TRANSGENIC TREES EXHIBITING INCREASED GROWTH, BIOMASS PRODUCTION AND XYLEM FIBRE LENGTH, AND METHODS FOR THEIR PRODUCTION

Abstract: Important aims in nearly all tree-breeding programs around the world are to produce plants with increased growth rates and stem volumes, and shorter rotation times. Such trees would yield more biomass per area unit. Here the present inventors have shown that when over-expressing a key regulatory gene in the biosynthesis of the plant hormone gibberellin (GA) in hybrid aspen (<i>Populus tremula x P. tremuloides</i>), improvements in valuable traits such as growth rate and biomass are obtained. In addition, these trees also have longer xylem fibers than unmodified wild type plants. Long fibres are very desirable in the production of strong paper, but it has not (as yet) proved possible to influence this trait by traditional breeding techniques. A further advantage of the present invention is that it may reduce or eliminate the use of growth influencing chemicals in forestry.

Description: Transgenic trees exhibiting increased growth, biomass production and xylem fibre length, and methods for their production The present invention concerns transgenic trees exhibiting improved properties, and in particular properties of economic importance, such as increased growth rate, biomass production and xylem fibre length; transgenic plants, seeds, plant cells and other types of propagating material, as well as methods for their production. Background A major disadvantage with traditional tree breeding, especially for forest tree species, is the slow progress due to their long generation periods. However, by taking advantage of recent developments in gene technology the time required to produce a new variety could be reduced significantly. In addition, a biotechnological approach would allow closer targeting of traits considered desirable by the forest and pulp industries, in specific tree species. To date, most applications of genetic engineering of trees have focused on modifying lignin biosynthesis, resulting in trees with less lignin or a modified lignin composition, earlier flowering, pest or herbicide resistance. In order to change growth and development processes in trees, the manipulation of plant hormone levels, or the hormone sensitivity, would also be of interest. However, as yet there has only been few examples of the modification of plant hormone levels in trees. These have mainly been accomplished by directly altering endogenous IAA biosynthesis or cytokinin biosynthesis or indirectly by modifying various hormone pools using the Agrobacterium rolC gene. Although such modifications in all cases lead to trees with altered growth characteristics and wood properties, so far no improvements with a clear practical application have been obtained. Gibberellins (GAs) are a group of more than 100 tetracyclic diterpenes, some of which are essential endogenous regulators that influence growth and development processes throughout the plant life cycle, e.g. shoot elongation, the expansion and shape of leaves, flowering and seed germination. The best examples illustrating the importance of GAs in control of shoot elongation are GA-deficient mutants of Arabidopsis, maize and pea. These have reduced levels of active GA(s) compared to wild type plants, resulting in a dw arfed phenotype due to a reduction in intemode length. The phenotype of such GA-deficient mutants can be completel restored by the application of an activ e GA. At the cellular le\ el. GAs ha\ e been tound to promote both cell division and cell elongation Biosynthesis of GAs in planta occurs through the isoprenoid pathway from mevalomc acid. Gibberellm lev els are mainly regulated by transcπptional control of gibberellm biosynthesis genes. In particular, the multifunctional enzyme gibberellm 20- oxidase ( GA 20-ox) is a key-enzyme in controlling GA biosynthesis (Fig. 1 ). It catalyses the stepwise conversion of the C-20 gibberellms. GA -, GA ς by three successiv e oxidations to GA 4 GA,, . which are the immediate precursors of the activ e gibberellms. GA, and GA . respectiv ely The expression of the GA 20-oxιdase gene is down regulated by the action of GA, 4 . suggesting that direct end-product repression is involv ed in regulation of the gene In addition, some authors have suggested that GA 20-oxιdase is photopeπodically regulated at the transcπption level. Application of chemicals that alter GA levels in the plant is a common practise in traditional agriculture and horticulture. Inhibitors of GA biosynthesis are especially commonly used as growth retardants in cereals and ornamental plants. In order to reduce the use of these chemicals, a biological approach like genetic modification of endogenous GA biosynthesis w ould have clear advantages. Using Arabidopsis as a model organism it has been shown that it is possible to change GA levels by modifying GA enzyme levels and that this results in plants with altered growth and development patterns Transgenic Arabidopsis expressing the GA 20-oxιdase in a sense oπentation shows earlier tloweπng and taller stems than wild type plants, whereas antisense plants have the reverse properties [Coles. J.P. el al. Modification of gibberellin production and plant development in Arabidopsis by sense and antisense expression of gibberellin 20-oxιdase genes. Plant J 17. 547-556 ( 1999)]. Ylodification of GA biosynthesis m a higher species, such as trees would be of additional interest since this would open up ways to modify wood. Previous hormone application studies have shown that GAs are required for the differentiation of xylem fibres, and that they have pronounced effects on the length of secondary xylem fibres and on both longitudinal and radial growth in hard wood species and conifers. Obviously there remains a need tor improved methods for modification of the grow th properties of trees, in particular properties of technical and economical interest, such as growth rate, biomass increase and fibre length. Likewise, there remains a need ot transgenic trees, exhibiting improv ed properties, such as increased grow th rate, stem v olume and xylem fibre length Consequently, the objectiv e of the present invention is to provide such improved methods and transgenic trees λnother objectiv e is to reduce or eliminate the use of growth influencing chemicals in forestry Prior art It has been shown (Huang, S et al Overexpression of 20-Oxιdase confers a gibberelhn-ov erproduction phenotype in Arabidopsis. Plant Physiol . 1998. v ol 1 18. p ^73-781 ) that the level of active GAs were raised following overexpression of GA 20- oxidase in Ai abidopsis thahana This finding is however not directly transferable on the present invention Most dicotyledons and all gymnosperm undergo some degree of secondary thickening. The amount of thickening depends on whether the mature plant is a herbaceous or a woody (arborescent) plant For example an Arabidopsis plant will only produce secondary thickening under special conditions whereas a woody species, for example Populus. will undergo a high degree of secondary growth The low degree ot secondary thickening m herbaceous species will also make it very difficult to predict how specific genetic changes in herbaceous species corresponds in woody species in regards to changes in wood formation. As an example, by over expressing the GA 20-oxιdase in Arabidopsis thahana the level of active GAs in the plant can be raised, as shown by Huang et al. (Supra). The transgenic plant phenotype included a dramatic cell elongation in all tissues. The petioles, inflorescence stems and leaves all showed cell elongation. It is clear from these results that the enhanced gibberellin levels have an effect on cell elongation from germination. However, from these results it is impossible to predict what the effect of GA 20-oxιdase over expression in a woody plant would be. Cell division in the cambial mersistem in a woody plant is under the control of several different hormones and physical constraints not found in an annual plant such as Arabidopsis thahana. It was therefore surprising that there w ould be increased cell elongation ot cells (fibres) originating from the cambium, and that it would be an increase in biomass caused by increased cambial cell di ision It would not be obvious to one skilled in the art to over express G \ 20-oxιdase to increase ood biomass and fibre elongation Increase in the diameter ot tree stems occurs primarily from meπstematic activity in the vascular cambium, a cylindrical lateral meπstem located between the xylem and phloem ot the stem, branches, and woody roots Two types ot cell division occur in the cambium additiv e and multiplicative Additiv e division involv es penclinal division of fusiform cambial initials to produce xylem and phloem mother cells that in turn divide to produce xylem and phloem cells Multiplicative division involves anticlinal division ot fusiform initials that prov ide tor circumferential expansion ot the cambium After xylem and phloem cells are cut off by the cambial mother cells, thev differentiate in an ordered sequence of events that includes cell enlargement, secondary wall formation, hgnification and loss of protoplasts These events do not occur stepwise. but rather as overlapping phases Summary of the invention It is an object ot the present invention to provide transgenic plants, seeds and plant cells, exhibiting impro ed growth parameters and in particular improved parameters ot economical interest, such as increased growth rate, biomass increase and xylem fibre length It is another object of the invention to provide a method tor the production and proliferation of said transgenic plants The above objects and others not explicitly mentioned, are fulfilled through a transgenic woody plant having a DNA sequence coding for the expression ot a polypeptide exhibiting GA 20-oxιdase activitv functionally inserted in the plant genome. Further embodiments are as defined in the attached dependent and independent claims, incorporated herein by reference Other features and advantages of the invention will be apparent form the following, non-limiting description and examples, and from the claims. Brief description of the drawings The invention will be described in closer detail below , in the descπption and accompanying examples and drawings, in which Figure 1 shows the biosynthetic pathw ays conv erting GA ι ; and GA,, to the biologicallv active products GA α and GA,. and their deactivated catabohtes. GA and GA,. GA 20-oxidase catalyses the oxidation at the C-20 carbon. Figure 2 shows a Northern analysis of ten GA 20-oxidase over-expressing lines (numbers 1 to 15) and the non-transformed control (C). 18μg of total RNA was loaded from each sample, and probed with a labelled fragment isolated from Arabidopsis gibberellin 20-oxidase {AtGAJOoxl ) cDNA or from an endogenous ubiquitm ( L ' BQ) EST sequence (ptτL BQ). Figure 3 shows the enhanced growth of transgenic hybrid aspen: the cumulative shoot elongation (upper diagram) and diameter growth (lower diagram) of various transgenic GA 20-oxidase over-expressing lines, after generation from tissue culture. potting and cultivation in a growth chamber for seven weeks (at time zero). Figure 4 is a photograph showing control and transgenic (line 1 1 ) plants after 12 w eeks in the growth chamber. Figure 5 illustrates the effects of GA 20-oxidase over-expression on (A) Cell lengths. ( B) cell numbers per internode. (C) number of xylem fibres and (D) xylem fibre length. Cell lengths and cell numbers were measured in fully elongated mtemodes of actively growing plants as was number of xylem fibres. Data for the GA 20-oxidase ov er-expressing lines are means for nine independently generated lines, respectively. Fibre length values for non-elongating plants represent means for three independently generated lines. The vertical bars represent SE. Figure 6 shows that there is enhanced diameter growth of transgenic hybrid aspen also in the second growth season. The cumulative diameter growth of control (\VT) and line 4 and 13 was determined under a period of 2.5 month, beginning after breaking ot dormancv. Description of the invention The present inv entors have surpnsmglv sho n that ectopic ov er-expression ot a GA 20-oxidase gene in trees, here exemplified by a temperate-zone deciduous tree, results in significant changes in giowth late stem volume and xvlem fibre length \ cDN λ sequence (SEQ ID NO 1 ) homologous to the 4ι abidopsis thahana G A. 20 oxidase sequence X83379 ( EMBL accession number) encoding a polypetide exhibiting GA 20 oxidase activ ity w as used Thus, the results obtained by the present inventors show that genetic modification ot GA le els in trees can be used to modify traits that are extremelv important tor the forest pulp and paper industries The results show that an endogenous elev ation ot the biologically activ e gibberellms GA and G A , in trees w ill accelerate growth This w as demonstrated bv expressing the 4tG420oxl gene from 4> abidopsis in hybπd aspen. Populus ti emula \ tremuloides. resulting in trees with faster height and diameter growth, larger leaves, longer xylem fibres and increased biomass This phenotype is partly reminiscent ot traits prev lously observed in transgenic Ai abidopsis plants over-expressing the same GA 20-oxidase (Coles. J P et al Modification ot gibberellin production and plant development in Arabidopsis by sense and antisense expression ot gibberellm 20-oxidase genes Plant J 17. 547-556 ( 1999) Such plants had longer hypocotyls. larger rosette lea es longer petioles, and accelerated tlow eπng compared to wild type plants However, studies regarding biomass increase and anatomical changes in transgenic 4ι abidopsis ha e not been reported It is highly doubtful if an increased biomass ould be achieved in transgenic 4ι abidopsis There have been suggestions that the GA 20-oxidase is a key enzyme in the regulation ot GA controlled growth [Hedden. P & Kamiya. Y Gibberellin biosynthesis Enzymes, genes and their regulation Annu Biol 48. 431-460 ( 1997)] The tact that transcπption of the GA 20-oxidase gene is feedback regulated by the action of GAs. suggests that constitutive over-expression ot the G A 20- oxidase gene will disturb the endogenous regulation of GA homeostasis Here, over- expression of GA 20-oxidase in hybπd aspen resulted in an increase m C, q -GAs involved in both the early 13-hydroxylatιon pathway and the non- 13-hydroxylatιon i path ay ( Fig 1 ) Thus, there as no consistent difference in le els between G A, and GA 4 . both ot hich are biologically activ e in Pomilus The present results theretore are in agi eement w ith prev ious G measurements on transgenic GA 20-oxidase overproducing 4ι abidopsis plants Furthermore, in both hvbπd aspen and [ι abidopsis thei e was a diamatic increase in the le els ot the deactiv ated end-product. GA, (Table 1 ) This has been suggested to be due to a slow er turnover ot GA than GA The relatively low lev els of the other end-product. G A 4 . formed by the non- 13- hydroxylation pathway can be explained bv a more rapid turnover ot this compound than GA, In contrast to the C -GAs. a decrease in lev els ot C -GAs w as observ ed in the transgenic plants This is consistent with the fact that C -GAs are. by definition substrates tor the GA 20-oxidase (Table 1 ) It is surprising that the elevated GA 20-oxidase expression has such marked effects in Populus tiemula \ emuloides This suggests that constitutive over-expression ot the GA 20-oxidase gene disturb the endogenous regulation of GA homeostasis In the present study the inventors found differences in GA levels between leaf and stem tissues, increases in lev els ot the activ e GA, and GA 4 being highest in stem tissue This could have been due to the 4ιG gene being expressed most strongly in the stem In hvbπd aspen, howev er, the CaiVIV 35S promoter is generally slightly more activ e in leaves than in the stem, although exceptions are known to occur Theretore. the higher GA, and GA 4 lev els in the stem are more likely due to differences in transport of GAs or in the biosynthesis of GA precursors However, it can not be ruled out that there may be a difference in GA 3β-hydroxylase or GA 2β-hydrox lase activity in the leaves In 4ι abidopsis it has been shown that 2β-hydroxylatιon is activated at the transcπptional lev el by acti e GAs The observ ed increase m height growth (Figure 3) in the transgenic GA 20- oxidase Populus trees was apparently not a consequence of increased cell elongation, as no significant changes in cell lengths were found (Figure 5A) However, observed differences in numbers ot cells per fully elongated mternode clearly indicate that elev ated GA levels affect cell div isions in stem tissue (Figure 5) This is an interesting aspect of the present invention, which opens up many possibilities of influencing the production of fibrous raw mateπal and biomass for different purposes. Although GAs hav e been found to affect cell elongation, it is also known GAs can induce mitotic activity in the subapical region of the stem It must be emphasised, howev er, that although samples were taken for cell length measurements from the same position for both control and transgenic plants (as defined by the number of internodes from the apex), it can not be ruled out that the cells in the transgenic plants could have been at an earlier developmental stage than in wild type plants, and they could still have been elongating. It has also been suggested that the action of GAs in meπstematic tissues extends the elongation zone of the organ being formed. ' 1 able 1. Concentration (ng g ' liesh weight) t)l GAs m stem/leal tissue from internodes 7 and 8 (see text foi details) ol tidiisgeniL hybnd aspen expic siiig ti e mji u xi cue ivieciii oi unee iiiueμeiiu cut mcdsuieiiie ins ii u — nυi JClCOldUIC Genotype GA, GA„ GA,, GA, GA,„ GA, GA, GA,, GA ( ontiol 578 15 322/041 315/121 n d In ά 045/043 084/088 063/063 163/123 150/94 I ni 1 1 16/079 029/028 085/055 320/064 536/144 971403 202/129 500/378 586/407 I nic 2 302/090 104/125 55/059 434/124 369/284 904/660 102/247 8 λlh 94 774/438 I me 4 106/071 027/024 081/063 523/086 503/284 11 3/476 117/1 58 784/478 532/529 I nic 7 100/07 029/028 121/060 538/078 474/236 207/629 140/135 603/469 684/327 I mo 9 249/073 062/026 1 19/056 567/073 600/216 970/513 542/235 1 46/4 1 700/496 1 me 11 167/062 057/028 204/060 431/142 577/331 107/530 142/181 943/428 726/426 I me π 091/078 026/025 090/064 658/064 164/345 198/646 293/322 798/385 759/551 I me 14 126/064 027/025 062/055 32/041 401/148 699/236 116/1 38 ι 52/469 786/375 Line 1 182/060 - 557/074 253/257 3 14/258 258/162 753/502 576/482 Studies inv olving the application ot plant hormones have show n that GAs can increase cambial activ ity in trees, especially in conjunction with IAA. Here, the engineered increase in GA lev els resulted in taster stem diameter gro th (Fig 4) An effect ot elev ated GA levels on secondary grow th was also observed w ith the increase in number ol xylem fibre cells and xylem fibre length (Fig. 5C. D). The length increase measured in the expeπments is about 8 ° 0 which must be held to be a significant increase, hav ing consequences tor the fibre properties and thus the potential uses tor the fibrous raw mateπal. This is consistent with application studies with both GAs and inhibitors ot GA bios} nthesis In Populus. fibre and v essel lengths increase in the transition zone betw een juv enile and mature w ood Theretore. the time it takes for a cell to mature within the cambial region, l e. the time spent in the different differentiation zones, will ultimately determine its size and cell w all thickness. If the action of GAs like GA, and/or G A, extends this transition time, this would explain both the increased fibre length in the mature parts of the transgenic plant and the lack of differences in xylem fibre length between transgenic and control plants in young stem tissue. The increase in leaf size in the transgenic plants was a result of an increase in both radial and longitudinal elongation (Table 2). At earlier stages the longitudinal growth w as more pronounced, resulting in long, narrow leaves. However, at later stages the leaf morphology became similar to control plants with a similar leaf width to length ratio. It has been shown that GAs promote leaf elongation e g in garden pea. The very long and narrow leaves observed in constitutiv e GA signal mutants, like sin mutants of barley and spλ mutants of Arabidopsis also indicate that GAs promote leaf elongation Howev er, little is still known on the more specific regulatory roles of GAs in leaf development. In Arabidopsis. leaf blade expansion is regulated by at least two independent and polaπsed processes length and width development, with ROTL ' DIFOLIA and ANGUSTIFOL1A genes playing specific roles I f is not known whether GAs modulate the cellular responses to these genes. However, it is noteworthy that leaf phenotypes m the transgenic GA 20-oxidase aspen change duπng development. suggesting that the two developmentally separate processes of leaf length and width growth are both affected bv GAs. but at different stages. Table 2. Moφhological characterisation of wild type and transgenic hybrid aspen expressing the AtGAJOoxl gene. The numbers of plants used for the measurements were 10.5 and 6 for the control, line 2. and line 11. respectively. In the biomass detemiination the numbers of plants sampled were " \ 2.3 and 23 for the control, line 2. line 11. and all sampled transgenics. pooled, respectively. For all statistical analyses ANOVA was used to compare the plants with respect to genotype. Statistically significant difference are indicated at the 1% (*) and 5 °o (**) probability levels (Fisher ' s PLSD). Genotype Control Line 2 Line 11 Internode length (cm =SE) 2.19=0.05 3.06=0.09* 3.07=0.09* Leaf length and width (cm ±SE) Leaf length 10 8.34 ±0.36 9.52=0.38** 9.17=0.28 Leaf width 10 7.25 ±0.41 6.28=0.20 5.85 ±0.24** Leaf length 14 9.52 ±0.45 11.18=0.60** 10.78 ±0.41 Leaf width 14 8.00 ±0.38 8.35 ±0.39 8.52 ±0.25 Leaflength lS 9.90±0.79 11.06=1.00 11.68 -0.56 Leaf width 18 7.48 ±0.64 8.80=0.47 8.88 ±0.43 Leaf length 20 7.75=0.34 11.94=0.44* 11.25 ±0.48* Leaf width 20 6.07=0.46 9.44=0.41* 8.85 ±0.41* Dry Weight Biomass (g) Control Line 2.11 Transgenes Leaf 5.45 ±0.51 6.38=0.58 6.14=0.28 Stem 4.58 ±0.43 10.42=1.01* 10.34 ±0.49* Root 43.05±4.71 38.36=2.63 37.81 ±2.06 Fresh Weight Biomass (g) Leaf 17.10=1.63 23.10=2.01** 22.05 ±0.91** Stem 12.04 = 1.19 31.39=2.88* 30.58=1.28* Biomass measurements in transgenic and w ild type plants revealed that an increase in GA 20-o.xιdase activ ity leads to a general increase in grow th. This effect as especially pronounced in the stem, indicating that GAs hav e a strong effect on stem grow th Spraying e.xpenments w ith v aπous GAs have prev iously show n that these hormones tend to increase shoot biomass in Populus at the expense of root grow th. Reductions in root formation due to increased GA levels in roots hav e earlier been show n in trees. In this study , poor root initiation w as the major problem for survival of the transgenic GA 20-oxidase plants in tissue culture and when planting in soil. At later developmental stages, root dev elopment w as still affected, but to a lesser degree (Table 2). It has also been show n that the effect of GAs on rooting v anes ith the stage of root development In conclusion, the present inventors have here demonstrated the important role of GA 20-oxidase activity in GA-controlled growth in Populus. It is surpnsing that the effects, hitherto only achieved, and in some cases only partially achieved, by external application, e.g. spraying, can be achieved by endogenous expression. It is also suφπsing that the mventiv e method can be applied to a higher species, here represented by hybπd aspen. Populus tremula \ P tremuloides One important advantage of the present invention is thus that it may make it possible to reduce or eliminate the use of growth influencing chemicals in forestry The present invention opens up ways to genetically engineer trees to grow faster and produce more biomass simply by increasing endogenous GA lev els. Interestingly, fibre lengths also increase as a result of the over-expression of the GAs. These results have both practical implications (assisting in the production of modified trees of interest to the pulp, paper and forest industries), and scientific importance, by allowing previously impossible studies on the mechanisms whereby GAs control growth and development in trees. Examples Experimental Protocol: Plant \ eetor Construction The 4ι abidopsis G 4 20-o\ιdase cD A construct cloned in bluescπpt pAt2301 [Phillips. A.L. et al Isolation and expression of three gibberellm 20-oxidase cDNA clones from Arabidopsis. Plant Physiol. 108. 1049- 105 7 ( 1995)]. recently renamed 4tGA20oxl [Coles. J P. et al. Modification of gibberellin production and plant development in Arabidopsis by sense and antisense expression of gibberellm 20-oxidase genes. Plant J 17. 54^-556 ( 1999)] was obtained as a gift from P Hedden. An upstream ATG sequence, preceding the translational start of the GA 20- oxidase enzyme, was removed by m vitro mutagenesis. A forward pπmer matching the 5 ' -end of the GA 20 oxidase gene, but lacking the extra ATG and carrying a Bam HI and Xba I site, and a reverse pπmer spanning an internal Hind III site in the GA 20 oxidase gene, were used in a PCR reaction. L sing AtGA20oxl as a template, a PCR product w as obtained and subcloned into the pOK.12 vector, generating plasmid pOK12.GA20ox5 " . An entire GA 20-oxidase cDNA lacking the extra ATG was then reconstituted, by digesting AtGA Ooxl isolating a fragment containing the 3 ' -end of the GA 20-oxidase gene, and hgation of this fragment into pOK12.GA20ox5 " . giving plasmid pOK.12 AtGA20ox l . The GA 20-oxidase cDNA was then isolated from pOK12 AtGA20ox l . by digestion with Bam HI. and hgated in sense oπentation into the Bam HI site of the binary v ector pPCV702.kana [Walden. R.. oncz. C. & Schell. J. Methods Viol. Cell. Biol. 1. 175- 194 ( 1990). The use of gene vectors in plant molecular biology. Methods Mol Cell Biol. 1. 175- 194 ( 1 990)]. subjecting it to the control of the CaMN 35S promoter. Plant Transformation and Growth Conditions Hybπd aspen Populus tremula L. x P tremuloides Michx. clone T89 was transformed and regenerated essentially as previously descπbed [Νilsson. O. et al. Spatial pattern of cauliflower mosaic v irus 35S promoter-luceferase expression m transgenic hybπd aspen trees monitored by enzymatic assay and non-destructive imaging T/ ansgenic Reasei ch 1 209-220 ( 1992)] Out ot 14 independent lines 10 w ere selected and multiplied by in o shoot culture on half-strength MS-medium containing minerals and v itamins [Vlurashige T & Skoog F A rev ised medium tor rapid giowth and bio-assay with tobacco tissue cultures Pln siologia Plantat ion 15 4 ^ 3-4 ^ 9 ( 1962)] Following loot initiation, the plants were dipped in Weibufix looting powder ( Svalot W eibull AB Hammenhog Sweden), and potted in a fertilised peat perhte mixture ' l ) and kept in a growth chamber at 18 C C under a photopeπod of 18 h and a relativ e humidity ot 90° o The photon flux density of the main light peπod ( 10 h) w s 1 75 umol m s ' at 400 - 7 50 nm (Osram Power Staw HQI-TS 400 W D lamps Osram Germanv ) and daylength extensions were giv en using low -intensity light (20 umol s ) Plants w ere watered daily, and repotted and fertilised with a complete nutπent solution ( SuperbaS Supra Hydro AB Landskrona Sweden) when needed After 108 days some plants w ere transferred to short photopeπod conditions ( 10 h) to induce growth cessation These plants were then cold acclimated and kept dormant at 8°C for 4 weeks, at which point samples ere taken tor fibre length measurements Growth Measurements and Sampling At se en w eeks of age. the plants were marked at an actively growing mternode at about the same position m all plants This w as used as a reference point for diameter growth measurements and for counting the internodes The diameter, height and number ot internodes of the plants were measured every 3' ' to 4' h day The number ot internodes w as counted from the top to the reference point the first mternode being defined as the one below the uppermost leaf at least 1 cm long Plants grown under long days ere harvested after 100 days, using seven control plants and 25 plants representing nine transgenic individual lines (line 6 was excluded) Internodes 7 and 8. with the upper leaves included, w ere sampled tor GA analysis and leaves from nodes 9 and 10 for northern analysis All tissues were frozen in liquid N, immediately after sampling For anatomical studies, the length of internodes 15 and 16 were measured, then excised and fixed immediately in F AA All parts remaining after sampling were separated into leaf. stem ana root fractions and used for fresh weight biomass determinations Dry weight w as determined after drying the samples at 55°C for 5 days Dormant plants consisting ot three controls and fiv e transgenes ( representing lines 2 1 1 and 14 ) w ere sampled at internodes 1. 10 and 20. counted from the leterence point These samples w ere used tor maceration and subsequent fibre length measurements Anatomical Characterisation Following conv entional chemical fixation samples ere embedded in LR White [Regan. S . Bourqum. V . Tuominen. H & Sundberg. B Accurate and high resolution in situ hybπdization analysis ot gene expression in secondary stem tissues. Plant J 19. 363-369 ( 1999)] Longitudinal sections tor cell counts (mternode 15 and 16) and transv erse sections tor fibre count ( mternode 30). 2 μm thick, were obtained and stained w ith toluidine blue Numbers ot epideπnal and pith cells in 1 mm of the longitudinal sections w ere determined for 3 sections per mternode Cell length and number ot cells per mternode were calculated as the av erages found in mtemodes 15 and 16 The number of xylem fibres was counted in three separate radial files (from pith to cambium) per indiv idual and the av erage tor each genotype (line 4. 1 1. 13 and control) was calculated Fibre Length Measurements For fibre length determination, tπmmed pieces ot outer xylem from the selected internodes w ere prepared The samples were macerated by boiling in a solution of 10% hydrogen peroxide and 50° o glacial acetic acid for 4-6 h WTien totally bleached, they were πnsed with distilled water three times, neutralised with sodium carbonate and washed again in w ater Finally, the fibres w ere separated from each other by shaking in water and measured using a fibre analyser (KAJAANI FiberLab, \ almet Automation ajaam Ltd. ajaam. Finland) On average, the lengths of 30 800 fibres per sample were measured. Northern Analysis Total RNA was extracted from leaves using a chloroform and hexadecy ltπmethylamonium bromide based method according to Chang et al [Chang. S . Puryear. J & Cairney. J A simple and efficient method for isolating RNA from pine trees Plant Viol Biol Rep 1 1 1 13-1 16 ( 1993 )] About 18 ug ot total RNA per sample as separated in a foπnaldehyde agarose gel according to Sambrook et al [Sambrook. J.. Fπtsch. E. & Mamatis. T Molecular Cloning 4 Laboraton Manual. Cold Spπng Harbor Laboratory Press. New York. 1989]. and blotted on to a nylon Hybond-N membrane (Amersham. Little Chalfont. UK) according to the manufacturer ' s protocol. A Bam HLXho I fragment recovered from pOK.12 AtGA20ox l and an ubiquitin-hke Expressed Sequence Tag ( EST A046p57) obtained in the Populus sequencing project [Sterky. F et al Gene discovery in the wood-forming tissues ot poplar: Analysis of 5.692 expressed sequence tags. Proc. Natl. Acad Sci I SA 95. 13330- 13335 ( 1998)] were used as probes for analysis of ectopic AtGAJOox I and reference gene expression. respectively Hybπdisation was performed in Church buffer [Church. G.M. & Gilbert. W.. Genomic sequencing. Proc \atl 4cad. Sci I SA %\ 1991 - 1995 ( 1984)] at 65=C overnight. The membrane was w ashed at 65 C C. two times three minutes in each of solution: 2 x SSPE. 0 1 % SDS: 1 x SSPE. 0. 1 % SDS: 0.5 x SSPE. 0.1 % SDS and 0.1 x SSPE. 0.1 % SDS. To visualise the expression patterns, the membrane was exposed to a phosphor imager (Molecular Imager GS-525. Bio-Rad Laboratoπes. Hercules. CA. USA) overnight. Quantification of Gibberellins Samples of 200-300 mg were ground in liquid nitrogen to a homogenous powder, and GAs were analysed as earlier descπbed by Peng et al. [Peng. J.R.. Richards. D.E.. Moπtz. T.. CanoDelgado, A. & Harberd. N P.. Extragemc suppressors of the Arabidopsis gai mutation alter the dose-response relationship of diverse gibberellin responses. Plant Pln siol. 1 19. 1 199- 1207 ( 1999)] with a few modifications. Volumes of the solvents used for extraction and partitioning, and that of the anion- exchange column, were reduced. Furthermore, the samples ere methylated pπor to the HPLC step, and finally they were analysed by GC 'MS-selected reaction monitoπng (SRiM) using a JEOL SX SX102A four sector mass spectrometer (JEOL. Tokyo. Japan). [ 2 H,]-GAs were used as internal standards. Results Generation of transgenic GA 20-oxidase hybrid aspen Out of 14 independently transtoπned lines. 10 were regenerated and selected for further analysis Southern analysis show ed that all the selected transgenic lines have one to three copies of the l gene inserted into their genomes, except for line 6. which has at least 4 copies (data not sho n) Northern analysis of RNA isolated from leaf tissue showed that the strongest expression of the AtGA20oxl gene was in lines 2. 7. 9. 14 and 15 Expression was slightly lower in lines 1. 4. 1 1 and 13. while expression was almost undetectable in line 6 (Fig 2) In general, the transgenic lines showed poorer rooting than the control plants, both under tissue culture conditions and when potted in soil When potted, for example. 100% of the control plants survived, compared to only 32% of the transgenic plants. GA levels GA content was determined both in leaves and internodes ot actively growing tissue. Transgenic plants showed high lev els of the 13-hydroxylated C μ ,-GAs (GA :( . GA, and GA and the non 13-hydroxylated C ,-GAs (GA, GA 4 and GA, 4 ) in both stem and leaves (Table 1 ) The increase ov er wild type levels was more pronounced in stem tissue than in leaf tissue. The lev els of the biologically activ e GA, and GA j in stem tissues ot lines were 22- and 24- fold higher, respectively, than in the control Furthermore, all transgenic lines showed lower levels of the substrates for the GA 20- oxidase. For example GA ι and GA. levels in stem tissues of line 7 were 17 % and 9 % of the contents in the control, respectively Shoot growth and morphology All transgenic lines showed faster than wild type height and diameter growth (Fig 3). giving the trees a characteπstic phenotype (Fig 4) Although levels of GA 20- oxidase mRNA vaπed between the transgenic lines (Fig. 2) there were no stπct correlation between expression levels and growth. A detailed study of two of the transgenic lines (Table 2) rev ealed that the difference in height growth was pπmaπly due to differences in mternode lengths No statistically significant differences in the number of nodes ( leav es) were observ ed ( data not shown). However, leaf dev elopment was different in the transgenic lines oung expanding leaves had a different moφhology and a higher leaf length to w idth ratio than leaves of control plants (Table 2) After node 14. I e. in the fully expanded leav es, there was no significant difference in this oφhυlogical ratio, but there was still a clear difference in leaf size between control and transgenic plants. The transgemcs had longer and broader leaves and. consequently, higher mean leaf fresh weights (Table 2). Furthermore, the petioles were longer in the transgenic lines than m the control (data not shown). There was a significant difference in shoot biomass between transgenic and control plants when measured either on a fresh or dry weight basis (Table 2) The transgenic plants had 64% higher shoot dry w eight, on average, than the control plants. This difference was especially pronounced m the stem, where the transgenic plants had 126 % higher dry w eight than the control plants Consequently, the transgenic plants also had an altered root:shoot weight ratio, of only about 2: 1 compared to 4- 1 for control plants. Although the initial rooting capacity was lower in the transgenic lines, there was no significant difference in root dry weight between transgenic and control plants (Table 2) Cell length and cell numbers in transgenic plants Expression of the AtGA20oxJ gene did not effect the lengths of epidermal and pith cells in presumably fully elongated internodes (see Expeπmental Protocol for definition) in the actively growing transgenic lines (Figure 5A). However epidermal and pith cell numbers in these elongated internodes were approximately 55 % higher than in the wild type control (Figure 5B). Cambial activ lty was also determined by counting numbers of xylem fibre cells in transverse sections (Fig. 5C). The transgenic lines show 71 % increase in number of xylem fibre cells compared to the control plants Xylem fibres were sampled at three different positions in dormant plants and their lengths were measured. No differences in fibre length from different heights in the trees w ere detected (data not shown), but the xylem fibres were approximately 8 % longer in the transgenic lines than in the control plants (Fig. 5C). Although the inv ention has been descπbed w ith regard to its preferred embodiments, which constitute the best mode presently know n to the inventors, it should be understood that v aπous changes and modifications as would be obvious to one hav ing the ordinary skill in this art may be made w ithout departing from the scope of the inv ention as set forth in the claims appended hereto SΞCϋΞNCΞ LISTING <11 C > Swetree C-encmics A3 < 120 > Trε sgemc trees and methods for tiieir production <130> H42953 <140> <141> <160> 2 <170> Patentln Ver . 2.1 <210> 1 <211> 1253 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Arabidopsis GA 20-oxidase cDNA construct <400> 1 aatctctcaa aatggccgta agtttcgtaa caacatctcc tgaggaagaa gacaaaccga SO agctaggcct tggaaatatt caaactccgt taatcttcaa cccttcaatg cttaaccttc 120 aagccaatat cccaaaccaa ttcatctggc ctgacgacga aaaaccttcc atcaacgttc 180 tcgagcttga tgttcctctc atcgaccttc aaaaccttct ctctgatcca tcctccactt 240 tagatgc tc gagactgatc tctgaggcct gtaagaagca cggtttcttc ctcgtggtca 300 atcacggcat cagcgaggag cttatatcag acgctcatga atacacgagc cgcttctttg 360 atatgcctct ctccgaaaaa cagagggttc ttagaaaatc cggtgagagt gttggctacg 420 caagcagttt caccggacgc ttctccacca agcttccatg gaaggagacc ctttctttcc 480 ggttttgcga cgacatgagc cgctcaaaat ccgttcaaga ttacttctgc gatgcgttgg 540 gacatgggtt tcagccattt gggaaggtgt atcaagagta ttgtgaagca atgagttctc 500 tatcattgaa gatcatggag cttctcgggc taagtttagg cgtaaaacgg gactacttta 660 gagagtt tt cgaagaaaac gattcaataa tgagactgaa ttactaccct ccatgtataa 720 aaccagatct cacactagga acaggacctc attgtgatcc aacatctctt accatccttc 780 accaagacca tgttaatggc cttcaagtct ttgtggaaaa tcaatggcgc tccattcgtc 840 ccaaccccaa ggcctttgtg gtcaatatcg gcgatacttt catggctcta tcgaacgata 900 gatacaagag ctgcttgcac cgggcggtgg tgaacagcaa gagcgagagg aagtcacttg 960 cattcttctt gtgtccgaaa aaagacagag tagtgacgcc accgagagag cttttggaca 1020 gcatcacatc aagaagatac cctgacttca catggtctat gttccttgag ttcactcaga 1080 aacattatag agcagacatg aacactctcc aagccttt c agattggctc accaaaccca 1140 t taagaaat aaaatattca tgtcttgtct tgttagttac tagtatcttc tttatatttc 1200 atgtatgtat atggtaatag gcaataacac cttttagcat ctcaaaaaaa aaaaaaaaa 1259 Leu Gly Val Lys Arg .Asp Tyr Phe Arg Glu Phe Phe Glu Glu Asn Asp 213 215 220 Ser lie Met Arg Leu Asn Tyr Tyr Pro Pro Cys lie Lys Pro Asp Leu 225 230 235 240 Thr Leu Gly Thr Gly Pro His Cys Asp Pro Thr Ser Leu T r lie Leu 245 250 255 His Gin Asp His Val Asn Gly Leu Gin Val Phe Val Glu Asn Gin Trp 260 265 270 Arg Ser lie Arg Pro Asn Pro Lys Ala Phe Val Val Asn lie Gly Asp 275 280 285 Thr Phe Met A.ia Leu Ser Asn Asp Arg Tyr Lys Ser Cys Leu His Arg 290 295 300 Ala Val Val Asn Ser Lys Ser Glu Arg Lys Ser Leu Ala Phe Phe Leu 305 310 315 320 Cys Pro Lys Lys Asp Arg Val Val Thr Pro Pro Arg Glu Leu Leu Asp 325 330 335 Ser lie Thr Ser Arg Arg Tyr Pro Asp Phe Thr Trp Ser Met Phe Leu 340 345 350 Glu Phe Thr Gin Lys His Tyr A.rg Ala Asp Met Asn Thr Leu Gin Ala 355 360 365 Phe Ser Asp Trp Leu Thr Lys Pro lie 370 375 <210 2 <211? 37" <212> PRT <213 ? .Artificial Sequence <220 > <223> Description cf Artificial Sequence: AraEidcpsis GA 20-oxidase cDNA construct <400> 2 Met Ala Val Ser Phe Val Thr Thr Ser Pro Glu Glu Glu Asp Lys Pro 5 10 15 Lys Leu Gly Leu Gly Asn He Gin Thr Pro Leu He Phe Asn Pro Ser 20 25 30 Met Leu Asn Leu Gin Ala Asn lie Pro Asn Gin Fhe He Trp Pre Asp 35 40 45 Asp Glu Lys Pro Ser He Asn Val Leu Glu Leu Asp Val Pro Leu He 50 55 60 Asp Leu Gin Asn Leu Leu Ser Asp Pro Ser Ser Thr Leu Asp Ala Ser 65 70 75 80 Arg Leu He Ser Glu Ala Cys Lys Lys His Gly Phe Phe Leu Val Val 85 90 95 A.sn His Gly He Ser Glu Glu Leu He Ser Asp Ala His Glu Tyr Thr 100 105 110 Ser Arg Phe Phe Asp Met Pro Leu Ser Glu Lys Gin Arg Val Leu Arg 115 120 125 Lys Ser Gly Glu Ser Val Gly Tyr Ala Ser Ser Phe Thr Gly Arg Phe 130 135 140 Ser Thr Lys Leu Pro Trp Lys Glu Thr Leu Ser Phe Arg Phe Cys Asp 145 150 155 150 Asp Met Ser Arg Ser Lys Ser Val Gin Asp Tyr Phe Cys Asp Ala Leu 155 170 175 Gly H s Gly Phe Gin Pro Phe Gly Lys Val Tyr Gin Glu Tyr Cys Glu 130 135 ISO .Ala Met Ser Ser Leu Ser Leu Lys He Met Glu Leu Leu Gly Leu Ser 195 200 205

Claims: Claims 1 A transgenic woody plant exhibiting at least one improv ed growth parameter characterized in that a DN A sequence coding tor the expression ot a polv peptide exhibiting G A 20-oxidase activ ity is functionally inserted in the plant genome 2 The transgenic w oody plant according to claim 1. characterized in that the improved growth parameter is one of increased growth rate, biomass production and/or xvlem fibre length 3 A transgenic woody plant ot the Populus species exhibiting at least one improved growth parameter, characterized in that a DNA sequence coding for the expression ot GA 20-oxidase is functionally inserted in the plant genome 4 The transgenic Populus sp plant according to claim 3. characterized in that the improv ed growth parameter is one of increased growth rate, biomass production and/or xylem fibre length 5 A transgenic Populus sp plant belonging to the species Populus tremula x P tremuloides exhibiting at least one improved growth parameter, characterized in that a DNA sequence coding for the expression ot GA 20-oxidase is functionally inserted in the plant genome 6 The transgenic Populus sp plant according to claim 5. characterized in that the improv ed growth parameter is one ot increased growth rate, biomass production and/or xylem fibre length 7 Seeds of a transgenic plant according to any one of claims 1 - 6 8 Cells of a transgenic plant according to any one of claims 1 - 6 9 Propagating mateπal of a transgenic plant according to any one ot claims 1 - 6 10. A transgenic plant according to any one of claims 1 - 6. characterized in that the DNA sequence coding for the expression of a polypeptide exhibiting GA 20- oxidase activity is SEQ. ID. NO. 1 or a homologue thereof. 1 1. Seeds of a transgenic plant according to claim ~ . characterized in that the DNA sequence coding for the expression of a polypeptide exhibiting GA 20-oxidase activity is SEQ. ID. NO 1 or a homologue thereof. 12. Cells of a transgenic plant according to claim 8. characterized in that the DNA sequence coding for the expression of a polypeptide exhibiting GA 20-oxidase activity is SEQ. ID. NO. 1 or a homologue thereof. 13. Propagating mateπal of a transgenic plant according to claim 9. characterized in that the DNA sequence coding for the expression of a polypeptide exhibiting GA 20-oxidase activity is SEQ. ID. NO. i or a homologue thereof. 14. A method of increasing at least one growth parameter in trees, characterized in that a DNA sequence coding for the expression of a polypeptide exhibiting GA 20- oxidase activity is functionally inserted in the plant genome. 15 The method according to claim 14. characterized in that the growth parameter is one of growth rate, biomass production and xylem fibre length. 16. The method according to claim 14. characterized in that a DNA sequence coding for the expression of a polypeptide exhibiting GA 20-oxidase activity is the sequence disclosed as SEQ. ID. NO. 1 or a homologue thereof

Inventors: ERIKSSON MARIA, ISRAELSSON MARIA, MORITZ THOMAS, OLSSON OLOF, ERIKSSON, MARIA, ISRAELSSON, MARIA, MORITZ, THOMAS, OLSSON, OLOF
Applicants: ERIKSSON MARIA, ISRAELSSON MARIA, MORITZ THOMAS, OLSSON OLOF, SWETREE GENOMICS AB, ERIKSSON, MARIA, ISRAELSSON, MARIA, MORITZ, THOMAS, OLSSON, OLOF
Application year: 2001
Main classification: C12N15
IPC codes: A01H5, C12N5, C12N15