Genetic Engineering Newsletter – Special Issue 7
July 2001
supported by
Gerling-Foundation, Triodos-Stichting, Mahle-Foundation, Zukunftsstiftung Landwirtschaft & Hatzfeldt Foundation
Transgenic woody plants in forestry and agriculture
CONTENTS
Preface
Ecological and genetic characteristics of woody plants
Methods of genetic engineering
Targets for forest-tree engineering
Increasing biomass production
Modifying lignin content and composition
Herbicide tolerance
Insect resistance
Abiotic-stress tolerance
Phytoremediation
Targets for fruit and wine production
Shortening of the juvenile phase
Pest resistance
Risk aspects and open questions
Vertical gene transfer
Horizontal gene transfer
Modifying the lignin composition
Insect resistance
Abiotic-stress tolerance
General risks
References
Preface
Transgenic woody plants are one target object of genetic engineering research quite unknown to the public. The current discussion about risks and benefits of genetic engineering is mainly focused on food crops and medical research. Despite the public ignorance this field of genetic engineering has developed rapidly since the first officially known field trial with transgenic poplars in 1988 in Belgium. In a study on GE technology in the forest sector commissioned by the WWF, Owusu (1999) summarizes 116 officially known field trials with transgenic trees. Up to now this number will definitely have increased. The first application for commercial cultivation of virus resistant GE papaya trees has already been approved in the United States (Haas 2001).
This Special Issue summarizes the different research emphases on transgenic woody plants, presents problems and limits of the new developments and discusses possible risks.
Ecological and genetic characteristics of woody plants
Trees and bushes are long-living organisms compared to the majority of economically used plants. Life cycles are much longer than for non-woody agricultural crops. The juvenile phase is quite long, it takes several years until trees or bushes flower. Thus, traditional breeding approaches, applied to trees and bushes, require a long time until they show success. Sometimes it takes decades to find out if a certain tree shows the desired characteristics or not (Tzfira et al. 1998). Such time-consuming breeding attempts are expensive and therefore mainly non-domesticated trees are used in forestry.
During their lifetimes woody plants, especially trees, have to cope repeatedly with different seasonal environmental stresses, for example climatic extremes or fluctuating pressures from a range of pest organism. This is another difference between forestry and agriculture.
Most tree species maintain very high levels of genetic diversity within populations, presenting a great potential of adaptation to changing environmental conditions (Mullin & Bertrand 1998). Among many tree species covering a large distribution area, like Scots Pine, Pinus sylvestris L., several well-adapted ecotypes and local races have evolved. Strict regulations about seed harvest and seed transfer guarantee the preservation of well-adapted genetic material used for planting stock, so that the genetic structure of planted stands resembles a wild population more closely than an agricultural crop.
Genetic engineering techniques offer a large field of new possibilities in the breeding of woody plants. Desired characteristics can be simply transformed to the target object, time consuming breeding experiments become unnecessary.
Methods of genetic engineering
Genetic transformation – the controlled introduction and expression of foreign genes – in trees and bushes is often carried out by using the soil bacteria Agrobacterium tumefaciens, a common and well-established method in genetic engineering. After the transformation process the successfully transformed cells will be cultivated until finally the transgenic tree has been regenerated (Tzfira et al. 1998). Naked-seed plant species (gymnosperms), for example the economically important conifers spruce and pine, are less amenable to Agrobacterium infection. In such species the transformation is carried out by particle acceleration (Kempken & Kempken, 2000). DNA-coated gold or wolfram particles are shot on cells. The particles are so small that they penetrate into the cells without damaging them. In general, embryogenic tissues or pollen grains are used as receptor cells. The transformed genes, mostly genes of herbaceous plants (very frequently genes of Arabidopsis, a common agricultural weed and the model species of plant genetic engineering research) are used in transformation experiments.
Targets for forest-tree engineering
The two most important targets in forest-tree engineering research are the increase in biomass production and changes in the wood structure which regard to the different purposes. Concerning wood structure research is focused on modifying lignin content and composition. Lignin has to be removed in an energy-consuming process that involves the use of polluting chemicals in pulp and paper production. Lower lignin content in favor of higher cellulose content would lower the costs of paper production.
Other targets are insect resistance, herbicide tolerance and tolerance to abiotic stresses like drought, frost, flooding, high salt content, etc.
Increasing biomass production
Obviously increasing growth rates and stem volumes as well as shortening rotation rates of forests are economically interesting targets. Growth and development phases in plants are regulated by hormones, especially the group of gibberellins play a key role in these processes. Key regulator genes in the biosynthesis of theses growth hormones, including the gene coding for gibberelin-20-oxidase, have been identified in Arabidopsis. Eriksson et al. (2000) transformed the Arabidopsis-gene coding for gibberelin-20-oxidase into poplar hybrids. The transgenic poplars were distinguished from non-transgenic poplars by increased growth rates. Additionally, the transgenic trees had more and longer xylem fibers. Long fibers are desired in the production of strong paper. Another proposal probably resulting in higher wood production made by Strauss et al. (1995) is to cause sterility by transgenic means. Transgenic sterility would save the energy normally used for flowering and may therefore now be used for higher growth rates.
Modifying lignin content and composition
Lignin occurs in high quantities in the secondary cell walls of fibers and in the water transport system in plants, the vessels and tracheids providing them with mechanical support and helping the plant´s defense system against disease-causing organisms (pathogens). During the process of paper production lignin has to be removed by heating wood fibers in sulphite leaches to get pure cellulose. The removal of lignin is the most expensive, most energy intensive and most environmentally damaging step in wood processing for pulp and paper production. Therefore, the paper industry has a great interest in reducing the lignin content in trees.
Different research teams have tried to reduce the lignin content in trees without any success. These attempts only resulted in striking changes in lignin composition, but with no effect on lignin content (Sederoff 1999). Finally Hu et al. (1999) transformed poplars with Agrobacterium tumefaciens carrying an antisense gene construct downregulating the synthesis of lignin without modifying lignin composition. These transgenic poplars exhibited up to 45% reduction of lignin and up to 15% increase of cellulose.
Herbicide tolerance
Herbicide-resistant crops are considered to be one of genetic engineering`s major successes. The herbicide application in forests is only useful for favoring the growth of tree seedlings by eliminating herbal plants competition. This is the reason why genes providing resistance to different herbicides like glyphosate or Basta have been transformed to trees (Tzfira et al. 1998).
Insect resistance
Insects are responsible for substantial losses in forest-tree species. Additionally, the common practice of planting dense forest plantation favors the rapid increase of insect population sizes. Up to now several insect-resistant trees have been produced carrying a gene of Bacillus thuringiensis which encodes for an insect toxin (Ewald & Han 1999, Wang et al. 1996, Tzfira et al. 1998).
Abiotic-stress tolerance
In general, most natural tree species are well adapted to their environment, exhibiting high ecological competence. However, certain abiotic-stress tolerance characteristics can become important for the reafforestation of difficult locations, like air-polluted locations, or for planting non-adapted exotic tree species. In this context Strohm et al. (1999) investigated if transgenic poplars overexpressing certain enzymes are less sensitive to acute ozone stress than the wild type. The transgenic poplars exhibit higher activities of the two enzymes glutathione synthetase and glutathione reductase which probably play a major role in the protection against extremely reactive oxygen molecules like ozone. However transgenic and non-transgenic trees have been equally damaged by ozone. There was no difference in the extent of leaf injury between transformed and non-transformed poplars.
Phytoremediation
Another possible target for transgenic trees is phytoremediation, the plant-mediated removal of toxic wastes from contaminated soil. Different pollutants, mainly heavy-metal ions or organic compounds, have been found to be degraded or accumulated by several bacterial and plant species. For example, transgenic Arabidopsis plants syntheticizing a certain gene product (coded by the merApe9 gene) demonstrate a higher resistance to mercury Hg2+ ions during germination and growth Rugh et al. (1996, cited from Tzfira et al. 1998).
Research is also done in the phytoremediation of organic pollutants. One case study examines a hybrid poplar that has been genetically engineered using a human cytochrome gene, P450 2E1, (described in Doyle et al., 2000) to detoxify a widespread industrial toxic chemical, trichloroethylene (TCE). As promoter the common 35S promoter from the cauliflower mosaic virus was used. The gene product modifies a range of compounds including trichloroethylene, ethylene dibromid, benzene, styrene, chloroform and others into less toxic or nontoxic substances by means of an oxidative reaction. The reaction products are finally translocated to the stems and leaves (http://www.ostp.gov/html/ceq_ostp_study6.pdf).
Targets for fruit and wine production
The most important target of genetic engineering in fruit and wine production is pest resistance. There is also great interest in shortening the juvenile phase of commercially used woody plants to reduce the waiting period for the first harvest. In future also abiotic-stresses tolerance could become interesting for fruit and wine producers (Mantinger, 1998).
Shortening of the juvenile phase
The genetic control of flower development has been intensively studied in Arabidopsis. The identified genes LEAFY (LFY) and APETALA1 (AP1) play an important role in the initiation of the flowering phase (Egea-Cortines & Weiss 2001). Peña et al. (2001) demonstrated that the permanent expression of these two genes under the control of the commonly used cauliflower mosaic virus 35S promoter significantly shortens the juvenile phase of citrus trees. Plants harboring one of the two genes flower and produce fruit within one year after germination. This accelerated juvenile period is heritable in crosses with non-transformed plants.
Pest resistance
One method used in plants inducing virus resistance is the so called coat protein-mediated resistance” (Hackland et al., 1994). This method can be compared to a vaccination in which plants are infected with cloned coat protein genes of mild viruses inducing resistance against severe viruses (Malinowski et al. 1998; Tennant et al. 1994). However several investigations demonstrate that total immunity of transgenic plants is rare (Sanders et al. 1992; Powell-Abel et al. 1986, Turner et al. 1987). Furthermore, the transfer of virus genes takes the risk of developing new virus particles combining parts of transfered virus gene sequences and parts of infecting virus gene sequences. In the worst case more aggressive and better adapted viruses can evolve. Such new viruses might also infect plants which have not been threatened by the old viruses.
Other research is carried out on resistance to disease-causing fungi and bacteria, like mildews (different fungi).
Identified resistance barley genes are used in these experiments.
Risk aspects and open questions
For the evaluation of the risks of the environmental release of transgenic trees and bushes, several aspects have to be considered which are characteristic for woody plants which distinguish them from most agricultural crops. In general, woody plants are long-living organisms which persist for long-term periods in the landscape; very often pollen and seeds of woody plants are dispersed over long distances; many trees and bushes cross with closely related species; finally, forest ecosystems are very complex – a multitude of interactions between the different organisms exists.
In comparison to other crops trees get very old. For example silver firs (Abies alba) can get more then 500 years old; poplar, the model object in genetic engineering of trees, can reach ages between 150 and 300 years (Oberdorfer, 1990). Bushes can reach high ages, as well, especially vegetatively propagating bushes like sloe (Prunus spinosa) or sea buckthorn (Hippophaë rhamnoides). Therefore, the possibility of dispersing new gene constructs by vertical or horizontal gene transfer exists over a long period of time after the release.
Vertical gene transfer
Many trees are pollinated by wind, in many cases also the seeds of trees are dispersed by wind. The model object of forest tree biotechnology, the poplar, is one of the species with wind-dispersed pollen and seed. In general, most of the pollen rain goes down close to the source of release (Ledig, 1998). But there are exceptions, for example in the case of pine trees considerable amounts of pollen can be found even in a distance of 300 m to their source. Depending on the weather pollen can get to very high layers of air, under such conditions pollen dispersal up to several hundred kilometers is easily possible (Di-Giovanni et al. 1996; Di-Giovanni & Kevan 1991). Thus, transfered gene constructs can be dispersed over long distances.
In addition to the long-distance dispersal, it is very likely that transfered gene constructs are also dispersed between closely related species and not only within a single species. Isolation barriers between different species are very often only partially effective. Many trees and bushes are able to cross with other species. This is also the case for a lot of economically interesting trees like poplar, pine and eucalyptus (Mullin & Bertrand, 1998).
Several woody plant species also use very effective mechanisms to propagate vegetatively. Especially poplars, as well as some willow species, are able to propagate by snapping twigs which drift down rivers and streams (James et al. 1998, Beismann et al. 1998).
Genetically engineered sterility seems to be an attractive method to avoid vertical gene transfer. But even if sterility can be caused by genetic engineering, it can not be guaranteed that this change is stable. For trees, instabilities of transformed gene constructs have been discovered only after a short time (Kumar. & Fladung 2001; Fladung & Muhs 1999; Fladung 1999).
Another possibility to avoid vertical gene transfer consists in transforming solely the chloroplast-DNA (the chloroplast-DNA is only found in chlorophyll producing cell organelles and not in the nucleus of the cell). In general, the chloroplast-DNA is only inherited by the mother individual, so it can not be dispersed by pollen. However, in naked-seed plant species (gymnosperms) including our economically important conifers, chloroplast-DNA is inherited by the father trees (Cato & Richardson, 1996), so the strategy of transforming only chloroplast-DNA avoiding vertical gene transfer is not applicable to conifers.
Depending on the desired target of the plantation there is also the possibility to use the transgenic trees only during their juvenile stage before they flower. However transgenic changes can also have undesired additional effects, so-called pleiotropic and position effects (see Genetic Engineering Newsletter Special No. 6), including shortening of the juvenile stage. This phenomenon could be observed for example in poplars (Fladung et al., 1997).
Horizontal gene transfer
Almost all woody species live in an intensive symbiosis (interaction) with fungi living in the soil. The two partners (the woody species and the fungi) form a so-called mycorrhiza, which is very important for the supply of nutrients and for the health of both partners. The fungi are seldom adapted to only one woody species. There are a lot of common fungi species which are able to live with very different woody species. They can even live in very different ecological locations. If horizontal gene transfer takes place between the transgenic woody species and the fungi, there is also the risk of transfering the gene constructs to other woody species. Horizontal gene transfer could be observed in greenhouse experiments between transgenic canola and Aspergillus niger, a common mould (Hofmann et al., 1994).
During the symbiosis the fungi takes organic nutrients from his host tree or host bush. Thus genetic changes in the host plants can also have an effect on the soil ecosystem. Unintentional changes in the synthesis of organic plant products, which are transported by the mycorrhiza into the soil, can change the composition of soil species or the extent of the mycorrhiza. Since organic plant products can also be directly released into the soil by root exudates, the whole soil flora and fauna can be effected. It is also possible that during the decomposition process a horizontal gene transfer takes place between decaying plant material and decomposing microorganism. Because of the long lifespan of trees the probability of the transfer of stable transgenes is higher in forestry than it is in agriculture.
Modifying the lignin composition
Lignin serves different functions; it stabilizes the plant and protects it against disease-causing organisms (Fink 1999). Modifying the lignin content or composition can have dramatic effects on these two important functions of lignin. These effects have to be investigated in long term research projects. It has to be considered that trees with a lower lignin content are more likely to be blown down by storms and have a lower resistance to disease-causing organisms.
Insect resistance
Transgenes coding for toxic proteins of Bacillus thuringiensis have already been inserted into trees to provide insect resistance. This genes have to be expressed the whole lifespan of the transformed tree to be of real utility. A severe problem is the risk of parallel development (coevolution) of toxin-resistant insects. Because of the long lifespan of trees and the short period for the developmen of insect generations the probability for an adaptation is quite high.
Chinese field trials on transgenic insect-resistant poplars revealed that the genetic transformation has been accompanied by several other unforeseen changes (Ewald & Han 1999; Wang et al. 1996). In some trees the synthesis of chlorophyll, an important molecule in the photosynthesis pathway, was disturbed. Additionally, successfully transformed trees have been damaged by previously harmless insects after two years.
Abiotic-stress tolerance
The integration of new abiotic-stress tolerance characters into woody plants, like drought or salt tolerance, will definitely have effects on the whole ecosystem, since these new characters will dramatically change the competitive situation. Transgenic competitive plants might even replace other rare plants. Experiments on changes in the competitive situation already carried out in the fifties by Ellenberg (1952, 1953), demonstrated how complex interactions between single species can be. Concerning such questions there is a great lack of knowledge and a high need of research.
General risks
In principle all transgenic changes might be accompanied by unintended effects, so called pleiotropic and position effects. Since the biotic communities (biocenosis) in which woody plants occur are very complex ecosystems, transgenic changes as well as their unintended effects might have many consequences. Woody plants are integral parts in quite complicated food webs. Many insects living and feeding on or in trees (for example bark beetles or aphids) are the feed for other animals. The interactions under the soil surface are quite complicated and unknown, too. The aspect of the long lifespan of woody plants is very important considering the risks of transgenic changes. Transgenic changes will have an impact over long time periods. Finally, trees show a great morphological variability depending on different environmental conditions. Therefore, the observed effects of transgenic changes can only be compared within similar locations.
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