The field of plant biotechnology is one of the fields of scientific research in which the most rapid advances have been made in recent years. Many opportunities for using biotechnology in plant breeding have been identified, most of which have application to the urgent problems of the improvement of crops in developing countries. This analysis is aimed at defining of the extent to which biotechnology might be applied to tree improvement programmes, especially in developing countries.
THE STATUS OF TREE IMPROVEMENT
The long history of selection and improvement of many agricultural crops is a contrast with that of forest tree species, where the shift from exploitation to domestication is much more recent. Sound management of genetic resources is critical to the optimisation of reforestation efforts, for both industrial and non-industrial applications. For convenience, “industrial” refers here to large scale plantations aimed at commercial timber production, while “non-industrial” plantings include multipurpose plantings and plantings where the focus is less commercial. It is appreciated that the boundary is diffuse.
For industrial forestry species, of which there are probably 100 million hectares of plantations worldwide (Mather 1990, Gauthier 1991), the most common approach to improvement is recurrent selection in genetically quite broad breeding populations. Four generations have been completed in Eucalyptus grandis and three in some Pinus species, but very few programmes are as advanced as these. The use of open-pollinated general combiner seed orchards is the most common approach to the capture of genetic gain, while a few advanced programmes are characterised by the deployment of superior full-sib families or clones. Reproductive biology and patterns of variation are reasonably well documented for the established industrial species. Vigour, form and wood quality are the priority selection criteria in most programmes, with disease- or insect-resistance of importance in only a few cases - in clear distinction from agricultural crop species, where disease- and insect- resistance predominate as selection criteria. The poplars are exceptional industrial species in many respects; hybridisation and clonal selection in much narrower populations have been the traditional approach to improvement, and, perhaps not coincidentally, disease resistance is a major selection criterion. Significant genetic gains are being achieved but, in particular for the long rotation species, there has been only a minor impact to date on the genetic quality of plantations. The major limitations to rapid improvement of most of the established industrial species are:
Long generation intervals, related to poor juvenile-mature correlations, and the long juvenile phase before flowering.
Low effectiveness of selection for many characters, due to low heritability or difficulty in assessment.
Through the use of open-pollinated seed orchards, the exploitation of only a part of the genetic variation available.
Major research priorities for these established industrial species should be the broader development of methods for the propagation of full-sib families and clones, the development of methods for early and more accurate selection and the promotion of precocious flowering. Such action must be coupled with efforts to optimise plantation establishment and management practices, so that potential research gains can be realized.
While only a small proportion of current industrial plantation programmes are located in developing countries, much of the estimated additional area of up to 100 million hectares required worldwide to meet future demand will be situated in tropical areas. Established, proven industrial species are likely to be used for most of this additional estate. A major practical tree improvement objective will be the establishment of sound breeding programmes for these new plantation operations.
In order to make use of a broader range of sites, and to supply products currently provided by harvesting of natural forests, it is likely that some of the additional plantation area will be established with tropical species which are not widely used or known at present. Selection criteria for these “new” industrial species will be similar to those for established species. Some are probably very amenable to improvement, while others present problems, e.g. flowering and seed problems, and insect susceptibility. Many potentially valuable species are simply not well known, in terms of their adaptation and biological and genetic features, and their gene pools are probably under threat. The broader implementation of good improvement programmes will be an important priority for these species. The testing of potentially useful species, characterization of mating systems, provenance collection, establishment of trials, implementation of gene conservation measures and commencement of other breeding work pose a very large task.
There is also a global requirement for several large programmes of non-industrial plantings over at least as large an area as the industrial plantations, mainly located in developing countries. Breeding programmes for non-industrial species differ markedly from those for industrial species in the wide variety, and the complexity, of selection criteria. Several of the taxa are highly variable and display features which render them particularly amenable to very rapid improvement by traditional means. Biological features of many potentially valuable non-industrial species, however, remain largely unknown. Although not well studied, some gene pools are under threat, and some species display unpredictable patterns of variation as a result of human activity. Although selection work has been undertaken in a few programmes, most non-industrial species remain at the species testing stage. The need to establish plantings on a wide range of sites, many of which are marginal or difficult, dictates that non-industrial plantings are likely to be reliant on a substantial number of the thousands of potentially useful species. The work involved in selecting the most promising species is formidable. The major emphases of improvement for non-industrial species are likely to be exploration, collection, taxonomic studies of variation, species and provenance testing, the assessment of reproductive features, and conservation activities. Interspecific hybridization may be of considerable value for several taxa. Where regionally consistent selection criteria can be defined and the tree crop is considered sufficiently important, clonal selection programmes are likely to be of value for species easily propagated vegetatively, and seed tree selection for species propagated by seed. Sophisticated breeding programmes are unlikely to be warranted for most species. It is possible that a “once only” or “quantum leap” approach to breeding may be more appropriate than programmes offering continuous incremental gains. Both types of approaches will necessitate incorporation of genetic conservation concerns into the breeding programmes to ensure lasting gains.
As pointed out by Kanowski (1993), tree improvement is greatly under-resourced. This is especially so in developing countries, where funding provided through national, regional, bilateral and international programmes is not sufficient to conduct properly the essential activities outlined above, and where the continuity of action necessary is frequently missing. 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, which constitute the major impediment to rapid improvement.
BIOTECHNOLOGIES AND THEIR APPLICATIONS TO TREE IMPROVEMENT
Cryopreservation and In Vitro Storage
For material displaying regenerative competence (e.g. embryogenic cultures), a research project directed towards the development of procedures for successful in vitro storage or cryopreservation of cultures for a new species might be expected to be short term (say three to five years), with a moderately high expectation of success.
Although increasingly used operationally for the storage of threatened germplasm of agricultural species, in vitro storage and cryopreservation have little to offer for this purpose in forest tree species. Gene pools of most of the established industrial species are reasonably well preserved in in situ and ex situ stands and, for the short and medium terms, in seed stores. Undoubtedly, an urgent gene conservation problem exists for many tree species, particularly among the tropical hardwoods and non-industrial species. Distributions of these species are poorly known, as are their biological characteristics. Although seeds of some species will be recalcitrant, many are orthodox. Major impediments to the conservation of forest tree germplasm include the availability of resources sufficient for only a very small fraction of the survey and collection work which would be required before any germplasm could be stored, and the unreliability of many existing seed storage facilities. The replacement of existing facilities with more sophisticated technology is not likely to make a positive impact on the gene conservation problem with tropical tree species. Even for the recalcitrant species, activities should favour the establishment of ex situ plantings which will facilitate evaluation of the material. In the longer term, cryopreservation and in vitro storage may have some application as a back-up conservation strategy for populations of well surveyed species which are recalcitrant such as poplars and walnut. Of course, ex situ conservation measures should themselves be regarded only as complementary to sound in situ programmes (see Wang et al. 1993).
Cryopreservation warrants much more attention as a means of maintaining juvenility and capturing genetic gains offered by clonal forestry with industrial species. The technology is thus applicable mainly where good breeding programmes are already in place, clonal forestry is a realistic goal, and “rejuvenation” is difficult - in particular for the conifers.
With the increasing emphasis on international cooperative breeding of both industrial and non-industrial forest tree species in tropical countries, international movement of material will be important. In the short term, such movement is likely to be mainly in the form of seed for species and provenance testing. Movement of vegetative material, however, is likely to become more important as breeding programmes advance, and in vitro approaches could find application in the longer term.
Techniques for the analysis of isozymes, Restriction Fragment Length Polymorphisms (RFLPs) and Random Amplified Polymorphic DNA (RAPDs), are now well established, and the adaptation of procedures to suit new species can be expected to be a relatively short term undertaking.
Markers have important immediate applications in supportive research for advanced breeding programmes with industrial species, mainly in relation to quality control, e.g. checking of clonal identification, orchard contamination and within-orchard mating patterns by “fingerprinting”. Isozymes will be satisfactory for many, but not all, of these purposes.
Markers also have important immediate application in supportive research for tropical hardwoods and non-industrial species, in particular for essential taxonomic studies and investigations of mating systems. Both isozymes and DNA markers will be useful for these purposes. Markers will also be useful for the quantification of genetic variation to aid in sampling strategies for gene conservation and breeding population collections, although, due to modest correlations with patterns of variation for adaptive traits, they must be used conservatively.
Realistically, application of marker-assisted selection in the short or medium term is likely to be very limited. Cheaper markers would be required and, even if these were available, the technology would apply mainly to advanced breeding programmes where the creation and maintenance of the appropriate population structures could be afforded, and where clonal forestry is achievable. A small number of programmes fall into this category and, for these, some effort aimed at developing marker-assisted selection can be justified. For most species, current resources would be better directed towards moving breeding programmes to this stage of advancement, rather than to the development of marker-assisted selection. Marker-assisted selection is unlikely to have much application to non-industrial species, although, by virtue of a very short generation time, taxa such as Gliricidia may be useful model species for experimentation.
The major current value of markers lies in (i) support to long term strategic research (ii) the great contributions which marker studies are making to advances in the understanding of basic genetic mechanisms and (iii) understanding genome organization at the molecular level. For forest tree species, the study of quantitative traits will be an important emphasis of this work in coming years. This work will be most efficiently concentrated on a few model species, e.g. Pinus taeda and in particular hybrids such as P. elliottii X P. caribaea.
In Vitro Selection
Many recent publications concerning crop plants have reported useful correlations between in vitro responses and the expression of desirable field traits (most commonly disease-resistance) although positive results are available also for tolerance to herbicides, metals, salt and low temperatures.
For the selection criteria of major general importance in forest tree species, in particular vigour, stem form and wood quality, however, poor correlations with field responses will limit the usefulness of in vitro selection. In vitro selection is therefore likely to have very limited application in forest tree species, and only to be of possible interest in a few programmes where there is a disease-resistance selection problem, but of no broad strategic value as a research objective.
Crops transformed with genes for insect and virus resistance and resistance to various types of herbicides are at or near commercial application, and plants into which these genes have been inserted include the poplars. Many projects are in progress with forest tree species, in particular for modification of lignin biosynthesis through antisense technology. Insertion of the insect or herbicide resistance genes currently available 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. Manipulation of more complex traits would be a much more formidable undertaking, and, although rapid progress is being made in this field, much research remains to be done. The availability of effective transformation techniques remains an obstacle, although improved techniques are being developed. Regeneration is difficult for some tree species, but the problem may be overrated; the non competence of mature material is not necessarily an obstacle to effective application of genetic engineering, provided that the juvenile material responds satisfactorily. An often overlooked research component is field testing which would be required before a responsible recommendation for large scale deployment of transgenic plants could be made. Such testing could be extensive and prolonged, depending on the species and genes involved. 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, e.g. in the poplars and some pines, eucalypts and tropical hardwoods. A single gene may be sufficient to confer resistance for very short rotation species, but a much more cautious approach should be applied with long rotation species. The work involved in introducing several different resistance genes, in sufficient numbers to ensure that insects do not acquire tolerance during a long rotation, should not be underestimated. The reduction of lignin biosynthesis is a valuable objective for the pulp species. The introduction of herbicide tolerance genes is of some interest, but in many programmes the advantage of using unguarded herbicide application may not be sufficient to pay for the research program. The extent to which genetic engineering permits substitution of environmentally “friendly” herbicides such as glyphosate for currently used residual herbicides could be an important factor. Cold tolerance genes are likely to be of some commercial value in many species, in particular the eucalypts. Much remains to be done, though, to establish that sufficient tolerance can be conferred using antifreeze proteins, and to extend the work to tree species. The 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 tree species is the state of knowledge of molecular control of the traits which 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 are of high quality with respect to other traits as well. The clonal test is the most logical basis for integration of genetic engineering into traditional tree improvement programmes. For these reasons, genetic engineering is most appropriately conducted with species where breeding programmes are advanced and sound clonal forestry programmes 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.
Variation induced during cell or callus cultures has been reported for many species. For some crops, variants have been produced showing economically useful levels of traits such as resistance to disease and increased levels of salt. The phenomenon is still not clearly understood, and persistence of modifications through subsequent sexual generations has been demonstrated in some cases but not in others. This is not a research area where favourable results could be predicted with any confidence, and use of the approach is furthermore dependent on being able to regenerate from the cells or callus. Research with new species will be more soundly based when the phenomena are better understood in the model species already under study.
Assuming stability of the characteristics, the approach is of most value where selection can be done at the cell level (e.g. by exposure to a phytotoxin or to high mineral levels), thus enabling screening of large numbers of genotypes, and where the level of the trait sought lies outside the range available naturally in the species. Cold tolerance in the eucalypts is an example which may meet these criteria. No immediate applicability is evident to the new tropical hardwoods or to non-industrial species, for which genetic variation naturally available is generally poorly defined.
This is a field with a long history of research. There have been several successes, notably with species of the Brassicaceae and Solanaceae. Severe limitations are imposed, however, by the taxonomic relationship of parents, and the requirement for regeneration from protoplasts. Expectations of success with a new pair of species could be expected to be low.
There is little apparent applicability of protoplast fusion to forest tree species, particularly those (e.g. some tropical hardwoods and non-industrials) for which investigations of the possibilities for traditional hybridization remain minimal. In the longer term, the objectives of protoplast fusion may be better served by manipulations at the DNA level.
Although anther culture has been used for the rapid production of homozygous lines in the breeding of some self-pollinated cereals and vegetable crops, few reports exist of regeneration from gametophyte cultures of forest tree species. Research with this objective is likely to be long-term with a relatively high risk of an unsuccessful result.
Induction of haploid plants has no immediate application in forest tree improvement programmes although such plants may be of some use in basic genetic studies, e.g. for studies of heterosis in forest tree species. As a long term strategic research objective for industrial species, induction of haploid plants should be of low priority until such time as methods for early selection and the promotion of early flowering are available. The breeding of non-industrial species is unlikely to warrant this level of sophistication.
In Vitro Embryo Rescue
This technique has been used, particularly in fruit trees, to grow embryos that normally would abort due to incompatibility between ovule and embryo development, and also for the rescue of the zygotic embryos of apomictic species. The techniques are not difficult, and development of protocols for a new species generally would be a minor research task of short duration and with a high expectation of success.
Embryo rescue has been used occasionally in forest tree species, but the requirement for such technology is likely to be limited, probably to a small number of hybrids for which embryo development in vivo is restricted. In the short term, research of this type is likely to have a very low priority. In the longer term, as barriers to natural hybridization become better defined, some work can be targeted at hybrids which species testing has identified as potentially of interest and for which other studies suggest that in vitro approaches might provide a solution.
Over 1 000 plant species have been micropropagated, including over 100 forest tree species. With sufficient research effort, successful protocols could be developed for most tree species. In general a research project of this type may be described as short to medium term with a moderately high expectation of success.
For most industrial species, the high cost of planting stock and insufficient data regarding field performance remain major obstacles to be overcome before broader use of micropropagules as direct planting stock could be contemplated. Micropropagation has an immediate application 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 which incorporate the identification of outstanding clones. This is currently the case in only a few programmes with tropical eucalypts, and perhaps also some poplar programmes, but potentially also could include other industrial species. Appropriate integration into breeding programmes is essential. Where clonal testing on a reasonable scale is possible and affordable, the current applicability of protocols 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. Breeding programmes with new industrial and non-industrial species are not sufficiently advanced to warrant much use of micropropagation for such purposes in the immediate future.
Micropropagation may have wider application for 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 protocols for those species for which such are not already available is therefore a useful research objective, but one which should not take priority over issues such as advancement of the breeding program.
Developments with somatic embryogenesis and artificial seed technology may overcome the planting stock cost limitation discussed above with considerable research, and enable direct use of such propagules in plantation establishment. For industrial species the development of these technologies is a useful long term research objective, but one which is best pursued with one or two appropriate model species such as Picea abies or Pinus taeda, while observing the same rules regarding numbers, extent and rotation of clones as in clonal forestry programmes in general.
In Vitro Control of the Maturation State
A long history of research with in vitro rejuvenation has led to some successes, but little evidence that rejuvenation can be completely, permanently and reliably achieved by this method. Similarly, sporadic reports of accelerated maturation through in vitro manipulations do not suggest that acceleration of ageing may be reliable obtained. Further empirical work with these objectives is likely to have a low probability of success. An understanding of the molecular basis of maturation is much more likely to lead to practical manipulation, but this work is in its infancy, and acceleration or reversal of maturation to precise levels remains a distant prospect.
The maintenance of juvenility is as useful as rejuvenation for many purposes for clonal forestry with industrial species, and is probably achievable using technologies such as cryopreservation or coppicing. Nevertheless, 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 where good breeding programmes are in place, and where other limitations to clonal forestry do not exist. Maturation is a much less important issue with many of the non-industrials. Manipulation of the maturation state to induce early flowering and reduce generation intervals is potentially of greater interest than rejuvenation, for industrial species at least, but is only of value where active breeding programmes are in place.
Biotechnology represents, for many agricultural crops, the best hope for meeting the urgent objectives of breeding programmes - preservation of gene pools of wild relatives, multiplication of elite cultivars, and in particular, the acquisition of virus and insect resistance. This applies also for many crops in developing countries, including cassava, a crop grown entirely by resource-poor farmers. By contrast, biotechnology offers little to meet the most urgent priorities in forest tree improvement, although forest tree species are the subject of very active biotechnology research programmes. This is because the major objectives of most tree improvement programmes differ greatly from those for crop species, their activities do not easily lend themselves to centralization due to the large range of species and end use objectives, and because of poor knowledge of most of these species.
The possibilities for technology replacement or supplementation in tree improvement programmes in the short term are:
For breeding programmes with established, biologically well-known industrial species:
The use of molecular markers in quality control in advanced breeding programmes, e.g. for checking of clonal identification, seed orchard contamination and within-orchard mating patterns by “fingerprinting”.
The use of micropropagation 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 worthy clones within the framework of sound clonal forestry strategies.
For breeding programmes with biologically less well-known “new” industrial and non-industrial species:
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.
Strategic research priorities relating to the application of biotechnology in tree improvement are:
Long-term generic research. Priority objectives are:
Genetic engineering for sterility. This is of high priority because it 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. Of high priority, this work is of relevance in particular for industrial species, but will pave the way also for applications of biotechnology to non-industrial trees.
Molecular studies of the maturation state. This is of high priority for industrial plantation species.
Development of somatic embryogenesis, in combination with artificial seed technology, as a clonal propagation method for industrial plantation species. This is of medium priority.
Research projects of the above type are most efficiently conducted with a small number of model species. The diffusion of resources and effort to many species is likely to impede progress.
Long-term specific research. Some objectives are:
Transformation with appropriate genes may be achieved within the short to medium term (say the next five to ten years), but must be followed by perhaps ten years of field testing before responsible commercial deployment could be recommended.
Marker-assisted selection, for species where breeding is advanced and where creation and maintenance of the appropriate population structures is feasible and affordable. It will probably be several years before this is possible on an effective operational scale.
Short- to medium-term research. Useful objectives are:
The examination of genetic correlations between regenerative competence and commercially important field traits. This is of high priority.
The development of cryopreservation methods as a means of maintaining juvenility, for advanced breeding programmes with industrial species.
The development of simple micropropagation techniques for species for which such is not already available (low to moderate 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). However, the possibilities of eventual regeneration of seedlots stored needs attention in this respect (see Wang et al. 1993).
Currently, there are few areas where biotechnology can profitably replace or complement existing technologies in tree improvement. The potential applications of several biotechnologies are interdependent e.g. the application of genetic engineering, and of cryopreservation for the retention of juvenility, are dependent on the availability of a suitable method for clonal propagation from cultured material. The applications of these technologies, and marker-assisted selection, are only appropriate to good clonal forestry programmes, and it can be argued that clonal forestry is the gateway to major applications of known biotechnologies in forest tree improvement. Sound clonal forestry is dependent on the existence of good breeding programmes to produce the desirable and cumulatively superior genotypes on a continuing basis. The funding of biotechnological research initiatives cannot be at the expense of the development of good genetic improvement programmes.
Apart from the large investments required for the development of most biotechnologies, and for appropriate testing, large investments in many breeding programmes are required to reach the appropriate levels of advancement, frequently with added levels of structural sophistication. As emphasized by Burdon (1992), a commitment to biotechnology must be part of a substantial increase in the total commitment to plantation forestry techniques and genetic improvement. This level of investment is not being made for most industrial species, and may never be affordable for most non-industrials. The gap between traditional methods and biotechnological approaches to forest tree breeding is therefore substantially a matter of financial investment.
The high costs of the required research programmes argue strongly for collaboration, rather than competitive proprietary biotechnology, as stressed by Burdon (1992). It is desirable at least that expensive and long-term generic research be conducted collaboratively by specialized laboratories and with appropriate model species.
Many highly profitable private forest plantation programmes exist in developing countries. Taking into account risks and potential benefits, managers of these programmes will make their own commercial decisions on the appropriate stage at which investment in biotechnology is appropriate, and on the level of investment. These industrial operations aside, funding available for genetic improvement in developing countries, in particular for new industrial and non-industrial species, is grossly inadequate for the urgent basic exploration, conservation and testing work which needs to be done, and for the training and facilities which are required. Funding currently used for these purposes should be partly redirected to biotechnology only to the extent that effective technology replacement is achievable in the short term. In this category are the use of molecular markers in studies of reproductive biology, in taxonomic investigations, and for the quantification of genetic variation. Massive increases in funding for tree improvement in developing countries may warrant more attention to biotechnology, but diversion of currently available funds to longer term biotechnological research initiatives is likely to have a negative impact on genetic improvement programmes for these species.