Introduction
Domestication of eucalypts for oil production in plantations is in its infancy. Tree breeders have only just started to make genetic changes to planting stock and in many instances seed for plan-tations still comes from natural stands. Eucalypts are largely outbreeding and genetically highly variable, which represents a huge opportunity for the tree breeder, whose main task is to exploit this variability through exploration, evaluation, selection and breeding. In this situation, large gains in heritable traits such as various growth and oil characteristics can be achieved simply and cheaply using relatively unsophisticated procedures. This is in marked contrast to many agricul-tural crops (e.g. barley, wheat and rice) that have been domesticated for thousands of years, are in many cases inbreeding species, and where changes such as polyploidy and mutation need to be artificially induced by breeders.
Tree breeding is worthwhile only if the subsequent economic returns are greater than its implementation costs. In the following discussion it will be assumed that developing better genotypes for oil production through selection and breeding is cost-effective. However, it is recognised that this argument may well be hard to sustain at times of low eucalyptus oil prices.
Many of the principal oil-producing species, for example, Eucalyptus globulus (China), E. smithii (Southern Africa) and E. citriodora (China, Brazil, India), are grown primarily for their wood;
eucalyptus oil is a valuable, but relatively minor, additional product. Improvement of character-istics affecting wood production such as growth rate, form and wood quality will always be a tree breeder’s first priority when working with these species. A benefit/cost analysis of incorporating selection for oil traits in such a programme may well return a negative result. Economic analysis should be applied to all breeding work to ensure that the benefits outweigh the cost of the programme.
Namkoong et al. (1980) suggested that genetic improvement programmes can usefully be grouped into three classes: those requiring low, medium or high intensity effort. The choice between these alternatives depends upon the resources (i.e. physical, financial and human) available and the potential economic benefits of the tree plantations (Harwood 1996). Eco-nomics may well dictate that a relatively simple, low-input strategy is appropriate for the genetic improvement of oil traits in eucalypts, and this will be the premise for further discussion in this chapter. Nevertheless, successful tree breeding, whether simple or complicated, needs scientific and technical expertise and a commitment to the provision of necessary resources in the long term. These resources will certainly include access to chemical analytical equipment such as gas chromatography (GC) and the expertise of an organic chemist when breeding for oil traits. The expense of screening large numbers of plants for oil content and composition is a consideration when determining an appropriate breeding strategy and plan.
Tree improvement programmes aim to develop new plantings superior to their predecessors in one or several key economic traits. The modus operandi of most contemporary programmes is to start with a carefully chosen breeding strategy implemented through a dependent breeding plan. The breeding strategy provides a philosophy of the management of genetic improvement while the breeding plan prescribes the ‘nuts and bolts’ for implementing the selected strategy.
Typically, the plan includes a set of objectives and a flow chart of what is to be done each month for several years ahead, and is subject to revision every 2–5 years (Eldridge et al. 1993).
This chapter has been prepared to serve as a guide to eucalyptus oil producers interested in improving the yield and quality of oil through selection and breeding. As will be seen, oil traits in eucalypts have been found to be under moderate to strong genetic control, with substantial levels of genetic variation, and this will facilitate the capture of useful gains from relatively sim-ple, low-input strategies appropriate to the present industry position of a static market and low prices. The chapter highlights the principal features of breeding strategies for improving oil traits in eucalypts and some of their key determinants, drawing on examples from the main oil-producing species where appropriate. In doing so, extensive use is made of three practical texts that are highly recommended to any newcomer to eucalypt breeding: Eldridge et al.
(1993), Williams and Matheson (1994) and Cotterill and Dean (1990). These in turn introduce the reader to other literature important to understanding tree breeding and forest genetics.
Basic concepts
Most worthwhile breeding strategies recommend starting with a well-adapted population with a broad genetic base. This base population is then subjected to a particular method and intensity of selection. Selected trees are then mated to maximise long-term genetic gain and minimise the effects of inbreeding within the limits imposed by human and economic resources.
Selection and mating are key activities in breeding. They accumulate genes which influence yield and adaptation, steadily increasing over successive generations the frequency of superior genotypes. Every successful breeding strategy, therefore, requires efficient methods of selecting superior genotypes. These methods include the progeny tests in which the selection is carried out, appropriate measurement techniques and selection technology (e.g. selection indices).
Mating can be done by open pollination or controlled pollination, carefully minimising the potential for inbreeding and allowing for genetic material from other sources to be incorporated.
In pursuing its principal functions of efficient selection and mating, a strategy should aim to assess the variation within a species, generate information about it and ensure that genetic resources and variation for future selections are conserved (Barnes 1987, Matheson 1990).
The cyclic or recurrent nature of the selecting, testing and mating processes which are part of an overall breeding strategy is illustrated in Figure 4.1. Every effective breeding strategy involves maintaining a hierarchy of three major types of population which can continue to meet the demand for genetically improved planting stock for the fourth population, the production population (i.e. commercial plantations) (Griffin 1989a, Matheson 1990, Eldridge et al. 1993).
These populations are the base, breeding and propagation populations. Awareness of the concept of maintaining distinct types of population within the cycle is essential in planning the operations of genetic improvement (Libby 1973), even if circumstances dictate that some populations are combined in the one planting.
Breeding objectives
When oil production is the sole objective of a eucalypt plantation, the usual goal of producers is to maximise the yield of high quality oil if it is economically feasible to do so. The yield of oil
from a given area of land depends on the species planted, available leaf biomass and the concen-tration of oil in those leaves at that point in time. Oil composition usually determines quality and will be an important consideration if it is liable to variation in the species under cultivation and affects the marketability or price of the oil produced.
A typical breeding objective for an oil-producing species would therefore be to increase the yield of high quality oil per unit area. This can be done by improving the yield of leaves, and the concentration of oil in those leaves, and ensuring that the quality of the oil produced maximises saleability and price.
Choice of base material
Correct choice of base material is critical to the success of breeding programmes. There are many examples in eucalypt breeding where breeding has begun with a poor choice of species or prove-nance and, in others, where the genetic base was much too limited (Eldridge et al. 1993). In the following discussion it is assumed that the choice of preferred species for oil production has already been made. The aim, then, is to point out important factors for consideration in acquir-ing suitable germplasm of a particular species with which to establish breedacquir-ing populations.
Examples pertaining to the main oil-producing species in use today (i.e. E. citriodora, E. dives, E.
exserta, E. globulus, E. polybractea, E. radiata, E. smithii and E. staigeriana), as well as some with high potential (e.g. E. camaldulensis and E. kochii), are given wherever possible.
Knowledge of the genetic structure of natural populations is important in determining strate-gies for seed collections for breeding programmes. Studies of the amount and distribution of genetic diversity in eucalypt species, as assessed by allozyme variation, have shown that, on average, 18 per cent of the genetic diversity in the twenty-five species studied to date occurs between populations (Moran 1992). The species with most genetic differentiation between populations are those with regional (i.e. moderately extensive) distributions but with small disjunct populations. Regionally-distributed eucalypts had, on average, a higher proportion of variation between populations (25 per cent) than localised or widespread (14 per cent) species.
Of the regionally-distributed species, those with disjunct distributions show greater
Base Progeny
testing Selection Mass Plantation
propagation Base
population
Breeding population
Propagation population
Oil-producing population
Selected trees
Seed orchards/clone
banks
Mating
Figure 4.1 Activity cycle for breeding and the hierarchy of the four populations relevant to breeding strategies and operations.
population differentiation (39 per cent) than those with continuous distributions (11 per cent).
Generally, however, most of the allozyme variation occurred within, rather than between, populations.
Genetic resources
After defining the objectives of the breeding programme, the next task in tree breeding is to establish a breeding population. Where selection aims to enhance oil production the natural inclination is to start by making selections in local plantations of the preferred oil-yielding species to establish a breeding population. In many instances this is a perfectly good strategy.
However, there are some pitfalls in relying solely on existing plantations (‘land races’) as the primary source of selections for breeding populations. The genetic history of many eucalypt plantings is obscure or unknown. Many have a narrow genetic base and have been derived from a provenance that gives mediocre performance in the environment of the planting site. This situation has existed with many species in numerous countries and trials of broadly based seed collections from the natural provenances of a particular eucalypt have often shown better adapta-bility and faster growth rate than the local land race, even after genetic improvement of the land race (see examples in Eldridge et al. 1993).
Any new programme should start with a thorough review of provenance performance within the region of planting. It may well be found that, given the uncertain origins of the land race and the lack of systematic provenance testing in the past, it is preferable to start again, virtually, by introducing broadly based provenance collections from natural stands of the target species. If there is a clear indication that some provenances or regions-of-provenance are better than others, collections can be concentrated in those areas. These, and collections representing the best ele-ments of the local land race, can then be used to establish large provenance-progeny trials. Such trials have multiple functions including ranking provenances and land races, serving as breeding populations for the first generation, providing resources for selection and breeding activities, and as commercial seed orchards. Several breeding strategies developed for wood-producing Eucalyptus species have advocated this approach, for example, E. globulus in China (Raymond 1988) and E. camaldulensis in Thailand (Raymond 1991).
Choice of trees within species
Chemical forms
An extreme type of variation within species, commonly found in Eucalyptus and in a wide variety of other plant families, is the occurrence of ‘chemical forms’. Penfold and Willis (1953) described these as
plants in naturally occurring populations which cannot be separated on morphological evi-dence, but which are readily distinguished by marked differences in chemical composition of their essential oils.
They do not differ qualitatively but show marked discontinuous, quantitative variation (Hellyer et al. 1969).
Penfold and Morrison (1927) first reported such forms in E. dives and called the variants
‘physiological forms’. In order to distinguish four separate and distinct oil forms in the species they called them ‘Type’, ‘Variety A’, ‘Variety B’ and ‘Variety C’ in order of discovery. The use of
this terminology has now been discontinued in favour of simply referring to the different types as chemical variants, chemical forms, chemovars or chemotypes, and highlighting the major oil component, for example ‘E. dives (cineole variant)’ (e.g. see Lassak 1988). Chemical forms do not appear to be the result of site differences, seasonal variation (Simmons and Parsons 1987), leaf ageing effects or hybridisation.
Chemical forms, as highlighted in Chapter 5, abound in several of the principal oil-producing eucalypts: E. camaldulensis has five chemical forms, E. citriodora four forms, E. dives five forms and E. radiata six forms. The compositional types of E. radiata are shown in Table 4.1. Each form may occur in separate, distinct populations (chemical races), but they can also be present together on the one site and maintain their chemical integrity despite interbreeding. Differences in chemical form do not appear to be associated with differences in oil yield.
In a study of fifty E. radiata trees in a single population containing three distinct chemical forms, Whiffin and Bouchier (1992) concluded that the forms appear to be the result of the actions of the enzymes which control terpenoid biosynthesis modifying a basic monoterpene pool. In E. camaldulensis, a high-spathulenol form was reported to occur at a frequency of one in ten trees amongst high-1,8-cineole forms at Petford in northern Queensland (Doran and Brophy
Table 4.1 The range in abundance of four key compounds in the oils of six chemical forms in a population of Eucalyptus radiata ( Johnstone 1984) 1,8-Cineole (%) -Phellendrene (%) Piperitone (%) Terpinen-4-ol (%)
2–12 4–27 21–55 02–26
7–27 3–33 0.8–10.5 02–37
4–27 3–23 0–19 12–36
9–23 1–7 1–6 14–28
30–60 3–20 0–6 02–23
58–76 1–20 1–3.5 1–6
Mt Buffalo Tumut
Orange
Yowrie
Figure 4.2 The natural distribution (black dots) of Eucalyptus radiata in southeastern Australia as plot-ted from authenticaplot-ted botanical specimens, showing the four regions Mt Buffalo, Orange, Tumut and Yowrie (circled) where Johnstone (1984) found most trees to give foliar essen-tial oils of the high-cineole form.
1990). In a progeny trial of Petford families, the high-spathulenol forms could not be distinguished from the high-1,8-cineole forms until plants were 18–25 months old, suggesting that diversion of the monoterpene pool is linked to maturation processes and the activation of specific enzymes (Doran 1992).
The potential importance of chemical forms, either positively as a rich source of desirable chemicals or negatively as adversely affecting the quality of oil, has been noted by Hillis (1986).
In choosing populations within a species as the genetic resources for a tree-breeding programme, it is preferable to concentrate on populations containing a single chemical form. In E. radiata, for example, Johnstone (1984) identified only four populations, from many sampled throughout the range of the species, where most trees were of the commercially valuable high-cineole form (Figure 4.2). These are the obvious populations to target in seed collections for base populations of this chemical form of the species. However, such a strategy may not be feasible when dealing with species or provenances where desirable (that is commercially valuable) and non-desirable chemical forms co-occur, as is the case with Petford E. camaldulensis. When dealing with popula-tions with multiple forms, seed trees of the desirable, commercial oil type should be selected and segregating progeny removed from the breeding population as soon as they can be confidently identified.
Provenances
When breeding a species for oil production, the primary goals are to maximise the yield of leaves that contain the highest possible concentration of oil of a composition which gains a premium price in the market place. The yield of leaves is usually highly correlated with diameter growth.
There is now ample evidence that much faster growth and greater oil yields of eucalypts in plantations can be obtained through careful selection of provenance and genetic improvement (Eldridge et al. 1993). In a study of provenance variation in E. globulus, Doran and Saunders (1993) found highly significant differences between provenances, and amongst families within provenances, for three principal oil traits (concentration of oil in the leaf, concentration of 1,8-cineole in the leaf and cineole content of the oil) and for stem diameter at breast height in trees aged 1.75 years (juvenile foliage) and 2.75 years (mature foliage) in provenance/progeny trials in New South Wales and Victoria respectively. The results of the younger trial are sum-marised in Figure 4.3. Families from the Otways/Lorne and Jeeralang provenances consistently ranked among the best in leaf oil concentrations and were among the fastest growing sources.
These would be the provenances favoured to provide candidates for breeding populations should breeding E. globulus for oil production in southern Australia be contemplated.
Families
Phenotypic (genotype environment) variation between trees in natural populations in such traits as oil concentration and oil composition is very large. Frequency diagrams for oil concen-tration and 1,8-cineole content in regions-of-provenance of two important oil-yielding species, E. polybractea from West Wyalong and E. radiata from Nerrigundah-Yowrie, are given in Figure 4.4. As such traits are highly heritable, it is desirable to screen trees in the native stands for these traits and select as seed trees for breeding populations only those that rank amongst the best for both traits. Breeding programmes for oil production using mallee eucalypts, including E. polybractea (Western Australia), and E. radiata (Australia and South Africa) have employed this strategy (Donald 1980, 1991, Bartle 1994, Doran et al. 1998).
Progeny
Even when trees have been selected in the natural stands for such traits as oil concentration and cineole content, their open-pollinated seed will give progeny which are highly variable in these traits. This variation provides further opportunity for selection between the breeding population and propagation population phases of the breeding strategy. Figure 4.5 illustrates the variation in oil yield per tree in one family of a southern New South Wales provenance of E. radiata (Doran et al. 1998).
Typically, there is substantial variation in commercial oil traits at all levels of the selection hierarchy in most, if not all, of the main oil-producing species. A commonly repeated scenario is
4 2 6 1 9 5 7 8 3 10
1,8-Cineole conc. (% w/w, dry matter basis)
4 6 3 1 5 7 8 9 2 10
Figure 4.3 Variation amongst provenances of Eucalyptus globulus at 1.75 years of age in (a) stem diameter, (b) oil concentration in leaves, (c) 1,8-cineole concentration in leaves, and (d) 1,8-cineole con-tent of oil. The caps above the bars represent the standard error of the difference (SED) between provenances. Provenance identification: 1Otways, 2 Jeeralang, 3 West Coast, 4 Cape Barren, 5King Island, 6 Flinders Island, 7 Moogara, 8 Geeveston, 9 Lorne, 10South Gippsland.
that reported for E. globulus (Doran and Saunders 1993) and illustrated in Figure 4.6. The high level of variation in oil concentration in all the provenances sampled can be seen. This variation provides the opportunity to select trees for this trait from almost the entire natural range of the species, despite the presence of significant provenance and family within-provenance variation.
Biological characteristics influencing choice of breeding strategy
Breeding system and inbreeding depression
Eucalypts have hermaphrodite, protandrous flowers and are pollinated by insects or birds (Griffin 1989a). They reproduce by a mixed mating system, with both outcrossing (where the
0 Oil conc. (% w/w, dry matter basis)
No. of trees Oil conc. (% w/w, dry matter basis)
No. of trees
Figure 4.4 Frequency distributions of oil concentration in foliage and 1,8-cineole content of oil in Eucalyptus polybractea from West Wyalong, New South Wales, and E. radiata from the Nerrigundah-Yowrie region of southeastern New South Wales.
pollen from one tree fertilises the flowers of another tree) and selfing (pollination of an indivi-dual tree or clone with its own pollen) (Moran 1992, Moran and Bell 1983). The proportions of outcrosses and inbreeds reported in seed collected from natural populations range from 45 per cent outcrossing in one population of E. pellita (House and Bell 1996) to 97 per cent outcrossing in E. camaldulensis (P.A. Butcher pers. comm.).
T1 T2 T3 T4 T5 T6 T8 T9 T11 T12 Tree number
Oil yield/tree (g)
0 50 100 150 200 250
Figure 4.5 Variation in oil yield per tree in a replicate of one family in a 23-month-old Eucalyptus radiata progeny trial.
12
10
8
6
4
2
0 5
Oil concentration (% w/w, dry matter basis)
1 2 10 9 3
Provenance
4 6 7 8
Figure 4.6 Variation in oil concentration in juvenile leaves in a 1.75-year-old Eucalyptus globulus provenance-progeny trial in southeastern New South Wales. The box represents the interquartile range, the whiskers represent the outerquartile range and the circles indicate
Figure 4.6 Variation in oil concentration in juvenile leaves in a 1.75-year-old Eucalyptus globulus provenance-progeny trial in southeastern New South Wales. The box represents the interquartile range, the whiskers represent the outerquartile range and the circles indicate