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The Case for Genetic Engineering of Native
and Landscape Trees against Introduced
Pest and Diseases

JONATHAN M. ADAMS,* GIANLUCA PIOVESAN,† STEVE STRAUSS,‡AND SANDRA BROWN,§ *Department of Natural Resources Science, University of Rhode Island, RI 02879, U.S.A. email [email protected]†Department of Forest Science, University of Tuscia, 01100 Viterbo, Italy, email [email protected]‡Department of Forest Science, Oregon State University, Corvallis, OR 97331–5752, U.S.A., email [email protected]§Winrock International, c/o 831 NW Sundance Circle, Corvallis, OR 97330, U.S.A., email [email protected] Abstract: Many important native forest trees and familiar landscape trees of the northern temperate zone
have been devastated by introduced pests and diseases. Without human intervention, many of these trees will
become extinct or endangered. As trade and travel increase, it is likely that further devastating epidemics will
occur. To undo the damage that has been done, we suggest limited, cautious transfer of resistance genes from
the original host species in the source region of the pest or disease. The transgenic trees can then be replanted
in forests or countryside to resume their original ecological niche. This method could have some advantages
over tree-breeding techniques, including introgression. For instance, fewer tree generations would be re-
quired and fewer unnecessary genes of the non-native tree species would be introduced. Furthermore, once
the technique is perfected it would be possible to separately add resistance genes to local land races of trees,
for reintroduction to their original habitats, without relying on intensive and lengthy local introgression pro-
grams. Practical problems with identifying and transferring resistance genes do exist, however, and soma-
clonal errors might lead to genetically engineered trees that do not resemble their parent in growth form. Nev-
ertheless, we believe that, with further work, this approach may offer a preferable alternative to introgression
with non-native trees.

El Caso de la Ingeniería Genética de Arboles Nativos y de Paisaje Contra Plagas y Enfermedades Introducidas Resumen:
Muchos árboles forestales nativos y árboles de paisajes familiares a la zona templada del norte han sido devastados por plagas y enfermedades introducidas. Sin la intervención humana, muchos de estos árboles seextinguirían o estarían amenazados. A medida que el comercio y los viajes se incrementan, es muy probable que ocur-ran epidemias devastadoras en el futuro. Para revertir el daño causado, sugerimos la transferencia limitada de genesresistentes de las especies hospederas originales en la región de inicio de una peste o enfermedad. Los árboles transgéni-cos pueden ser plantados en bosques o en el campo para que reactiven sus nichos ecológicos originales. Este métodopuede tener algunas ventajas sobre las técnicas de reproducción de árboles, incluyendo la introgresión. Por ejemplo, serequeriría la introducción de menos generaciones de árboles y menos genes innecesarios de las especies de árboles nonativos. Más aún, una vez que la técnica se ha perfeccionado, sería posible agregar de manera separada genes de resis-tencia a razas locales de árboles, para la reintroducción en sus hábitats originales, sin depender de programas de intro-gresión local intensiva y prolongada. Sin embargo, existen problemas prácticos de identificación y transferencia degenes de resistencia y algunos errores somato-clonales podrían conducir a la producción de árboles genéticamente mod-ificados que no se parezcan a sus padres en cuanto a la forma del crecimiento. A pesar de todo, creemos que con traba-jos a futuro, esta técnica puede ofrecer una alternativa preferencial a la introgresión con árboles no-nativos. Conservation Biology, Pages 1–7Volume 16, No. 4, August 2002 Genetic Engineering of Trees Against Disease Introduction
too late to be effective. However, the use of a hypoviru-lent strain of the Chestnut blight fungus (Chryphonec- In both Europe and North America, common native tria parasitica) has apparently diluted the virulent trees of forests, cities, and the countryside have been strain and thus reduced disease symptoms in some areas devastated by introduced pests and diseases. These in- in Europe (Cortesi et al. 1996; Heiniger et al. 1997). Un- clude the elms (Ulmus) of both Europe and North Amer- fortunately, the American strain of the fungus has so far ica and the North American chestnut (Castanea den- not been susceptible to this treatment. Attempts to tata). In the case of the chestnut, eastern U.S. forests breed resistance into chestnuts from related species have lost one of their dominant trees (Bailey 1995).
(e.g., the east Asian species Castanea mollissima) via It is likely that these types of catastrophic pandemics hybridization may ultimately prove effective, but this will continue. In North America a repeatedly introduced work has already taken decades and—depending on Cerambycid beetle from Asia ( Anoplophora glabripen- how extensive backcrossing and selection is (American nis) threatens to wipe out buckeyes (Aesculus) and the Chestnut Foundation 2000)—may result in a species maples ( Acer) that dominate the eastern deciduous and with other significantly altered physiological and mor- mixed forests (Cavey et al. 1998). In California, ever- green oaks ( Quercus) and some related species are be- In the case of the European elms, some isolated indi- ing killed by a new species of the Oomycete fungus viduals resistant to Ophiostoma ulmi have been located.
(Phytophthora) thought to have been imported on Because of the trees’ long lifespan, however, breeding rhododendrons (Rizzo & Bailey 2000). Although not a reliably resistant strains from so few and isolated individ- native species, Cupressus sempervirens has been widely uals will take many decades and will itself produce a ge- planted in the western Mediterranean region and is be- netic bottleneck that may expose the resistant trees to ing devastated by the introduced fungus Seridium car- other epidemics. Many of the clones of elms that for- dinale, which probably originated in California (Anselmi merly provided a characteristic and attractive appear- & Govi 1996 ). This fungus has also damaged native ance to European landscapes show no resistance, so stands of Cupressus macrocarpa in the United States. A there is almost no prospect of their return as full-sized similar case in Europe is the sycamore or plane tree (Pla- trees without slow artificial hybridization with other elm tanus orientalis and Platanus hybrida) and Ceratocys- strains. Unfortunately, as with chestnut, this may also al- tis fimbriata f. platani, which causes a fungal disease.
ter their growth habit and adaptive qualities. We should The fungus arrived in Mediterranean areas from the also consider the possibility that the disease itself may United States, probably during World War II (Anselmi & return as a slightly different strain, as apparently hap- Govi 1996). The disease kills both cultivated and wild pened with elms over the course of nearly 60 years in trees, whereas the North American sycamore (Platanus Europe. When Ophiostoma novo-ulmi appeared, it occidentalis) appears to be naturally resistant. With the killed some elm species and cultivars that were resistant additional climatic stress of global warming, tree popula- to O. ulmi ( Brasier & Webber 1987; Namkoong 1991; tions may become even more susceptible to outbreaks Smalley & Guries 1993; Brasier 2001). In Italy, extensive of introduced pests and diseases (e.g., McCarty 2001).
tree-selection work on Cupressus has produced five In each of these cases, the origin of the pest outbreak clones resistant to Seridium cardinalem (Santini et al.
appears to be the introduction of geographically alien 1997), but this involved a considerable loss of genotypic microorganisms. It appears that the resistance of trees in the source regions evolved over millions of years of ex- In the United States, the wild chestnut population has posure to these agents, whereas in the region into now been devastated by chestnut blight, and there is lit- which the pest has been introduced there has been no tle prospect of populations returning of their own ac- selection for resistance so the trees are killed en masse.
cord even on a time scale of thousands of years. Strong In some cases, particularly for the insect pests, natural genetic resistance does not appear to exist in the wild enemies were left behind as they changed locales, populations of Castanea dentate, and the situation ap- which may also be a contributing factor. Unfortunately, pears to be similar to that of American elms (Brasier it is likely that the accelerating movement of plants, 2001). The prospects for finding resistant individuals goods, and people around the world will increase the among populations of North American maples or Califor- frequency of tree-disease pandemics, with increasing ef- nian oaks are unknown, but we should consider the pos- fect on native forests, cultivated landscapes, and the ani- sibility that resistance is rare or nonexistent.
mals and other organisms that depend on trees for food Given the widespread mortality that is occurring and and habitat (Kennedy 2001; Wingfield et al. 2001).
will probably continue to occur among temperate and In the regions affected by these widespread forest dis- tropical tree populations, how should conservationists eases, the response has generally been insufficient to respond? One option is to let nature take its course, al- prevent damage or restore species. In most cases, selec- though in fact this is not “nature” but instead a problem tive felling of infected trees has proven too patchy or caused by human meddling. Another option is to hope Conservation BiologyVolume 16, No. 4, August 2002 Genetic Engineering of Trees Against Disease that either traditional plant-breeding methods, mycol- kinds of diseases—and in diverse kinds of plant species ogy, or entomology can provide naturally resistant ( Young 2000)—scientists have an excellent idea of what strains of trees or biological control options for virulent such genes are likely to look like. In fact, as a conse- pathogens or insects. Breeding resistant trees from quence of highly conserved amino acid motifs in many source populations, although preferable if there are no forms of these genes, it is now possible to isolate them associated problems, is not a possibility when resistance in large quantities in a single day using PCR (polymerase alleles are rare or absent. Biological control is likely to chain reaction) with degenerate primers. But it is also involve the introduction of new species of fungi and in- known that these PCR–derived “resistance gene ana- sects with unknown capacities for interacting with flora, logs” (RGAs) are so prevalent in the genome (Michle- despite the study they undergo prior to introduction.
more & Meyers 2001) that it is necessary to narrow Moreover, these new species often fail to become estab- down the search to a small region via traditional genetic lished or provide effective levels of control ( Turnbill & mapping or direct biochemical methods first to avoid being overwhelmed with possibilities. The RGAs appearto comprise as much as 2% of all genes in the Arabidop-sis genome (Michlemore & Meyers 2001).
The Opportunity Offered by Genetic Engineering
Because most genes for resistance to obligate patho- gens appear to have a role in sensing or signaling patho- It may be necessary to consider what some environmen- gen presence to mobilize plant-defense metabolism, it talists would regard as unthinkable: genetic engineering should be possible to isolate genes based on their pro- to add resistance genes to trees for reintroduction to na- tein products’ biochemical interaction with pathogens.
tive forests. This technology is new, particularly for wild This theoretical possibility has not yet been demon- trees, and the science on which it is based—tree and strated, however, so it may be necessary to rely on some plant genomics—is limited. Although genetic engineer- kind of genetic mapping approach, usually involving in- ing is credited with the dramatic rescue of one tree spe- terspecies hybrids between resistant and susceptible cies from disease—Hawaiian papaya (Carica papaya) species. This has the advantage of advancing not only from papaya ringspot potyvirus (PRSV ) (Lius et al.
genetic engineering, but a conventional backcross ap- 1997)—the majority of diseases threatening forest trees proach to resistance breeding as well, as is being pur- are not viral in origin. In addition, a general set of strate- sued in the chestnut; thus, costs for the two alternative gies that appears to be effective for engineering viral re- sistance, the expression of virally derived transgenes in Under backcross breeding, the hybrid and mapping plants, is not effective for fungal pathogens. No generic populations are the actual vehicle for moving resistance technology for overcoming fungal pathogenicity has genes; under genetic engineering, different kinds of ge- been described. Thus, is it reasonable to consider that netic crosses could also be used whose only purpose is genetic engineering and genomics in trees could pro- to identify resistance genes. This might be the case, for vide significant new means for producing resistant trees example, when resistance exists in a species that is not in the face of devastating diseases? To produce resistant fully interfertile with the threatened species or when a strains, what will be needed, how long will it take, and chromosomal difference prevents normal segregation in what will the costs and potential tradeoffs be? hybrid offspring with the threatened species, complicat- Although there have been many reports of increased ing mapping and effective backcross-mediated introgres- fungal disease resistance due to single transgenes in the sion. The genes could still be mapped in intra- or inter- laboratory, so far there has been only one case of signifi- s p e c i f i c c r o s s e s i n v o l v i n g o t h e r s p e c i e s w h e r e cant field resistance (Gao et al. 2000), and this was for a resistance to the pathogen segregates.
specific pathogen and crop species. We therefore be- Two key requirements of the genetic engineering ap- lieve it is wise to assume that restoring forest species via proach are that a large number of genetic markers must genetic engineering will not result from generically ef- be available for creating a dense genome map and that a fective “supergenes,” but instead it is likely to require sufficiently large group of segregating progeny must be the use of resistance genes isolated from their own ge- created so that the location of the gene(s) can be deter- nomes or from a related tree species in the same genus mined precisely. High-density maps of resistance genes or family. Such genes, particularly when compared with have been created in poplars (Populus) through the use alternatives like general fungal toxins, would also have of amplified fragment-length polymorphisms ( AFLPs; the advantage of raising far fewer ecological concerns Cervera et al. 1996 ). It is likely to continue to be the about their nontarget effects. The problem is how to method of choice for a number of years because it re- identify, physically isolate, and transfer such genes in an quires no prior knowledge of the genome. A mapping efficient and physiologically safe manner.
approach also assumes that only one or a few major re- Because of recent advances in identifying common sistance genes segregate. If resistance turns out to be a DNA sequence motifs in genes for resistance to diverse polygenic trait ( many loci encode partial resistance), it Conservation BiologyVolume 16, No. 4, August 2002 Genetic Engineering of Trees Against Disease is unlikely that mapping can be precise enough to physi- velopmentally plastic poplars. Researchers and indus- cally isolate genes or that a sufficient benefit could be tries circumvent somaclonal variation as an important obtained by transfer of one or a few of them. Finally, it factor simply by producing large numbers of indepen- also requires that resistance genes be at least partially dently transformed transgenic lines and screening them dominant. Transformation typically inserts single, novel carefully. Only a portion are considered for in-depth gene copies (although it may do this at multiple loca- studies or commercial development. To produce a well- tions in the genome) so the gene must confer resistance adapted wild tree, the production of a large population when hemizygous (no alternative allele present at the lo- of transgenic progeny is also important. After the pri- cus) or heterozygous. Fortunately, a majority of genes mary transgenic trees flower in the field, natural selec- for resistance to obligate plant pathogens show domi- tion plays the largest role in sorting out the transfor- mants that are most fit. To help produce the levels of Once a small region of the genome, generally one centi- genetic variability found in natural tree populations, a Morgan (1% recombination) or less, is identified as con- single backcrossing between transformants and a range taining a resistance gene, it is feasible to consider isolat- of surviving wild-type individuals from within each local ing and transferring it. In a species with a small genome or regional population would also be appropriate. Natu- and in which gene transfer is efficient (e.g., poplars), ral selection in each population reintroduced to the wild large pieces of the genome that overlap the mapped lo- would then ensure that resistance genes are favored cus, typically on the order of 100 kb, can be tested di- while substantial genetic variability at other loci is main- rectly. This requires cloning of the genome in a bacterial artificial chromosome ( BAC) library capable of contain-ing such large fragments and suitable for Agrobacteriumgene transfer ( Hamilton 1997 ). For other species, how- Research Challenges for Effective Genetic
ever, additional genetic information is desirable to subdi- Engineering of Resistance in Trees.
vide this large region further so that the task of genetransfer is applied to a small, well-characterized area. To Unless there is a breakthrough in direct isolation of resis- subdivide this area, the BAC clones of interest are first tance genes based on their molecular interaction with subcloned and studied via mapping or sequencing. Re- pathogens, the map-based cloning method described gions that appear promising can then be used to isolate above would be extremely difficult to apply to most the active forms of these genes from cDNA libraries of trees species today. The key obstacles are the absence of expressed genes, or the RGA-containing regions can be genomics tools and efficient methods of gene transfer.
subcloned and transferred directly. This resistance The increasing automation of genome mapping and se- gene–isolation method would be particularly useful in quencing, however, may make it possible to create the species such as conifers that have very large, repetitive required genomic tools, at least for a number of key- genomes. In many cases, however, resistance genes are stone angiosperm genera in the temperate zone. Physi- present in tandem repetitive structures, any member of cal maps and associated sequence-tagged markers made which could encode the desired resistance. It is there- in one species would be likely to work throughout many fore desirable to transfer linked groups of genes. Thus, a genera. These maps would facilitate the pinpointing of gene-transfer method that is highly efficient at transfer- sections of the genome likely to contain resistance ing large fragments may be essential. This strategy also genes. In addition, because resistance genes are often has the advantage of transferring not one but a family of clustered on particular chromosomes, the maps would related resistance genes, providing some buffer against serve as a guidepost, helping to identify the regions pathogen evolutionary change. If more than one locus in the genome contains resistance genes, it is highly desir- Given the rate of advancement in genome technology, able to transfer both, which can be done successively or and the importance of keystone tree species to forest ec- at the same time via cotransformation.
osystems and human economies, it is now in the realm The ultimate test of success is the production of resis- of possibility that complete genome sequences, and/or tant, phenotypically normal transgenic plants. In pop- or large expressed sequence databases, might be created lars, which have had more transgenic trees produced for our most important tree genera or families within and studied in the field than any other tree species ( Tz- one to two decades. This would allow RGAs and tightly fira et al. 1998), somaclonal variation (unintended phe- linked polymorphic genetic markers such as SNPs (sin- notypic or genetic abnormality) appears to be small gle nucleotide polymorphisms) or SSRs (simple se- (Strauss et al. 2001). However, these studies have quence repeats) to be directly studied and isolated. It spanned only a few years. It is unclear if somaclonal vari- would also allow many resistance genes whose se- ation is significant over longer time periods or in other quences are today unknown to be inferred directly by a tree species. It is possible that other species will show combination of map position and sequence comparison less tolerance of genomic perturbation than do the de- Conservation BiologyVolume 16, No. 4, August 2002 Genetic Engineering of Trees Against Disease Although a large number of tree genera have been In addition to the possibility of significant somaclonal transformed, the frequencies of gene transfer and recov- variation, the main disadvantage of genetic engineering ery of transgenic plants are too low in most genera to is the increased information and technical capability re- support the transfer of large DNA fragments and the pro- quired, which presently limits the number of species, duction of large transgenic populations that will be genotypes, and genes that it can be applied to.
needed ( Brunner et al. 1998; Tzfira et al. 1998). The re- The extent to which the use of genetic engineering covery of transgenic plants is today a highly empirical, becomes successful will be a direct result of the re- species- or genotype-specific enterprise and generally re- search effort applied. Given the degree of opposition quires the use of accessory genes that are undesirable in that genetically engineered crops have met with from the wild (e.g., antibiotic or herbicide resistance genes).
many elements of the environmental movement, an un- But there are signs that this may change radically over fortunate consequence might be the cessation or curtail- the next decade if research continues. New methods are ment of funding for tree biotechnology research that being developed to insert genes that directly promote could save many valued native species from either ex- regeneration of transgenic cells themselves, rather than tinction or near extinction. With the rapid pace of ad- relying on toxin resistance, and a variety of systems have vances in genome research, and the near certainty of been developed that can excise permanently any acces- continuing pandemics of tree diseases, we believe that sory genes employed (e.g., Ebinuma et al. 1997). Thus, developing a background of genomics tools, including the ability to transform trees may become a more effec- the capability for a genetic engineering approach for use tive and generalized technology in the future. This will when necessary, is an appropriate precautionary strategy.
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Adams et al. (00-523)
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