The development of in vitro rooting systems for cold-tolerant Eucalyptus grandis x nitens clones and the assessment of the hydraulic efficiency of roots produced by in vitro vs. cutting propagation.

Abstract

Hybrid clones of the fast-growing Eucalyptus grandis and cold-tolerant E. nitens (GN clones) have been identified by the South African Forestry Industry as being highly suitable for plantations in cold-dry marginal areas. However, one of the main problems regarding their propagation is the difficulty in rooting of cuttings, both in vitro and ex vitro. The aims of this investigation, therefore, were (1) to develop widely applicable and efficient in vitro rooting system(s) for these commercially important clones, and (2) to assess some physiological characteristics of the roots produced. Adventitious shoots (15-20 mm in length) were obtained (l0 shoots/explant) from axillary buds on Murashige and Skoog's (MS) medium containing 0.01 mg.l-1 NAA, 0.01 mg.l-1 IBA and 0.2 g.l-1 FAP. The effect of various medium components, as well as modification of culture environment on in vitro rooting, were investigated. The highest rooting frequencies in clones GN121 (75%) and GN107 (65%) were achieved on l/4 MS with additional 0.22 g.l-1 CaCl2..2H2O and 0.18 g.1-1 MgS04.7H2O, 0.1 mg.l-1 IBA, 0.1 mg.l-1 biotin, 0.1 mg.l-1 calcium pantothenate, 15 g.1-1 sucrose and 4 g.l-1 Gelrite. Best culture conditions were an initial 72-hours dark incubation followed by a 16-hours day/8-hours night photoperiod at a PPFD of 37 µmol.m-2.s-1 and 23°C day/21°C night for seven days, after which the PPFD was increased to 66 µmol.m-2.s-1 at 27°C day/21°C night for 18 days. Towards the development of a more widely applicable in vitro rooting protocol for GN clones, the use of Agrobacterium rhizogenes strains was investigated. Production of transgenic roots was observed on carrot discs and shoots from seedlings of Eucalyptus grandis and E. nitens, but not on shoots of GN clones. Therefore, a method needs to be established for the successful transfer and integration of the Ri plasmid of Agrobacterium into the hybrid plant genome for induction of transgenic roots. The quality of roots produced in vitro and from cuttings was assessed by examination of root anatomy and hydraulic characteristics. Adventitious roots were prepared for measurement of hydraulic conductivity by detopping explants, then filtered, acidified distilled water was drawn through undisturbed potted root systems under partial vacuum, causing no damage to the roots. Initial studies showed that tissue culture-derived roots exhibited a higher specific root mass hydraulic conductivity than those derived from cuttings (6.46 x 10-6 vs. 3.06 X 10-6 g.kPa-1.s-1.g-1 dry root), probably due to root architecture. Curves relating vulnerability to water potential were constructed and both types of roots showed vulnerability to cavitation at high water potentials. Differences were also observed in staining reactions (safranin and fastgreen) which might suggest differences in presence and level of secondary metabolites in these roots at the juvenile stage. Applications of the developed protocols and future research strategies are discussed.