Stephen J. Trueman

Maturation and related aspects in clonal forestry – Part II: reinvigoration, rejuvenation and juvenility maintenance

Several techniques have been developed for reinvigorating, rejuvenating or maintaining the juvenility of plants. None of these techniques is as effective as natural rejuvenation whereby the most mature plant parts, the reproductive organs, produce the most juvenile plant parts, the embryos, through gametogenesis and sexual reproduction. The most common criteria for identifying reinvigoration or rejuvenation are based on morphology, morphogenic capacity and the ability to produce cones or flowers. Doubts remain over whether true rejuvenation (reduced ontogenetic age) can be achieved by artificial methods such as serial vegetative propagation, or whether these methods merely provide reinvigoration (reduced physiological age) by temporarily removing environmental and physiological constraints to growth. Rejuvenation and reinvigoration are difficult to distinguish from each other but there is some evidence that artificial methods provide at least partial rejuvenation of plants. This review examines methods for delaying the maturation of juvenile plants and for reducing the ontogenetic or physiological age of more-mature plants. The review discusses theoretical and practical aspects of juvenility, rejuvenation and reinvigoration in clonal forestry, and identifies research areas in maturation that could lead to more-effective methods for establishing high-productivity plantations.

Maturation and related aspects in clonal forestry—Part I: concepts, regulation and consequences of phase change

Progression from the juvenile to mature phase in woody plants is accompanied by changes in characteristics as diverse as adventitious rooting capacity, leaf morphology, canopy architecture, wood anatomy and reproductive development. Many concepts of phase change, the intensity and duration of changes that occur during the phase transition, and the practical consequences of plant maturation for growth and development, are poorly understood. Little is known about the physiological and environmental control of maturation in woody plants compared with herbaceous plants, and reliable markers of phase state have only been developed for a few species, mainly conifers. Understanding the mechanisms and forms of phase change is a prerequisite for achieving maturation or rejuvenation for applications such as seed production or clonal propagation. This review describes concepts, terminology and consequences of phase change, combining theoretical and practical aspects of tree maturation that relate to clonal forestry.

Nutrient responses differ between node and organogenic cultures of Corymbia torelliana × C. citriodora (Myrtaceae)

Hybrids between Corymbia torelliana (F.Muell.) K.D.Hill & L.A.S.Johnson and C. citriodora subsp. variegata (F.Muell.) A.R.Bean & M.W.McDonald are used extensively for plantation forestry in subtropical eastern Australia; however, plantation establishment has been hampered by inadequate seed supply and variable amenability to propagation as rooted cuttings. The present study investigated node-culture and organogenic-culture methods for in vitro propagation of two families of juvenile Corymbia hybrids. The effects of nutrient and auxin concentrations on root formation, shoot elongation and shoot proliferation, and subsequent conversion to plantlets in an in vitro soil-less system, were assessed. The response to the nutrient concentration differed between organogenic and node cultures. Half-strength, compared with full-strength, medium with auxin increased shoot proliferation by 58% for one family in the node culture, in which many shoots form roots and elongate rapidly in the absence of cytokinin. However, full-strength medium increased shoot proliferation by 213% over that in the half-strength medium for the other family in the organogenic culture, in which shoots do not form roots in the presence of cytokinin, and nutrient uptake occurs via callus and stem tissue rather than roots. The auxin, naphthalene acetic acid, sometimes stimulated root formation and shoot elongation in the node culture, although it had no effect on the final number of shoots. Most shoots (57–100%) subsequently formed roots in the in vitro soil-less medium. The high rates of proliferation and plantlet conversion in the present study provide the means, not only for simultaneous laboratory storage and field-testing of Corymbia clones before selection of desired genotypes, but also for en masse plantlet production of selected clones or families for plantation establishment.

Clonal maturation of Corymbia torelliana × C. citriodora is delayed by minimal-growth storage

Summary Minimal-growth storage may be a simple method to preserve germ plasm while tree varieties undergo clonal selection, but there has previously been little evidence to demonstrate that minimal-growth storage delays maturation. This study assessed the maturation of 10 Corymbia torelliana × C. citriodora clones whose shoots had been stored for 12 months using three different methods: (1) as ex-vitro stock plants in the nursery; (2) in-vitro at 25°C; and (3) in-vitro under minimal-growth conditions at 14°C. Minimal-growth storage increased the subsequent frequency of adventitious rooting in cuttings of 6 of the 10 clones, providing increases in rooted cutting production of between 53% and 163% over ex-vitro nursery storage. Minimal-growth storage also increased adventitious root number in two clones and root mass in four clones. C. torelliana × C. citriodora clones underwent maturation during ex vitro stock plant archiving in the nursery, but in-vitro storage under minimal-growth conditions was highly effective in delaying clonal maturation.

Nutrient partitioning among the roots, hedge and cuttings of Corymbia citriodora stock plants

Many eucalypt species are difficult to propagate as rooted cuttings. The mineral nutrition of cuttings is a key factor that limits adventitious root induction but little is known about partitioning of nutrients by eucalypt stock plants. This study determined N, P, K, Ca, B, S, Mg, Mn, Zn, Al, Fe and Na concentrations in the root system, pruned hedge and harvested cuttings of stock plants of the eucalypt, Corymbia citriodora. Between 17% and 31% of total plant mass was collected as cuttings at each harvest. The mobile nutrients, N, K and S, were highly concentrated in the cuttings and were removed in high amounts (e.g. 27-6%> of total plant N) at each harvest, whereas less-mobile nutrients such as Ca and Zn were less concentrated in the cuttings than other plant parts. Adventitious rooting of eucalypt cuttings has been related to B concentration but this study revealed that B was much more highly concentrated in the hedge than the cuttings. Management of N and K concentrations for shoot production, and B concentrations for adventitious rooting, may be critical for sustaining rooted cutting production by C. citriodora.

Cytokinin concentrations for optimal micropropagation of 'Corymbia torelliana x C. citriodora'

Summary The eucalypt Corymbia torelliana × C. citriodora is grown in subtropical Australia, India and Brazil, but plantation establishment has been limited by inadequate seed supply and low amenability to propagation using conventional cuttings. We have recently developed tissue culture methods for propagation, storage and distribution of this hybrid, using low to moderate concentrations (0–2.2μM) of the cytokinin benzyladenine (BA) for proliferating shoots in vitro. In this study, we determined the effects of higher BA concentrations (2.2–17.8μM) on shoot proliferation and the subsequent conversion of shoots into plantlets in five full-sibling C. torelliana × C. citriodora families. We found that 4.4μM BA provided consistently high shoot proliferation in all families (137 ± 46 to 858 ± 175 shoots from each seed in 21 weeks), complete survival of clones (100%), very high conversion of shoots into plantlets (91 ± 4% to 99 ± 1%) and excellent formation of adventitious roots (3.6 ±0.1 to 4.5 ± 0.1 roots per plantlet). This method will allow simultaneous archiving and testing of C. torelliana × C. citriodora germplasm in the laboratory, nursery and plantation as part of a clonal forestry or vegetative family forestry program.

Designing food and habitat trees for urban koalas: graft compatibility, survival and height of tall eucalypt species grafted onto shorter rootstocks

The Corymbia and Eucalyptus species eaten by koalas are generally large trees, but these are often unpopular with urban landowners and councils because of the dangers of limbs falling from a great height. We aimed to develop shorter koala food and habitat trees for urban areas by heterografting tall eucalypt species onto rootstocks of shorter species and comparing their survival and growth with homografted trees and control ungrafted trees. In total, 12 of 14 interspecific scion/rootstock combinations were grafted successfully in the nursery but graft compatibility and field survival depended on taxonomic relatedness. The six interspecific combinations that had multiple surviving trees at 5 years after planting were all between species within the same taxonomic section or between a species and its own interspecific hybrid. Almost all trees died from grafts between species in different taxonomic sections. In most cases, the height of surviving interspecific grafted trees did not differ from control intraspecific grafted trees or from ungrafted trees of their scion species. Grafting elicited a ‘thrive or not survive’ response that diminished its usefulness for producing shorter trees. However, one combination, E. moluccana/E. behriana, had field survival of 40% and reduced height (4.0 m vs 9.9 m). These could be valuable habitat trees for koalas and other fauna in urban areas.

Designing food and habitat trees for urban koalas: identifying short ecotypes of Corymbia intermedia

The eucalypt trees eaten by koalas are generally tall, but urban landholders prefer to plant shorter trees that pose less danger of limbs falling from a great height or damaging powerlines. Our aim was to develop shorter eucalypt trees to provide food and shelter for koalas and other fauna in urban areas. We identified short ecotypes of Corymbia intermedia (R.T.Baker) K.D.Hill & L.A.S.Johnson growing naturally on exposed coastal headlands, and tested whether their seedlings were shorter than the seedlings of nearby tall ecotypes when planted in cultivation. Trees raised from the short ecotypes were 22–43% shorter than trees raised from the tall ecotypes, being around 5–7 m tall rather than 8–12 m tall after 8 years. This demonstrated that there was a genetic basis for the short stature of C. intermedia trees on coastal headlands. These shorter C. intermedia trees could be valuable food and habitat trees for urban koalas and other fauna.

Antibacterial Properties of Flavonoids from Kino of the Eucalypt Tree, Corymbia torelliana

Traditional medicine and ecological cues can both help to reveal bioactive natural compounds. Indigenous Australians have long used kino from trunks of the eucalypt tree, Corymbia citriodora, in traditional medicine. A closely related eucalypt, C. torelliana, produces a fruit resin with antimicrobial properties that is highly attractive to stingless bees. We tested the antimicrobial activity of extracts from kino of C. citriodora, C. torelliana × C. citriodora, and C. torelliana against three Gram-negative and two Gram-positive bacteria and the unicellular fungus, Candida albicans. All extracts were active against all microbes, with the highest activity observed against P. aeruginosa. We tested the activity of seven flavonoids from the kino of C. torelliana against P. aeruginosa and S. aureus. All flavonoids were active against P. aeruginosa, and one compound, (+)-(2S)-4′,5,7-trihydroxy-6-methylflavanone, was active against S. aureus. Another compound, 4′,5,7-trihydroxy-6,8-dimethylflavanone, greatly increased biofilm formation by both P. aeruginosa and S. aureus. The presence or absence of methyl groups at positions 6 and 8 in the flavonoid A ring determined their anti-Staphylococcus and biofilm-stimulating activity. One of the most abundant and active compounds, 3,4′,5,7-tetrahydroxyflavanone, was tested further against P. aeruginosa and was found to be bacteriostatic at its minimum inhibitory concentration of 200 µg/mL. This flavanonol reduced adhesion of P. aeruginosa cells while inducing no cytotoxic effects in Vero cells. This study demonstrated the antimicrobial properties of flavonoids in eucalypt kino and highlighted that traditional medicinal knowledge and ecological cues can reveal valuable natural compounds.