Case Study 1: Eucalyptus grandis Trees  with Improved Timber for Cellulose Pulp  & Paper Production  Dr. Giancarlo Pasquali – Centro de Biotecnologia, Universidade Federal do Rio Grande do  Sul – Porto Alegre, RS, Brazil  PART I: THE NON-TRANSGENIC PLANT GENERAL DESCRIPTION OF THE PLANT. Eucalyptus grandis Hill ex Maiden (Myrtaceae - Myrtle family) is native to the east coast of Australia. Its common name is rose gum or flooded gum (a misnomer). E. grandis is one of the premier forest species in the Australian States of Queensland and New South Wales where it grows 43 to 55 m tall and 122 to 183 cm in diameter (1). Its form is excellent with tall, straight, clean holes up to two-thirds of the total height. The bark is thin and deciduous, shedding in strips to expose a smooth surface marked with flowing patterns of silvery white, slaty gray, terra cotta, or light green. Occasionally a "stocking" of light-gray, platelike or fissured bark persists over the basal 1 to 2 m on the trunk. Juvenile leaves are ovate. Adult leaves are stalked, lanceolate to broad lanceolate, 10-16 cm long, 2-3 cm wide, glossy dark green. Flower clusters (umbels) at leaf base are 2.5 to 3 cm long including flattened stalk of 13 mm. Buds are pearshaped with a blunt-pointed conical lid of 10 mm by 5 mm, usually with whitish waxy coating. Seed capsules are short-stalked, pear-shaped or conical, slightly narrowed at rim, thin, 8 mm long and 6 mm wide, with whitish waxy coating, narrow sunken disk, and 4-6 pointed thin valves slightly projecting and curved in, persisting on twigs back of leaves. Wood is pink to light reddish brown, moderately hard. The fast growth rate in tropical regions and the high quality timber makes E. grandis one of the most economically important trees around the world. In Brazil and South Africa, E. grandis and mainly its hybrids, especially (E. grandis x E. urophylla), has its timber industrially employed for cellulose pulp and paper production. Lowering lignin and increasing cellulose contents would strikingly improve paper productivity, reducing industrial costs in (i) energy and chemicals for pulping (separation of lignins from cellulose) and (ii) treatment of industrial effluents needed to be detoxified before their return to nature. REPRODUCTIVE BIOLOGY OF THE SPECIES. E. grandis, like all eucalypts, bears perfect flowers. Buds form in axillary umbels with usually seven buds per cluster. Each flower consists of a central style surrounded by stamens standing

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about 8 mm tall and forming a bloom about 20 mm in diameter. The puffy clusters of creamy white blooms are attractive and conspicuous but not of horticultural quality. E. grandis blooming season in Brazil varies according to geographical location. Flower development in southeast Brazil, for instance, takes approximately one year from floral initiation to fruit maturation. Initial floral buds usually appear in November/December. Green buds are observed until February. Mature buds and main flowering occur from January to April. Green fruits without radial splits occur from March to June and those with radial splits in July and August. Mature fruits occur mainly from September to January. Foraging insects, particularly honeybees (Apis melifera), pollinate the flowers. In an individual flower, the stigma is not receptive until after pollen shed, but because each tree blooms serially, there is ample opportunity for self-fertilization. In seed orchards, selfing occurs with a frequency of up to 5%, although some authors have described selfing of 10 to 38 %. Detrimental abnormalities are observed in self-crossed-derived seedlings, with depressed height growth reaching 8 to 49 percent compared to crossed progenies (2). Flowering precocity is strongly inherited; a few families bloom at plantation-age 1 year, many more at age 2, and 97 % of the orchard at age 3. From 2 to 3 weeks after blooming, the stamens and style wither and fall away, leaving a woody, urn-shaped seed capsule closed by four to six valve covers. The capsules are about 8 mm long by 6 mm in diameter. Most umbels carry five to seven capsules to maturity. Reproduction by natural vegetative fragmentation is not documented for E. grandis or other Eucalyptus species. CENTER OF ORIGIN AND CENTER(S) OF GENETIC DIVERSITY. Over its central range in east Australia, E. grandis grows on alluvial or volcanic loams in valleys and flats within 160 km of the coast, straddling the Queensland-New South Wales border from latitude 26 to 33° S. Two outlier populations extend the range to the Atherton Tablelands at latitude 13° S. (1). E. grandis is also found in natural forests in islands of Indonesia and Timor. The climate in the Australian native range of E. grandis is humid subtropical with mean minimum temperatures during the coldest month ranging from 2 to 10° C and mean maximums near 29° C during the hottest month. Rainfall averages 1,020 to 1,780 mm; it is concentrated in the summer, but monthly precipitation during the dry season is at least 20 mm (4). Coastal areas are generally frost-free, but higher altitude, inland areas experience occasional frosts (3). E. grandis is one of the most important commercial eucalypts, with more than one-half million hectares (1.3 million acres) planted in tropical and subtropical areas on four continents. Massive planting programs have been carried out in the Republic of South Africa and Brazil, and there are substantial plantings in Angola, Argentina, India, Uruguay, Zaire, Zambia, and Zimbabwe (4).

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MEANS OF DISPERSAL AND ESTABLISHMENT Foraging insects, mainly honeybees, are responsible for the pollination of ~ 95 % E. grandis flowers. Different groups have studied pollen flow in artificial populations of Eucalyptus species in different countries. Using molecular markers and paternity analysis, (5) described that E. grandis pollen parents located within a population in Australia were between 0 and 192 m from the respective mother trees, with an average pollination distance of 58 m. Pollination of mother trees was outcrossed (> 95%), not by nearest neighbors, and displayed a preference for interprovenance matings within the population. Progeny that could not be assigned pollen parents within the population (46%) were assumed to have resulted from pollen immigration from external sources. Seed capsules are mature for harvest 6 or 7 months after flowering. However, the capsules remain closed on the tree for at least one year after maturity. The valves of the capsules dry out, open, and release seeds. Capsules scattered loosely on a dry surface release their seeds after about 2 hours in full sun. Individual trees bear from 3 to 25 sound seeds per capsule, with an average near 8 and a much greater mass of infertile ovules called "chaff." Fertile seeds are tiny, only about 1 mm in diameter. Chaff particles are lighter colored and only minutely smaller and lighter than seeds. Seed production is reliable year to year, but there is great tree-to-tree variation in the quantity, purity, and viability of seed crops. Seeds have been successfully stored for 20 years by either freezing at -8° C or refrigerating at 10° C. E. grandis seeds require no presowing treatment. Germination of E. grandis is epigeal and takes place in 7 to 14 days after sowing (6). Moist, bare soil is required for natural regeneration; fire, erosion, and flood deposits provide satisfactory seedbeds. In commercial forests, the species is almost always regenerated by planting. Seedlings are usually raised to 20 to 30 cm tall, which takes 3 to 5 months (7). Due to the sensitivity to desiccation, seedlings are normally grown in containers. Rigid containers with multiple cavities from which the seedlings are removed with roots and soil intact are almost always used in large operations. Seedlings are also grown in plastic nursery bags. In the absence of frost and drought, seedlings can be planted throughout the year. In many areas seedling production and planting must be carefully timed. INTRA-SPECIFIC, INTER-SPECIFIC AND/OR INTER-GENERIC HYBRIDIZATION: Geographical distance and morphological flower barriers are the natural conditions to impede E. grandis hybridization with other Eucalyptus species. Eucalyptus flowering occur quite simultaneously independently of the species, especially in non-native tropical regions like Brazil. Foraging insects, especially bees, are the main pollinators of most Eucalyptus species of commercial interest, including E. grandis. Therefore, time of flowering and plant-specific pollinators are not ecological barriers for Eucalyptus hybridization when planted in closer proximities. Human-mediated, intended hand hybridization is being intensely explored for more than a 1,3 century in Eucalyptus forestry. Hybrids of E. grandis and other Eucalyptus species are 3

considered the best trees for cellulose and paper production in Brazil and South Africa, among other countries. A significant number of E. grandis hybrids are fertile and produce equally viable progenieslike (E. grandis × E. urophylla), (E. grandis × E. camaldulensis), and (E. grandis × E. tereticornis) (reviewed in 8). Although natural or hand pollination is frequent in non-native regions for Eucalyptus, the Brazilian Eucalyptus forestry intended for cellulose pulp and paper production has characteristics that should be considered in risk assessments: • •

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All Eucalyptus species are considered exotic in Brazil; E. grandis (and other Eucalyptus species grown in Brazil) is a tall and large tree, easterly found even before it starts flowering and spreading pollen or seeds; Plants sexually compatible with Eucalyptus, even belonging to the Myrtacea family, are not described or known in the Americas; Vast areas of Eucalyptus commercial plantations exist in Brazil since 1940´s (470.000 ha between 1909 and 1966) and most of them are still employed for Eucalyptus timber production; A significant percentage of the areas is planted with clonal plants, drastically reducing the number of viable seeds produced; For cellulose pulp and paper production, eucalypts are harvested after 7 years, branches, leaves and barks are maintained in the field and the trunks are ground to small pieces and cooked under high temperatures and in the presence of alkalis for pulping; Although thousand tons of seeds fall from trees during the growing period, spontaneous, voluntary plants are very rare within the commercial plantations; Any voluntary plant that succeed surviving is eliminated from the plantations either by hand picking or during the mechanical harvesting; Simultaneously of months later the harvesting process, new Eucalyptus plants are planted between remaining trunks in the area. After 7 years, remaining trunks were completely decomposed and their spots are ready for receiving new plantlets.

PART II: THE RECEIVING ENVIRONMENT CULTIVATION OF THE CASE STUDY PLANT SPECIES IN BRAZIL Although reproduction by natural vegetative fragmentation is not documented for E. grandis or any other Eucalyptus species, clonal propagation (cuttings, macro and micropropagation) is the method of choice for multiplying seedlings in Brazil. Vast areas (stands) of highly selected Eucalyptus genotypes are planted in Brazil in so called “clonal forests”. Outcrossings among clonal individuals have the same effect on progeny as self crossings. Seeds and progenies derived from self-crossed trees have much lower viability than outcrossed-derived individuals.

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E. grandis and its hybrids are cultivated from Rio Grande do Sul til Amapá., in areas of good humidity, i.e, closer to the Atlantic coast. The case study plant (E. grandis) is being tested in field trials in the states of São Paulo and Minas Gerais. PRESENCE OF ANY SEXUALLY COMPATIBLE RELATIVES IN THE RECEIVING ENVIRONMENT. Only Eucalyptus trees of the same species or compatible species like E. urophylla, E. tereticornis, E. camaldulensis, among others, are sexually compatible with E. grandis in the Brazilian environment. ECOLOGICAL INTERACTIONS IN THE RECEIVING ENVIRONMENT (E.G., IDENTIFY SIGNIFICANT PEST COMPLEXES). Eucalyptus species have been grown for more than 120 years in Brazil on millions of ha in and have not demonstrated any invasive characteristics. In addition, Eucalyptus species, including E. grandis, have been grown in Southern United States for decades and are not considered invasive by the Florida Department of Agriculture, which has a very aggressive non-invasive species policy. There is no evidence that any of the Eucalyptus species commercially explored for cellulose pulp and paper production have escaped from cultivation and caused any problems in Brazil. Insect pests like ants, termites, beatles and different types of catterpillars are common in Eucalyptus plantations. This insects use to feed on native Myrtaceae plants and the large, continuous and homogeneous plantations of Eucalyptus distributed all around Brazil allowed an ideal environment for them to overpopulate. Chemical control of insect pests is sometimes necessary during the seedling or shrub phases of Eucalyptus growth. Biological control and integrated management is performed in fields of adult grown plants. The most prominent diseases affecting Eucalyptus plantations in Brazil are caused by fungi: • • • • •

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Rust (Puccinia psidii); Canker (Cryphonectria cubensis, Valsa ceratosperma, Botryosphaeria ribis; Oidium sp.; Grey mold (Botrytis cinerea); Damping-off (Cylindrocladium candelabrum, C. clavatum, Rhizoctonia solani, Pythium spp., Phytophthora spp. and Fusarium spp.)´ Leaf spots (C. candelabrum, C. ilicicola, C. parasiticum, C. pteridis and C. uinqueseptatum).

Control of fungal diseases by fungicides is economically forbidden. The generations and selection of resistant cultivars is the method of choice to reduce phytopathogenic limitations.

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PART III: THE TRANSGENIC PLANT Data here presented and part of the text were extracted from reference (9): Li, L., Zhou, Y., Cheng, X., Sun, J., Marita, J.M., Ralph, J., Chiang, V.L. (2003) Combinatorial modification of multiple lignin traits in trees through multigene cotransformation. Proc. Natl. Acad. Sci. USA 100, 4939-4944. THE PURPOSE OF THE TRANSFORMATION As stated before, lowering lignin and increasing cellulose contents in timber would strikingly improve paper productivity, reducing industrial costs in energy and chemicals for pulping and treatment of industrial effluents. Tremendous efforts have been devoted to developing genetically engineered trees, with the emphasis on reducing lignin quantity, to improve woodpulp production efficiency (10–14). However, lignin chemical reactivity also is a critical barrier to wood-pulp production, because lignin removal from wood is either initiated by chemical degradations or, in most cases, accomplished entirely through chemical reactions. Thus, the current tree biotechnology emphasis on low lignin quantity must be expanded to include greater lignin reactivity and, ultimately, a combination of low and reactive lignin traits. Genetically modified (GM) E. grandis trees were generated with these purposes and proved to exhibit both advantages: lower lignin and higher cellulose contents. THE ANTICIPATED CULTIVATION REGION (E.G., SMALL SCALE IDENTITY PRESERVED CROP OR WIDELY CULTIVATED CROP ETC.) Not applicable. A SUMMARY OF THE INTRODUCED GENETIC ELEMENTS AND THE SOURCE OF THESE (E.G., DONOR ORGANISM ETC.). Lignin in angiosperm trees is polymerized from the guaiacyl and syringyl monolignols (Fig. 1) with a syringyl/guaiacyl (S/G) ratio of ~2-2.5 (15–19). Wood-pulping kinetics further revealed that every unit increase in the lignin S/G ratio would roughly double the rate of lignin removal (19), supporting the idea that combinations of S/G-ratio augmentations and lignin reductions may offer far-reaching opportunities for maximizing wood-pulp production efficiency. However, in trees genetic reduction of lignin, which has been achieved through the suppression of the monolignol pathway gene encoding either 4-coumarate-CoA ligase (4CL) (11) or caffeoyl-CoA O-methyltransferase (12), had no significant effect on the S/G ratio. Attempts to modify the S/G ratio in trees could not succeed in lignin reduction (20,21). These results argue that lignin quantity and the S/G ratio are regulated independently during lignin biosynthesis in trees. Considerable evidence is now available that shows that in angiosperm trees, the syringyl monolignol pathway (Fig. 1) branches out from the guaiacyl pathway through coniferaldehyde and is regulated in sequence by three genes encoding coniferaldehyde 5-hydroxylase (CAld5H) (22), 5-hydroxyconiferaldehyde O-methyltransferase (23), and sinapyl alcohol dehydrogenase (24). Enzyme kinetics further demonstrated that CAld5H has a 6- to 50-times-slower turnover 6

rate than the other two syringyl pathway enzymes (22–24), pointing to a key role for CAld5H in limiting syringyl monolignol biosynthesis and, therefore, lignin S/G ratio. Thus, simultaneously up-regulating CAld5H and down-regulating 4CL gene expression may lead to a concurrent lignin-reactivity augmentation and quantity reduction in plants. Although multigene manipulation in plants could be achieved by repetitive gene insertion or crosspollination (refs. 25–27; see ref. 28 for review), these approaches are impractical for trees, which have long life cycles. E. grandis GM trees were generated by Agrobacterium-mediated transformation. One genetic construct harboring three transgene cassettes was obtained (Fig. 2) and DNA elements are listed in Table 1. The resulting A. tumefaciens binary plasmid was named p4CL-C5H. E. grandis 4CL xylem-specific promoter (Eg4CL-P) and terminator (Eg4CL-3´) were employed to drive the expression of the E. grandis 4CL gene in antisense orientation and the E. grandis CAld5H gene in sense orientation. The neomycin phosphotransferase (nptII) gene, driven by the cauliflower mosaic virus 35S (CaMV 35S) promoter and the nopaline synthase terminator (3´nos) was used as antibiotic selectable marker for plant cells. After transformation of young leaf segments of E. grandis and selection of resistant tissues, 15 phenotypically normal transgenic plantlets were recovered, expressing each or both transgenes of interest in different levels. The transgenics showed that antisense down-regulation of 4CL gene expression selectively mediated lignin reduction, whereas overexpression of the CAld5H gene specifically induced S/G-ratio augmentation. These independent effects became additive, with transgenic trees simultaneously expressing two transgenes exhibiting strong lignin reductions and drastic lignin S/G-ratio augmentations. Additionally, 4 independent transgenics increased the level of cellulose deposition in cell walls as a compensation of ligning content reduction.

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Fig. 1. Proposed principal biosynthetic pathway for the formation of monolignols in woody angiosperms.

C4H, cinnamate 4-hydroxylase; C3H, 4-coumarate 3-hydroxylase; 4CL, 4-coumarate CoA-ligase; CCoAOMT, caffeoyl CoA O-methyltransferase; CCR, cinnamoyl-CoA reductase; CAld5H, 5-hydroxyconiferaldehyde O-methyltransferase; SAD,sinapyl alcohol dehydrogenase; CAD, cinnamyl alcohol dehydrogenase.

Fig. 2. Schematic representation of p4CL-C5H T-DNA. DNA elements are described in Table 1. The Eg4CL terminator is indicated simply by 3´.

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Table 1: Summary of DNA Components in the T-DNA of binary plasmid p4CL-C5H.

Genetic Element

Size1 kb

LB

0.45

A restriction fragment from the octopine Ti plasmid, pTi15955, containing the 24 bp T-DNA left border used to terminate the transfer of the T-DNA from Agrobacterium tumefaciens to the plant genome (Barker et al., 1983).

3'nos

0.26

A 3' nontranslated region of the nopaline synthase gene which functions to terminate transcription and direct polyadenylation of the nptII mRNA (Depicker et al., 1982; Bevan et al., 1983).

nptII

0.79

The gene isolated from Tn5 (Beck et al., 1982) which encodes for neomycin phosphotransferase type II. Expression of this gene in plant cells confers resistance to kanamycin and serves as a selectable marker for transformation (Fraley et al., 1983).

35S

0.32

The 35S promoter region of the cauliflower mosaic virus (CaMV) (Gardner et al., 1981; Sanders et al., 1987).

Eg4CL-P

0.62

E. grandis xylem-specific promoter of the 4CL (Fig. 1) encoding gene, driving the transcription of aEg4CL gene.

aEg4CL

1.8

E. grandis 4CL coding sequence in antisense orientation.

Eg4CL-3´

0.3

3' nontranslated region of the E. grandis 4CL gene which functions to terminate transcription and direct polyadenylation of the aEg4CL mRNA.

Eg4CL-P

0.62

E. grandis xylem-specific promoter of the 4CL (Fig. 1) encoding gene.

EgCAld5H

1.6

E. grandis CAld5H coding sequence in sense orientation.

Eg4CL-3´

0.3

3' nontranslated region of the E. grandis 4CL gene which functions to terminate transcription and direct polyadenylation of the aEg4CL mRNA.

LB

0.36

A restriction fragment from the pTiT37 plasmid containing the 24 bp nopaline-type T-DNA right border used to initiate the T-DNA transfer from Agrobacterium tumefaciens to the plant genome (Depicker et al., 1982).

1.

Function and Source

Sizes are approximations.

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INHERITANCE AND STABILITY OF EACH INTRODUCED TRAIT (E.G., SEGREGATION ANALYSIS, STABILITY OF THE INSERT ETC.). PCR analysis and Southern blot hybridization were conducted in order to check T-DNA integration and gene copy number. Fig. 3 illustrates the result of PCR analysis specifically for the Eg4CL-P/aEg4CL and Eg4CL-P/EgCAld5H fusion fragments. All 15 transgenic line exhibited a band corresponding to the 4CL or CAld5H transgene constructs. Only transgenic lines 8, 9, 10 and 15 exhibited bands that corresponded to both gene constructs.

Fig. 3. PCR analysis of the integration of antisense Eg4CL and sense EgCAld5H transgenes in various transgenic E. grandis plants (lane numbers represent different transgenic lines). A 1-kb DNA ladder was used for both panels (lane M). The PCR fragments seen are 1.66 and 1.61 kb in size, encompassing a portion of the promoter-gene fusion. Such transgene fragments were absent from the control (lane C).

Results of Southern blot analysis for all 15 transgenic lines confirmed that only lines 8, 9, 10 and 15 contained both intact transgene constructs (result not shown). Additionally, it revealed that only line 9 exhibited one single copy of the T-DNA, as depicted in Fig. 4. The existence of a single hybridization band in DNA samples digested with SacI (aEg4CL) and BclI (EgCAld5H), and multiple bands for the other represented restriction enzymes are in agreement with the expected pattern of digestion of binary plasmid p4CL-C5H T-DNA.

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Fig. 4. Molecular characterization of E. grandis transgenic line 9. Genomic DNA gel blot analysis. E. grandis genomic DNA (10 µg/lane) was digested with restriction enzymes and hybridized with 32P-labeled promoter-aEg4CL (A) and promoter-EgCAld5H (B) fragments.

DIFFERENCES IN GENETIC AND PHENOTYPIC VARIABILITY FROM NON-TRANSGENIC CROP. After 1 year cultivation in greenhouse, transgenic lines (T0) were propagated by macrocuttings. A total number of 900 clonal plantlets were specifically derived from transgenic line 9. PCR analysis of all clonally propagated plants revealed essentially the same pattern of DNA amplification as the matrix plant (result not shown). After acclimation, plantlets were planted in open field and distributed along one hectare (ha). Transgenic plantlets were randomly distributed in the area along with 200 non-transgenic (control) E. grandis plants. Each plant was 2.5 m distant from its neighbors. Five lines of non-transgenic E. grandis plants of the same age surrounds the test field. A square of 100 m of grassland, free of any cultivated plant, surrounds the test field. The test field is located in Pedra Azul, MG, inside 30,000 ha of Eucalyptus sp. commercial fields. After 5 years in the test field, no statistical differences were observed in the parameters listed below between transgenic line 9 plants and non-transgenic plants (border or inside trees): • • • • • • • • • • • •

Growth rate Height Foliage area Trunk diameter General tree architecture Bark appearance and thickness Leaf morphology Flower morphology Flower viability (fertilization tests) Fruit morphology Seed number, morphology and germination Pollen number and viability

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Ald5H gene and enzymee expressionss, Nevertheeless, significcant differennces in gene 4CL and CA lignin content and qu uality, as welll as cellulosse content were w observedd between GM G and non-GM i Fig. 5 andd Table 2. plants, ass illustrated in

Fig. 5. Thee effects of dow wn-regulation n of 4CL and up-regulation u of CAld5H in n E. grandis pllants (transgen nic line 9) on 4CL 4 and CAld5H enzyme activities a and lignin l accumu ulation and S/G G ratio. (A) Protein P gel-bloot (10 mg of prottein extracts per p lane) analyysis of xylem 4CL 4 and CAld d5H protein leevels by usingg anti-4CL and d anti-CAld d5H antibody probes. p (B) 4C CL and CAld55H enzyme acttivities in stem m developing xylem x tissue. Crude C protein (20-40 mg) was assayed for 4C CL and CAld5H activities with w caffeate and a coniferald dehyde, respectiveely, by using HPLC/MS. H Errror bars repreesent standard d deviation vaalues of three replicates. r (C)) The levels of lignin reduction and lignin S/G S ratio increease in stem wood w of transggenic lines as compared c with h the control. Table 2. Chemical C comp positions in steem wood of coontrol and traansgenic E. graandis.

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In conclusion, E. grandis trees derived from transgenic line 9 have a reduction in lignin content of up to 50% (40% average), an increase of the S/G ration of 54% (average), and an increase in the cellulose content of 22% (average). Therefore, timber derived from GM trees is much easily pulped due to the lower content and higher reactivity of lignin, besides yielding higher levels of cellulose for paper production. DIFFERENCES IN MODES AND/OR RATE OF REPRODUCTION FROM NON-TRANSGENIC CROP (E.G., ANY AVAILABLE OUT-CROSSING DATA ETC.). Transgenic E. grandis trees were not intentionally crossed or hybridized. Therefore, no T1 progeny was generated. Propagation of GM (and non-GM) plants was performed vegetativelly, via microcuttings. As referred below (session 11), no expected differences in the mode of reproduction are expected from GM trees. EXPRESSION LEVELS OF NOVEL PROTEINS IN DIFFERENT TISSUES OVER TIME. As depicted in Fig. 5, the expression levels of 4CL and CAld5H enzymes were clearly modified when comparing GM-derived and non-GM-derived plant protein extracts. General protein content between GM and non-GM extracts was indistinct. Enzyme activities of all other enzymes of the lignin biosynthetic pathway represented in Fig. 1 and cellulose synthase were statistically equivalent. Assays were performed every year for matrices plants over the 7 years of their existence (results not shown). DIFFERENCES IN AGRONOMIC CHARACTERISTICS FROM NON-TRANSGENIC CROP. No differences in forestry characteristics were observed between GM and non-GM trees. Please refer to session 5 above. DIFFERENCES IN DISEASE AND/OR PEST SUSCEPTIBILITY FROM NON-TRANSGENIC CROP. Although not proved statistically, we observed a slight trend towards a higher susceptibility of the GM plants to foraging insects and to the fungus Ceratocystis sp. POTENTIAL IMPACT ON NON-TARGET ORGANISMS IN THE RECEIVING ENVIRONMENT: Not applicable since genes employed and proteins involved are not aimed for pest or disease control. ANY AVAILABLE EXPOSURE DATA (E.G., POLLEN MOVEMENT, PROTEIN DISSIPATION, ETC.). Evaluations of pollen and seed characteristics like weight, shape, color, protein content, starch content, DNA content and viability revealed no differences between GM and non-GM trees. Fertilization of non-GM and GM-flowers with GM and non-GM pollen was successfully performed, with no differences the number of mature fruits generated or number of seeds per fruit. Evaluations on seed germination were also conducted with essentially equivalent results for GM, non-GM or “hybrid” seeds.

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REFERENCES FOR ANY RISK ASSESSMENTS UNDERTAKEN IN OTHER JURISDICTIONS. This is the first report of transgenic E. grandis trees with modified 4CL and CAld5H gene expressions. It is therefore the first line of experiments aiming risk assessments for the transgenics.  

 

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