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IMPROVEMENT OF POPULUS THROUGH GENETIC ENGINEERING A.S. RISHI, NEIL D. NELSON AND ARUN GOYAL1* Biotechnology Initiative, Forestry and Forest Products Division, Center for Applied Research and Technology Development (CARTD), Natural Resources Research Institute, and 1Department of Biology, College of Science and Engineering, Department of Biochemistry and Molecular Biology, School of Medicine, University of Minnesota-Duluth, Duluth, MN 55812, USA Received on 20 Oct., 2000, Accepted on 10 April, 2001

SUMMARY Genetic engineering has provided tools to improve Populus through introducing genes from heterologous sources and also through down-regulating metabolic pathways. This has resulted in the improvement of several useful traits within a short period of time. Considerable success has been achieved in improving traits such as insect resistance, herbicide tolerance, accelerated flowering, introducing male sterility, phytoremediation of toxic compounds, and modifying wood quality by manipulating the lignin biosynthesis pathway. Producing high-value recombinant proteins and biomolecules in transgenic poplar is an emerging field of high priority. Key Words: Genetic engineering, Populus, transformation.

INTRODUCTION Populus is a genus that is widely distributed over the Northern Hemisphere, grown for its economic use for wood, fiber, and energy (Zsuffa et al., 1984). Based on morphology, geographical localization, and hybridization pattern, the genus Populus is divided into five sections: Leuce, Aigeiros, Tacamahaca, Leucoides, and Turanga. Populus wood is used by the paper/pulp and wood industries for manufacturing paper and other commercial products. Populus could also play a significant future role in environmental protection by remediating contaminated soil and ground water. This mini-review deals with the application of genetic engineering to improve Populus species and further exploring its commercial potential. The sections Aigeiros, Leucoides, and Tacamahaca are found to be compatible for hybridization purposes. The artificial breeding program of Populus began in 1912 at the Kew Botanical Garden and since then has been carried out to improve important desirable traits for

growth, yield, abiotic and biotic stress tolerance, and wood and fiber quality (Henry, 1914, Hall et al., 1989). Populus improvement through conventional breeding is limited by long generation times, difficulty in large-scale screening, and lack of novel genes within the germplasm. These constraints could be overcome by using genetic engineering techniques. Since the first report of transgenic plant production in early 1980’s, several plant species have been transformed with useful genes from diverse sources, thus widening the gene pool of the germplasm. The gene transfer techniques have helped in reducing the time required for improvement and have become a good alternative or addition to conventional breeding programs. Several important traits including insect resistance, herbicide and stress tolerance, altered physiology, reduced lignin content, and phytoremediation have been introduced from diverse sources through genetic engineering. Thus, gene transfer technology has considerable potential for accelerating tree improvement and offers the possibility of directly altering specific parts of the genome, either by manipulating the expression pattern of endogenous genes or by introducing useful genes from other organisms.

*Corresponding author : Arun Goyal Indian J. Plant Physiol., Vol. 6, No. 2, (N.S.) pp. 119-126 (April-June, 2001)

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Transgenic plants are obtained by introducing the genes to plant cell(s) and regenering plants from the transformed tissue. Hence, regeneration of plants from living cell(s) forms the basis of production of transgenic plants. Earliest’in vitro regeneration of plantlets from Populus was reported in P. tremuloides (Winton 1968, Wolter 1968). The regeneration of Populus through somatic embryogenesis and organogenesis has been reported (Park and Son 1997, Kang and Chun 1997), and Populus has become a model system for forest biotechnological research (Leple et al. 1999) and in some respects for plant biotechnology.

neomycin phosphotransferase (npt-II), hygromycin phosphotransferase (HPT), phosphinotricin acetyl transferase (bar) and acetolactate synthetase (crs1-1), that confer resistance to antibiotics/chemicals (kanamycin, hygromycin, phosphinotricin, and chlorsulfuron respectively) have been successfully used along with the gene of interest to select the transformed tissue in Populus (Fillatti et al. 1987, De Block et al. 1990, Brasileiro et al. 1992, Howe et al. 1994, Confalonieri et al. 1994). The detailed protocols for transformation of Populus tremula have been reported (Tzifra et al., 1997; Han et al., 2000).

Transformation of plants can be achieved by either direct gene transfer or by using Agrobacterium-mediated gene transfer. The latter method is preferred for many plants including Populus, for its advantages in providing single copy, whole gene integration, and better expression of the transgenes. The application of the direct gene transfer method in Populus has been reported in a few cases, both through electroporation and electric discharge particle acceleration (Chupeau et al. 1994, McCown et al. 1991). However, the most common method for introducing genes of interest is thorough Agrobacterium-mediated gene transfer. The Agrobacterium-mediated gene transfer has been used to transform a number of hosts including dicots (Baron and Zambryski 1996), monocots (Hiei et al. 1997), and even fungi (Gouka et al. 1999). Populus has been known as a natural host for Agrobacterium (DeCleene and DeLey 1976); however, the first transgenic poplar was reported in P. alba x P. grandidentata (Fillatti et al. 1987). The transformation procedure using Agrobacterium has been critically evaluated for obtaining higher transformation frequency among the Populus species. Different genotypes, explant types, and Agrobacterium strains were found to influence the transformation frequency (Kubisiak et al. 1993, Confalonieri et al. 1995, Han et al. 1996). Further use of acetosyringone to induce vir genes in the transformation protocol has provided higher transformation frequency (Confalonieri et al. 1995). Recently, use of matrix attachment regions (MARs) along with the desired gene has resulted in higher transformation frequency and better transgene expression in poplar (Han et al. 1997). Selecting the transformants is a critical feature in obtaining transgenic plants, and different antibiotics/chemicals have been used to select the transformed cell(s). Several marker genes, such as

Considerable success in transformation of Populus has led to the focus of improvement of traits through genetic engineering. Identification of useful genes from different sources, and introducing them to the Populus genome, has resulted in significant improvement of different traits. A few examples of improvement of traits through genetic engineering include insect resistance, herbicide and stress tolerance, altered physiology, reduced lignin content, and phytoremediation.

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Insect pests, both lepidopteran (Hyphantria cunea , Clostera anastomosis, and Lumantria dispar) and coleopteran (Chrysomela scripta, Chrysomela tremulae), cause serious defoliation resulting in slow growth of Populus (Kulman, 1971). Several proteins such as proteinase inhibitors, lectins and crystal toxins of Bacillus have insecticidal properties against diverse insects with different modes of action (Hofte and Whiteley 1989, Gatehouse et al. 1994, Sudhakar et al. 1998). Transgenic plants containing genes encoding these macromolecules are protected against target insects (Hilder et al. 1987, Estruch et al. 1997). Expression of different proteinase inhibitor genes (Kunitz proteinase inhibitor (Kti3), serine proteinase inhibitor (PIN2), cysteine proteinase inhibitor) in transgenic poplar resulted in less feeding, slower larval development, and some mortality (Leple et al. 1995, Cornu et al. 1996, Heuchelin et al. 1997, Klopfenstein et al. 1997, Confalonieri et al. 1998). Different crystal toxin genes of Bacillus thuringiensis have been used to incorporate insect resistance in Populus. Partially modified cryIA(a) crystal toxin gene in transgenic poplar resulted in a decreased survival rate for forest tent caterpillars and reduced larval weight in gypsy moth (McCown et al. 1991, Robison et al. 1994, Kliener et al. 1995). Transgenic

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hybrid poplar expressing the cryIB gene showed reduced larval feeding and decreased larval weight of cottonwood leaf beetle, however did not result in significant mortality rates (Francis, 1996). In another study transgenic P. tremula x P. tremuloides containing cryIIIA were found to be toxic to coleopteran insects, showing increased mortality of Chrysomela tremulae larvae (Cornu et al. 1996). Transgenic poplars highly resistant to cottonwood leaf beetle were obtained when a modified version of cryIIIA gene (Mycogen USA) driven by the constitutive 35S CaMV promoter flanked by matrix attachment regions was used (Strauss 1999). These studies indicate that a higher level of expression of crystal toxin gene(s) is required in the transgenic plants for better protection against the target insect pests. Use of a strong promoter and changing the bacterial gene to plant-preferred codon sequence should result in higher expression levels. Weeds compete with crop plants for nutrition and other resources, thereby reducing productivity. In forestry weeds are a significant problem in the early establishment stages of plantations. Strategies to specifically inhibit the growth of weeds without affecting the growth of the crop plants have been adopted through genetic engineering of the target crop plants. In this strategy, genes that confer resistance to a specific herbicide are introduced into the crop plant, and on application of the herbicide the weeds are suppressed without affecting the growth of the transgenic plant. The first transgenic Populus was developed using a mutant bacterial gene, aro-A, that confers resistance to the herbicide glyphosate, and the transgenic plants exhibited lower sensitivity to glyphosate (Fillatti et al. 1987). Increased tolerance to glyphosate was obtained using a strong promoter and the gene product targeted to the chloroplast. (Donahue et al. 1994). Recently, expression of two genes CP4 and GOX that can detoxify and reduce the binding to the herbicide resulted in poplars that are highly tolerant to glyphosate (Strauss 1999, Meilan et al. 2000). Phytoremediation is a novel technique that uses the ability of certain plants to accumulate and/or degrade contaminants of soil and groundwater. Hybrid poplars have been shown to mineralize chlorinated compounds such as tricholorethylene (TCE), carbon tetrachloride (CT), and other halogenated aliphatic hydrocarbons (Newman et al. 1997, Gordon et al. 1998). Two genetic

engineering approaches are currently being used by Gordon’s group at the University of Washington, Seattle, to enhance the TCE metabolism in transgenic poplars. In the first approach transgenic tobacco plants expressing the cytochrome P450 IIE1 have shown enhanced TCE metabolism (Doty et al. 2000). Transgenic poplar plants containing the same construct are currently being evaluated for phytoremediation of TCE. In the second approach, transgenic poplar expressing the β-mammalian hemoglobin gene that has the ability to enhance oxidative metabolism, are currently being evaluated (http:// es.epa.gov/ncerqa/final/gordon.html). The results of these studies could have a great impact in decontaminating soil and water pollutants through the genetic engineering approach and extensive use of poplar to prevent pollution. Lignin, a complex phenolic heteropolymer, is present in the wood of trees and has an important biological role (Higuchi, 1985). In plants lignin acts as a barrier for invading pathogens, and provides rigidity and solute conductance. (Walter et al., 1992). The amount and the composition of lignin monomers in wood affect cellulose extraction and the quality of paper making. Modification of lignin biosynthesis, to either reduce the lignin content or to modify the composition, is a promising strategy that could increase profitability and reduce the use of chemicals that are not environmentally friendly. In this approach, antisense technology is used to produce transgenic plants, wherein some of the genes that are involved in lignin biosynthesis are downregulated. Lignin biosynthesis is a complex biosynthetic process that involves several enzymes (Baucher et al. 1998, Whetten and Sederoff 1995). A number of genes involved in lignin biosynthesis have been isolated from different Populus species (Bugos et al. 1991, Dumas et al. 1992, Subramaniam et al. 1993, Allina and Douglas 1994, Leple et al. 1995, Osakabe et al. 1995, Van Doorsselaere et al. 1995). As mentioned above, antisense technology has been used to modify the lignin content by blocking the expression of different enzymes involved in the lignin biosynthetic pathway. Transgenic aspen and poplar containing a bi-specific caffeic acid/5-hydroxyferulic acid-O-methyl transferase (COMT) gene in antisense orientation resulted in a reduced COMT activity and a difference in lignin composition without altering the lignin content (Boerjan et al. 1997). However, in another

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study transgenic aspen containing the complete coding sequence of COMT cDNA (Bugos et al. 1991) resulted in reduced COMT activity due to homologous transgenemediated sense suppression of COMT in developing xylem, resulting in a red-brown wood color. The results from the above study indicate that although the lignin content of these plants did not vary, the structure of lignin in the transgenic plants was modified, with a reduced S/G ratio of the lignin monomers (Tsai et al., 1998). Transgenic hybrid poplar expressing cDNA of cinnamyl alcohol dehydrogenase (CAD) in antisense orientation resulted in a 70% reduction in CAD activity, with a slight reduction of lignin content, but a significantly higher content of free phenolic units (Boerjan et al. 1997). This facilitates solubilization and fragmentation of lignin during kraft pulping (Lapierre et al., 1999). Recently, downregulation of 4-coumarate coA ligase (4CL) using PtCL1 in antisense orientation in transgenic aspen resulted not only in a reduction of lignin content to 45 %, but also increased growth and cellulose content by 15% (Hu et al. 1999). Overexpression of ferulate 5-hydroxylase (F5H) in transgenic tobacco and poplar resulted in an enhanced lignin syringyl monomer content and altered extractability of lignin (Franke et al, 2000). All these studies indicate that by downregulating the lignin biosynthetic pathway at different stages it is possible to alter the composition of the lignin monomer and in one case to reduce the lignin content of the wood. The successful application of genetic engineering through metabolic engineering will have high potential in easier extraction and removal of lignin by the pulp and paper indstry. Flowering in higher plants is essential for sexual reproduction which creates genetic variation. In nature transfer of genetic material occurs through pollen by cross-pollination to other individuals of the same and related species. Regulations and the public prefer that transgenes remain within the transgenic crop. Hence, there is a necessity to check the transfer of transgenes by aborting the formation of pollen. Genetic engineering approaches have been successfully used to create male sterile plants in some crop plants. The basic approach is to introduce a toxic gene under the control of an anther/ tapetum-specific promoter, whose expression leads to disruption of the anther formation (Goldberg 1988, Mariani et al. 1990). Genes coding for barnase and diphtheria

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toxin are ideal candidates that have been used to engineer sterility in plants (Day et al. 1995, Mariani et al. 1990). The promoter TA29, APETALA3, and other floral-specific promoters are valuable in controlling the organ-specific and tight expression of the toxin gene to produce sterile plants. Another strategy is to suppress or block the expression of the functional genes involved in the developmental process of flowering. This involves gene silencing either by co-suppression of the transgenes or through antisense technology. Several homeotic genes, APETLA1 (AP1), LEAFY (LFY), and APETALA2 (APT2), involved in the developmental process of flowering have been identified in Arabidopsis and their homologs in Populus (Sheppard et al. 1996, Strauss 1999). Use of these homologs in transgenic plants will provide a clear understanding of the success of this strategy to engineer male sterility. Accelerated flowering could help in accelerating tree breeding programs, especially in Populus. The knowledge of the genes involved in flowering could provide ways to cause flowering to occur earlier in the life cycle of the tree. Constitutive expression of genes involved in flowering could result in a shorter period of sexual immaturity in trees. Constitutive overexpression of the LFY gene of Arabidopsis resulted in precocious flower development in transgenic aspen (Weigel and Nilsson 1995). The PTLF gene, a homolog of LFY, when overexpressed constitutively accelerated the flowering time in Arabidopsis, but not in Populus (Strauss 1999, Rottmann et al. 2000). A number of genes that are involved in flowering and their promoter elements are currently being isolated, characterized, and evaluated (Strauss 1999). Genetic control of flowering and approaches for the development of sterile plants have been discussed in detail (Strauss et al. 1995). FUTURE PROSPECTS The introduction of novel genes into Populus genome through genetic engineering has resulted in improvement of useful traits that was not possible through conventional breeding programs. Although transgenic poplars are not yet commercially deployed, several genetically modified poplars are being currently evaluated in field trial studies. Modification of the Bt cry toxin genes by using plant preferred codons could improve the expression levels to

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protect from insect damage. Use of more than one insecticidal gene, where the additional gene codes for a different mode of action, a strategy known as gene pyramiding, will produce better resistance to the target insects and also delay the development of resistance in the target insects. The use of genetically modified Populus to decontaminate toxic wastes will have a high priority because of poplar’s broad adaptation to varied agroclimatic regions and other biological advantages. Considerable advances have been made to reduce the lignin content in Populus through metabolic engineering by altering the lignin biosynthetic pathway and could lead to reduced use of chemicals in the pulp and paper industry, thereby increasing profitability and reducing pollutants that must be treated. Major emphasis has been focused on flowering to induce sterility and to cause early flowering. Induction of sterility has a high priorit due to the concern of transfer of transgenes to wild populations of Populus. Isolation of novel genes and promoters within Populus germplasm will provide critical capability to understand several biochemical pathways and to modify them for commercial purposes. Biotechnology has great potential and will continue to play a major role in the improvement of forest trees within a short span of time. Several modern technical advances in high-throughput analysis will help in rapid screening to identify and isolate novel genes and promoter sequences for commercial exploitation. Production of recombinant proteins and biomolecules in plants is an emerging field of transgenic research known as “Molecular Farming”. There are considerable potential benefits of using Populus in producing useful chemical products for various industrial applications, that will allow Populus to be grown by farmers as a cash crop.

ACKNOWLEDMENTS This work is supported by a Natural Resources Research Institute (NRRI), University of Minnesota (UMD) research grant for biotechnology research. The authors thank Dr. Michael Lalich, Director of NRRI, and Dr. Thys Johnson, former Assistant Director of NRRI, for their support of this research program. We thank Dr. Durba Ghoshal for critically reading the manuscript.

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Indian J. Plant Physiol., Vol. 6, No. 2, (N.S.) pp. 119-126 (April-June, 2001)