bud-break values on this medium (84-97%) were not significantly different to those achieved directly on a multiplication medium (80-91%) (MS, 0.1 mg/l biotin, 0.1 mg/l calcium pantothenate, 0.2mg/l BAP, 0.01mg/1 NAA). Shoot multiplication yields for GN clones (4-13 shoots/bud) and GU clones (2-6 shoots/bud) were achieved on a medium consisting of MS, 0.1mg/1 biotin, 0.1 mg/l calcium pantothenate, 0.2mg/1 BAP and 0.01 mg/l NAA. As genotypic effects were highly significant among NH clones, a single multiplication medium for all clones of this hybrid could not be determined. The best method of elongation for clones of all three hybrids involved culturing shoots on MS, 0.1 mg/l calcium pantothenate, 0.1mg/1 biotin, 0.35mg/1 NAA, 0.1mg/l kinetin and 0.1mg/1 IBA, under photoperiod conditions, rather than total darkness, for 6 weeks. This resulted in 82.3-86.6% elongation and shoot lengths increasing by 22.9-35.2 mm for GN clones, 80.2-82.3 % elongation and an increase in length of 24.7-32.2 mm for NH clones and 70.8-78.1 % elongation, and shoot elongation of 21.6-29.3 mm for GU clones from passage 1-2. For all the above stages, media contained 20/25 g/l sucrose and 3.5g/l Gelrite, and cultures were maintained at 25°C ± 2°C day/ 21°C night with a 16 h light/ 8 h dark photoperiod (PPFD 66µmol/m2/s). In terms of rooting, cultures on different media were initially subjected to a 72 hour period of total darkness at room temperature, then a 16 h light/8 h dark photoperiod (PPFD 37µmol/m2 /s) at 24°C day/ 21°C night for 7 days. This was followed by a 16 h light/ 8 h dark photoperiod (PPFD 66µmol/m2/s) at 25°C ± 2°C day/ 21°C night for 21 days. Tested clones of the three hybrids were all rooted successfully (56-93% rooting in GN clones, 36-76% rooting in NH clones and 46-96% rooting in GU clones) on a medium containing ¼ MS, 0.1 mg/l biotin, 0.1 mg/l calcium pantothenate, 0.1mg/l IBA, 0.22g/1 CaCI2 .2H20, 0.185g/l MgS04.7H2O, 15g/l sucrose and 3.5g/1 Gelrite. Predicted yields from the established protocol are also presented (168-667 plants of E. grandis x nitens (GN), 35- 854 plants of E. grandis x nitens (NH) and 54-349 plants of E. grandis x urophylla from 100 initial nodal explants, depending on the clone). Hence, the established protocols can be used successfully for some of the clones, but the implementation of specific media and methods to obtain high yields may still be necessary for certain clones.