The development of tissue culture techniques to study wood formation in Eucalyptus globulus Labill

© 1999 Dr. Mathew A. LeitchSeveral studies were conducted firstly to determine the longitudinal pattern of cambial development during the year in Eucalyptus globulus Labill.; secondly, to determine if procedures could be developed to culture explanted material from various regions along the main stem axis free of contamination; thirdly, to determine if these procedures could be used to study the regulation of cambial activity and subsequent xylem cell differentiation by various plant growth hormones in vitro; and lastly to determine whether variations in cell development would occur when callus produced on the primary explant in vitro was subcultured and grown independently. For the first study, trees (from Australian Paper Plantations in Morwell, Victoria, Australia) were harvested throughout the year and stems were sectioned at 6 percentage heights of total tree height for microscopy. The remaining studies utilised portions of the trees used in the first study and from the same family of trees following the first study. Billets from the main stem axis between 45 - 60 % and 98 - 100 % of total tree height were removed and main stem 'chips' (MSC, 2-3 cm axial x 2 cm tangential x 0.5 cm radial) from the former and apical stem segments (ASS, 0.8 - 1.1 cm axial) from the latter were grown on a defined sterile culture medium. The pattern of cambial activity and wood formation in E. globulus varied over the length of the stem throughout the year. Below 50 % of total tree height cell numbers (dividing and expanding xylem cells) were greatest during rnid-winter and early to mid-summer with a decrease in numbers occurring just before and following the summer growth period (November and April; respectively). Above 50 % of total tree height cell numbers were greatest in October and in January/February with a decrease in numbers occurring in July, August, December and in April following summer growth. Variability in cell properties along the length of the stem were also evident in this study. Following the development of procedures for sterilisation of main stem segments, removal and sterilisation of primary explants and testing of media, in vitro experiments were conducted. In vitro, cambial activity and xylem cell differentiation occurred in MSC cultures on three defined media while in the ASS cultures this occurred on 19 defined media preparations. Main stem chip cultures produced more dividing and expanding cells in the cambial region than ASS cultures; however, ASS cultures produced more secondary-walled cells than MSC cultures. Radial and tangential cell size (as viewed in transverse section) for fibres displaying secondary cell walls was greatest for MSC cultures. Double-cell-wall thickness (DCWT) was variable between the two sampling regions and treatments within each region. Ray width in the cambial zone was greater in almost all MSC cultures when compared to ASS cultures. Apical stem segments were also used in a preliminary Agrobacterium-mediated transformation experiment. Using two constructs and three infiltration methods greater than half the ASS used survived the infiltration procedure. Eucalyptus delegatensis was cultured using the ASS procedure to test the system on a species other than E. globulus. ASS remained free of contamination and displayed some cambial activity in the form of cell division, radial expansion of cambial derivatives and some secondary wall formation. Callus proliferating from ASS and MSC cultures was either thin-walled, nonpitted parenchyma in a random pattern, or thick-walled, simple-pitted parenchyma and fibre-like cells displaying localised patches of organisation. Callus values were generally noted as less when a strong in vitro cambial response occurred. Subculturing callus to a media containing GA3 and 2,4-D led to organised radial files of fibre-like cells, rays, parenchyma, vessel element-like cells and a cambial layer exterior to these cells and interior to a phloem-type layer. Secondary wall formation occurred in parenchyma and fibre-like and vessel element-like cells which occasionally displayed a three-layered secondary wall containing bordered pits. This research has provided tissue culture procedures for a variety of experimental outcomes using material from two regions of the main stem axis of E. globulus Labill. These procedures have the potential to become a rapid system of testing hormonal and nutrient effects on wood formation and also have potential as a system to screen genes to be used in transformation experiments for the genetic improvement of wood traits