Russell J. Haines is with the Forest Research Centre of the Queensland Department of Forestry in Gympie, Australia. He was the recipient of the FAO Andre Mayer Research Fellowship in 1992.
Cultured nodes and axilliary meristems of Araucaria cunninghamii, a valuable plantation conifer
A rooted axillary shoot of Araucaria cunninghamii
A summary of current research into biotechnology applications for forest tree improvement and recommendations for the prioritization of research objectives in the sector: This article is based on a longer work soon to be published as an FAO Forestry Paper.
In no other field of scientific investigation today are such rapid advances being made as in plant biotechnology. Biotechnology comprises any technique that uses living organisms to make or modify a product, to improve plants or animals or to develop micro-organisms for specific uses. The public imagination has been caught by such highly publicized developments as the tomato that can be frozen and the cassava and other agricultural crops that have been genetically engineered for insect and virus resistance, and which are now in or near commercial release.
The potential benefits of plant biotechnology in forestry are perhaps even greater than in agriculture because of the possibility of gaining time in certain tree improvement processes. The challenges facing foresters regarding production or outturn, whether of wood or other products, are no less urgent than those facing agriculturists.
Tree improvement research falls into two categories: supportive research, e.g. the collection of data on reproductive biology and genetics necessary to support effective breeding; and strategic research, aimed at the development of better breeding methods. Many strategic research projects concerning biotechnology have been initiated, in the opinion of some (e.g. Sedgley and Griffin, 1989) at the expense of other urgently required tree improvement activities. Clearly, the careful prioritization of research objectives is important and biotechnologies should only be used where there is an intimate basic knowledge of the species being experimented. Nonetheless, if basic biological information and knowledge are available and if sound tree improvement programmes are in place, biotechnology can be a powerful tool. This analysis is directed towards the definition of important biotechnological research priorities in forest tree improvement.
The general objective of a genetic improvement programme should be the sustainable management of genetic variation to produce, identify and multiply for the operational planting of well-adapted genotypes of the desired quality. Typically, this incorporates:
· the establishment of initial populations, including species and provenance testing, as well as the development of breeding and gene conservation populations;
· population improvement, frequently including recurrent cycles of selection and recombination;
· the derivation and multiplication of strains to be used operationally.
In principle, the above applies to both industrial (i.e. biologically well-known species planted on a large scale) and non-industrial species, although the practicalities differ to some extent. The current status of tree improvement and associated research trends have recently been reviewed (FAO, in press; Kanowski, 1993). Significant genetic gains are being achieved in breeding programmes for established industrial species, and the broadening of such efforts will be important. Major limitations to rapid improvement with most of the established industrial species are:
Tissue cultures in an incubation cabinet
· long generation intervals, related to poor juvenile-mature correlations (i.e. that the characteristics of young trees are not necessarily accurate indicators of those found in mature individuals) and the long juvenile phase with respect to flowering;
· the low effectiveness of selection for many characters as a result of low heritability or difficulties in assessment;
· through the use of open-pollinated orchards, the exploitation of only a part of the genetic variation available.
Major research priorities for established industrial species should be the broader development of methods for the propagation of full-sib families (i.e. multiple examples from a single, known male-female pair) or clones, the development of methods for early and more accurate selection and the promotion of precocious flowering. To utilize a broader range of sites, and to supply products currently provided by harvesting in natural forests, a significant proportion of new plantation areas will probably be established with tropical species that are not widely used at present. Some may be very amenable to improvement, while others present problems, e.g. flowering and seed problems and susceptibility to insects and disease. The distribution and potential uses of many species are not well known and gene pools are probably under threat. The implementation of improvement programmes will be an important priority for these species. The testing of potentially useful species, the characterization of mating systems, provenance collection, the establishment of trials on different sites, the implementation of gene conservation measures and the commencement of other breeding activities pose large challenges.
Several non-industrial taxa are highly variable and feature early and prolific flowering - conducive to very rapid improvement by traditional means. The biological features of many potentially valuable non-industrial species, however, remain largely unknown. Some gene pools are under threat. Although selection work has been undertaken in a few programmes, most non-industrial species remain at the species-testing stage. Although the genetic improvement of non-industrial species is similar in principle to that of industrial trees, and some similar limitations apply, special difficulties are presented by:
· the need to establish plantings on a wide range of sites, many of which are marginal or problematic;
· the multiplicity of selection criteria (e.g. fuelwood or mulching quality!;
· the variability of selection criteria from one grower to the next;
· the low value of forest products in some systems; and
· the difficulty of transferring breeding results to operational plantings, e.g. where growers have a strong economic preference for raising their own planting stock.
A rejuvenated shoot from an old sequoia tree
The work involved even in selecting the most promising species is formidable and, for reasons presented above, improvement beyond species and provenance testing may be difficult to justify for many non-industrial trees. Major priorities for the improvement of non-industrial species are likely to be taxonomic studies of variation; species and provenance testing; the assessment of reproductive features; and conservation activities.
As pointed out by Kanowski (1993), tree improvement does not receive adequate financial or human resource inputs. This is especially true in developing countries where funding provided through national, regional, bilateral and international programmes is not sufficient to conduct the essential activities properly. The problems are further compounded by inadequate levels of training and facilities in most areas. For the non-industrial species in particular, it is these resource limitations and the variability in user requirements, rather than biological constraints' that constitute the major impediments to rapid improvement.
The author has reviewed the current status of biotechnology, and applications in tree improvement in detail in a work soon to be published (FAO, in press). The following paragraphs summarize the most important biotechnologies.
Cryopreservation and in vitro storage
This comprises the maintenance of cells, tissues or organs in cultures where growth is slowed (e.g. by the reduction of light, temperature or nutrients) or suspended (by immersion in liquid nitrogen). Many technical difficulties are involved, particularly in the subsequent regeneration of plants from the cultures, but recent results are generally encouraging. Regeneration from cryopreserved tissues has been induced for more than 70 plant species, including coconut, rubber, cocoa and coffee, and for several forest tree species. These results have led to hopes that the technologies may have a number of applications in tree improvement.
Gene conservation. Although increasingly used for the storage of threatened germplasm of agricultural species (Engelmann, 1991), in vitro storage and cryo-preservation have little to offer for this purpose with regard to forest trees. Gene pools of most of the established industrial species are reasonably well preserved in stands, both in situ and ex situ, and in seed stores. Undoubtedly, the gene pool of many tree species is threatened, particularly among the tropical hardwoods and non-industrial species. Distributions of these species are poorly known, as are their biological characteristics. Major impediments to the preservation of forest tree germplasm are: the inadequacy of resources for the survey and collection work that would be required before any germplasm could be stored; and the unreliability of many existing seed storage facilities. Even for the recalcitrant (hard-to-store) species, priority would be better directed to the establishment of ex situ plantings, which should facilitate urgently needed evaluations of the material. In the longer term, cryo-preservation and in vitro storage may have some application as a backup conservation strategy, but only for populations of well-surveyed, recalcitrant species.
Maintenance of juvenility. Suspension of the growth processes also implies the maintenance of the maturation state previously attained in the tissues - without any of the uncertainty associated with alternative strategies such as long-term hedging or serial propagation. Cryopreservation therefore warrants much more attention as a means of maintaining juvenility during simultaneous clonal testing and thus capturing genetic gains offered by clonal forestry with industrial species. The technology is therefore applicable mainly in cases where good breeding programmes are in place, where clonal forestry is a realistic goal and where "rejuvenation" is difficult -particularly for the conifers.
Use of molecular markers
The use of molecular markers involves the examination, using sophisticated biochemical techniques, of variations in cellular molecules such as DNA and proteins. As an alternative to traditionally measured features such as vigour, stem quality and various morphological aspects, molecular markers offer the advantages of being unaffected by the environment or the developmental stage of the plant while also being very numerous. These characteristics have led to a number of potential applications in tree improvement.
Genetic fingerprinting. The inherent characteristics of molecular markers render them much more useful than morphological traits in establishing the identity of a particular tree or tracing its genetic relationship to other trees. For example, using molecular markers, it was possible to identify each of 39 peach cultivars individually (Ballard et al., 1992). Markers have important immediate applications in supportive research for advanced breeding programmes with industrial species mainly for quality control, e.g. checking clonal identification, orchard contamination and within-orchard mating patterns by "fingerprinting". Markers also have important immediate applications in supportive research for tropical hardwoods and non-industrial species, in particular for essential taxonomic studies and investigations of mating systems.
Quantification of genetic variation. Molecular markers are potentially more useful for quantifying genetic variation than are traits such as vigour and stem form, for which environmentally induced variation is frequently a confounding factor (i.e. it is not clear if traits are produced genetically or by external factors). Markers have been used to compare within- and inter-population variation in several tree species (Muller-Starck, Baradat and Bergmann, 1992). The quantification of genetic variation to aid in sampling strategies for the development of gene conservation and breeding populations of new industrial and non-industrial species is a potentially useful application of molecular markers. Markers may, however, provide underestimates of genetic variation with respect to traits (e.g. vigour and stem quality) that are more subject to evolutionary pressures and, therefore, will need to be used with caution.
Marker-assisted selection. This refers to indirect selection on the basis of markers shown to be associated with commercially important genes. Unaffected by the environment or developmental stage, markers offer the possibility of highly effective and early selection, long the hope of forest tree breeders (e.g. selection for wood quality at the young seedling stage). Although the possibilities are very attractive, there are limitations that will prohibit application in the short or medium term (Strauss, Lande and Namkoong, 1992): i) marker analysis is currently too expensive to permit the screening of large populations of seedlings; ii) associations between markers and economically important traits have to be established separately for different families, thus, even when cheaper markers are available, marker-assisted selection will apply mainly to advanced and sophisticated breeding programmes - those for which the creation and maintenance of the appropriate pedigree structures can be afforded and where clonal forestry is achievable. For most species, current resources would be far better directed towards moving breeding programmes to this stage of advancement, rather than to the development of marker-assisted selection.
The major current value of molecular markers lies in long-term strategic research; marker studies are making great contributions to advances in the understanding of basic genetic mechanisms and genome organization at the molecular level. An important emphasis of this work in coming years will be the study of quantitative traits of forest trees, of which a few model species will receive most attention, for example loblolly pine (Pinus taeda).
This comprises the insertion of novel genes into a plant or else the modification of existing genes through manipulation of the DNA molecule. Crops to which genes for insect, virus and selected herbicide resistance has been added are being or are near to being applied commercially. A tree crop into which these genes have been inserted is the poplar. Many projects are under way for forest trees, the reduction of lignin biosynthesis, for instance, but numerous technical difficulties remain to be solved. The insertion of currently available insect- or herbicide-resistant genes into a new species would constitute a major research undertaking, and successful application would be dependent on being able to regenerate from the transformed cells. The manipulation of more complex traits would be an even more formidable undertaking and much research remains to be done. An often overlooked research component is the extensive testing that would be required before a responsible recommendation for the large-scale deployment of transgenic plants could be made. Research projects of this type are necessarily intensive and must be regarded as long-term with only a modest expectation of success.
Insect resistance is of potential value, for example in poplars and some tropical hardwoods. However, the work involved in introducing several different resistance genes, sufficient to ensure that insects do not acquire tolerance during the rotation, should not be underestimated. The reduction of lignin biosynthesis is a very valuable objective for the pulp species. The introduction of herbicide-tolerant genes is of some interest but, in many programmes, the advantage of practicing unguarded herbicide applications may not be sufficient to pay for the research programme. Cold-tolerant genes are likely to be of some commercial value for many species, in particular the eucalypts. Much remains to be done, however, to establish that sufficient tolerance can be conferred using antifreeze proteins and to extend the work to tree species.
Prevention of the escape of genes into wild populations is likely to become an important concern, and sterility should be an early target of genetic engineering work with forest tree species. The major factor limiting application of genetic engineering in forest trees is the state of knowledge of molecular control of the traits that are of most interest - those relating to growth, adaptation and stem and wood quality. Genetic engineering of these traits remains a distant prospect.
It is important that genetically engineered genotypes be of high quality with respect to other traits as well. The clonal test is the most logical basis for the integration of genetic engineering into traditional tree improvement programmes. For these reasons, genetic engineering is most appropriately conducted with species for which breeding programmes are advanced and clonal forestry can be realistically contemplated. Research on this subject should not assume a high priority with species for which natural variation available within the taxon remains poorly investigated.
This refers to in vitro plant propagation methods. The principal approaches are axillary budding (actually a miniaturization of propagation with cuttings): the induction of adventitious buds on non-meristematic tissue (i.e. inducing a shoot where one would not normally develop): and somatic embryogenesis (where individual cultured cells or small groups of cells undergo development resembling that of the zygotic embryo). As an alternative to other vegetative propagation methods, the attraction of micropropagation lies in its ability to multiply elite clonal material very rapidly. More than 1 000 plant species have been micropropagated, including more than 100 forest tree species (Bajaj. 1991; Thorpe, Harry and Kumar, 1991). Successful experimental practices probably could be developed for most tree species.
For most industrial forest plantation species, the costs of planting stock and insufficient data regarding field performance remain major obstacles to be overcome before a broader use of micropropagules as direct planting stock may be contemplated (Haines, 1992). Micropropagation has an immediate application, however, in integrated clonal propagation systems featuring the commercial planting of cuttings harvested from rapidly multiplied, micropropagated stool plants of the selected clones. This approach is of value only in very advanced breeding programmes incorporating the identification of outstanding clones currently only a few programmes are at this level. Appropriate integration into breeding programmes is essential. Where clonal testing on a relatively large scale is possible and affordable, the current applicability of techniques mainly to juvenile material is not necessarily an impediment to the capture of good gains through clonal forestry. This conclusion, however, is dependent on the ability to store juvenile material for the period of a clonal test. Genetic variation in response, often substantial, is not likely to be a major problem where clonal testing can be preceded by screening for responsive genotypes, although demonstrating the absence of adverse correlations with economic traits is important. Breeding programmes new industrial species and non-industrials are not sufficiently advanced to warrant much use of micropropagation in the short term.
Field testing of loblolly pine (Pinus taeda) tissue culture plants and Dings of the same family. Plantlets display slightly more mature features
Micropropagation may have a wider application in the multiplication of stool plants of industrial species as breeding programmes become more advanced and other limitations to clonal forestry (e.g. maturation problems) are overcome. For some non-industrial tree species, micropropagation may ultimately have a role in the multiplication of selected varieties prior to release. Development of simple micropropagation techniques for those species for which such methods are not already available is therefore a useful research objective but should not take priority over issues such as advancement of the breeding programme.
Work done with some crop species indicates the possibility of encapsulating somatic embryos to form artificial seeds which can then be handled like conventional seeds. With considerable research, developments in this area may overcome the constraint of planting stock costs (discussed above) and enable the direct use of such propagules in forest plantation establishment. For industrial species, therefore, the development of these technologies is a useful long-term research objective but one which is best pursued with one or two model species, for example Picea abies and Pinus taeda.
In vitro control of the maturation state
There have been several reports of cultured mature buds displaying a reversion to a more juvenile state in response to the culture techniques and conditions. This has led to hopes that in vitro rejuvenation may be the solution to the poor rooting and vigour displayed by shoots collected from trees of selectable age for many forest plantation species. The major limitation to this approach is that there is little evidence of complete, permanent and reliable rejuvenation. In fact, some studies have clearly demonstrated the effect to be a temporary response to the culture condition. Further empirical work with this objective has a low probability of success. An understanding of the molecular basis of maturation (e.g. Hutchison and Greenwood, 1991) is much more likely to lead to practical manipulation, but this work is in its infancy and the reversal or promotion of maturation to precise levels remains a distant prospect.
For clonal forestry with industrial species, the maintenance of juvenility is about as useful as rejuvenation for many purposes (Haines, 1992) and it is probably able to be achieved using technologies such as cryopreservation or coppicing. Nevertheless, a more fundamental control of the maturation state remains one of the most valuable objectives of long-term strategic research in forest tree improvement with industrial species. Rejuvenation is most applicable to efforts where good breeding programmes are in place and where other limitations to clonal forestry do not exist.
The preceding analysis suggests that the short-term possibilities for applying biotechnology in supportive research in forestry are:
· the use of molecular markers for quality control in advanced breeding programmes with established industrial species, e.g. for checking clonal identification, orchard contamination and within-orchard mating patterns by fingerprinting;
· the use of markers in essential taxonomic studies and investigations of mating systems;
· the use of markers for the quantification of genetic variation to aid in the design of sampling strategies for gene conservation and breeding population collections for breeding programmes with "new" industrial and non-industrial species.
Strategic research priorities relating to the application of biotechnology in tree improvement can be grouped into three broad areas:
Long-term generic research. This is most efficient if conducted collaboratively with a small number of model species, thus avoiding the diffusion of resources and efforts. High priority should be accorded to:
· genetic engineering for sterility - this will underlie many of the eventual applications of genetic engineering;
· the use of molecular markers and DNA transformation techniques to investigate genetic processes at the molecular level, in particular those relating to complex traits such as growth, adaptation and stem and wood quality, is of particular relevance to industrial species, but will also pave the way for the application of biotechnology to non-industrial trees;
· molecular studies of the maturation state for industrial plantation species.
A somewhat lower priority should be given to the development of somatic embryogenesis in combination with artificial seed technology as an inexpensive method of clonal propagation.
Long-term specific research. Two high priority areas are:
· genetic engineering of useful traits, including lignin reduction in pulp species; cold tolerance, particularly in eucalypts; and insect resistance, e.g. in poplars and perhaps Meliaceae (when appropriate breeding programmes are in place). Transformation with appropriate genes (the introduction of several genes in the case of insect resistance) may be achieved within the short to medium term (the next five to ten years) but must be followed by perhaps ten years of field testing before responsible commercial deployment may be recommended;
· marker-assisted selection, for species where breeding is advanced and where the creation and maintenance of the appropriate population structures are feasible and affordable - it will probably be ten years before this is possible on an operational scale.
Short- to medium-term research. Areas that warrant attention include:
· the examination of genetic correlations between regenerative competence and commercially important field traits (high priority);
· the development of cryopreservation methods as a means of maintaining juvenility in advanced breeding programmes with industrial species (high priority);
· the development of cryopreservation as a backup measure for gene conservation in proven species for which breeding programmes are in existence and for which seed recalcitrance has been demonstrated (moderate priority);
· the development of simple micropropagation techniques for species where none is yet available (low to moderate priority).
Modern biotechnology should be perceived as a new group of tools or means to be used as adjuncts or complements to conventional technologies in solving problems and meeting the needs of human beings. A balance should be maintained between modern biotechnological and conventional research, and the development and application of biotechnology should be driven by needs and not by technological capability. The use of modern biotechnologies should be promoted for more efficient solutions to problems already on the agenda and within the framework of the existing priorities of individual countries. Thus, the funding of biotechnological research initiatives cannot and must not be at the expense of conventional genetic improvement programmes.
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