# Patent

## Title
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 (Populus tremula×P. tremuloides), improvements in valuable traits such as growth rate and biomass are obtained. In addition, these trees also have longer xylem fibres than unmodified wild type plants. Long fibers 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.

## Background
RELATED APPLICATIONS
This application is a continuation-in-part of U.S. patent application Ser. No. 10/221,296 filed Sep. 6, 2002, which is the National Stage of International Application No. PCT/SE01/00451, filed Mar. 2, 2001, which claims the benefit of U.S. Provisional Application Ser. No. 60/240,319, filed Oct. 13, 2000 and Swedish Patent application 0000751-8, filed Mar. 7, 2000, each of the foregoing which is hereby incorporated by reference.


FIELD OF THE INVENTION
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 have 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 dwarfed phenotype due to a reduction in internode length. The phenotype of such GA-deficient mutants can be completely restored by the application of an active GA. At the cellular level, GAs have been found to promote both cell division and cell elongation.
Biosynthesis of GAs in planta occurs through the isoprenoid pathway from mevalonic acid. Gibberellin levels are mainly regulated by transcriptional control of gibberellin biosynthesis genes. In particular, the multifunctional enzyme gibberellin 20-oxidase (GA 20-ox) is a key-enzyme in controlling GA biosynthesis (FIG. 1). It catalyses the stepwise conversion of the C-20 gibberellins, GA12/GA53, by three successive oxidations to GA9/GA20, which are the immediate precursors of the active gibberellins, GA4 and GA1, respectively. The expression of the GA 20-oxidase gene is down regulated by the action of GA1/4, suggesting that direct end-product repression is involved in regulation of the gene. In addition, some authors have suggested that GA 20-oxidase is photoperiodically regulated at the transcription 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 would have clear advantages. Using Arabidopsis as a model organism it has been shown that it is possible to change GA levels by modifying GA 20-oxidase enzyme levels and that this results in plants with altered growth and development patterns. Transgenic Arabidopsis expressing the GA 20-oxidase in a sense orientation shows earlier flowering and taller stems than wild type plants, whereas antisense plants have the reverse properties [Coles, J. P. et al. Modification of gibberellin production and plant development in Arabidopsis by sense and antisense expression of gibberellin 20-oxidase genes. Plant J. 17, 547-556 (1999)].
Modification of GA biosynthesis in 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 for improved methods for modification of the growth 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 of transgenic trees, exhibiting improved properties, such as increased growth rate, stem volume and xylem fibre length. Consequently, the objective of the present invention is to provide such improved methods and transgenic trees. Another objective 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-Oxidase confers a gibberellin-overproduction phenotype in Arabidopsis, Plant Physiol., 1998, vol. 118, p 773-781) that the level of active GAs were raised following overexpression of GA 20-oxidase in Arabidopsis thaliana. 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 of secondary thickening in 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-oxidase in Arabidopsis thaliana 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-oxidase over expression in a woody plant would be. Cell division in the cambial meristem in a woody plant is under the control of several different hormones and physical constraints not found in an annual plant such as Arabidopsis thaliana. 
It was therefore surprising that there would be increased cell elongation of cells (fibres) originating from the cambium, and that it would be an increase in biomass caused by increased cambial cell division. It would not be obvious to one skilled in the art to over express GA 20-oxidase to increase wood biomass and fibre elongation.
Increase in the diameter of tree stems occurs primarily from meristematic activity in the vascular cambium, a cylindrical lateral meristem located between the xylem and phloem of the stem, branches, and woody roots. Two types of cell division occur in the cambium: additive and multiplicative. Additive division involves periclinal 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 of fusiform initials that provide for circumferential expansion of the cambium. After xylem and phloem cells are cut off by the cambial mother cells, they differentiate in an ordered sequence of events that includes cell enlargement, secondary wall formation, lignification and loss of protoplasts. These events do not occur stepwise, but rather as overlapping phases.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide transgenic plants, seeds and plant cells, exhibiting improved growth parameters and in particular improved parameters of economical interest, such as increased growth rate, biomass increase and xylem fibre length. It is another object of the invention to provide a method for 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 of a polypeptide exhibiting GA 20-oxidase activity 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 description and accompanying examples and drawings, in which
FIG. 1 shows the biosynthetic pathways converting GA12 and GA53 to the biologically active products GA4 and GA1, and their deactivated catabolites, GA34 and GA8. GA 20-oxidase catalyses the oxidation at the C-20 carbon.
FIG. 2 shows a Northern analysis of ten GA 20-oxidase over-expressing lines (numbers 1 to 15) and the non-transformed control (C). 18 μg of total RNA was loaded from each sample, and probed with a labelled fragment isolated from Arabidopsis gibberellin 20-oxidase (AtGA20ox1) cDNA or from an endogenous ubiquitin (UBQ) EST sequence (pttUBQ).
FIG. 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).
FIG. 4 is a photograph showing control and transgenic (line 11) plants after 12 weeks in the growth chamber.
FIG. 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 internodes of actively growing plants as was number of xylem fibres. Data for the GA 20-oxidase over-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.
FIG. 6 shows that there is enhanced diameter growth of transgenic hybrid aspen also in the second growth season. The cumulative diameter growth of control (WT) and line 4 and 13 was determined under a period of 2.5 months, beginning after breaking of dormancy.



DESCRIPTION OF THE INVENTION
The present inventors have surprisingly shown that ectopic over-expression of a GA 20-oxidase gene in trees, here exemplified by a temperate-zone deciduous tree, results in significant changes in growth rate, stem volume and xylem fibre length. A cDNA sequence (SEQ. ID. NO. 1) homologous to the Arabidopsis thaliana GA 20 oxidase sequence X83379 (EMBL accession number) encoding a polypeptide exhibiting GA 20 oxidase activity was used. Thus, the results obtained by the present inventors show that genetic modification of GA levels in trees can be used to modify traits that are extremely important for the forest, pulp and paper industries.
The results show that an endogenous elevation of the biologically active gibberellins GA1 and GA4 in trees will accelerate growth. This was demonstrated by expressing the AtGA20ox1 gene from Arabidopsis in hybrid aspen, Populus tremula×tremuloides, resulting in trees with faster height and diameter growth, larger leaves, longer xylem fibres and increased biomass. This phenotype is partly reminiscent of traits previously observed in transgenic Arabidopsis plants over-expressing the same GA 20-oxidase (Coles, J. P. et al., Modification of gibberellin production and plant development in Arabidopsis by sense and antisense expression of gibberellin 20-oxidase genes. Plant J. 17, 547-556 (1999). Such plants had longer hypocotyls, larger rosette leaves, longer petioles, and accelerated flowering compared to wild type plants.
However, studies regarding biomass increase and anatomical changes in transgenic Arabidopsis have not been reported. It is highly doubtful if an increased biomass would be achieved in transgenic Arabidopsis. 
There have been suggestions that the GA 20-oxidase is a key enzyme in the regulation of GA controlled growth [Hedden, P. & Kamiya, Y. Gibberellin biosynthesis: Enzymes, genes and their regulation. Annu. Rev. Plant Physiol. Plant Mol. Biol. 48, 431-460 (1997)]. The fact that transcription of the GA 20-oxidase gene is feedback regulated by the action of GAs, suggests that constitutive over-expression of the GA 20-oxidase gene will disturb the endogenous regulation of GA homeostasis. Here, over-expression of GA 20-oxidase in hybrid aspen resulted in an increase in C19-GAs involved in both the early 13-hydroxylation pathway and the non-13-hydroxylation pathway (FIG. 1). Thus, there was no consistent difference in levels between GA1 and GA4, both of which are biologically active in Populus. The present results therefore are in agreement with previous GA measurements on transgenic GA 20-oxidase overproducing Arabidopsis plants. Furthermore, in both hybrid aspen and Arabidopsis there was a dramatic increase in the levels of the deactivated end-product, GA8 (Table 1).
This has been suggested to be due to a slower turnover of GA8 than GA1. The relatively low levels of the other end-product, GA34, formed by the non-13-hydroxylation pathway can be explained by a more rapid turnover of this compound than GA8. In contrast to the C19-GAs, a decrease in levels of C20-GAs was observed in the transgenic plants. This is consistent with the fact that C20-GAs are, by definition, substrates for the GA 20-oxidase (Table 1).
It is surprising that the elevated GA 20-oxidase expression has such marked effects in Populus tremula×tremuloides. This suggests that constitutive over-expression of 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 levels of the active GA1 and GA4 being highest in stem tissue. This could have been due to the AtGA20ox1 gene being expressed most strongly in the stem. In hybrid aspen, however, the CaMV 35S promoter is generally slightly more active in leaves than in the stem, although exceptions are known to occur. Therefore, the higher GA1 and GA4 levels 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-hydroxylase activity in the leaves. In Arabidopsis it has been shown that 2β-hydroxylation is activated at the transcriptional level by active GAs.
The observed increase in height growth (FIG. 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 (FIG. 5A). However, observed differences in numbers of cells per fully elongated internode clearly indicate that elevated GA levels affect cell divisions in stem tissue (FIG. 5). This is an interesting aspect of the present invention, which opens up many possibilities of influencing the production of fibrous raw material and biomass for different purposes.
Although GAs have 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, however, 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 intemodes 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 meristematic tissues extends the elongation zone of the organ being formed.







TABLE 1







Concentration (ng g−1 fresh weight) of GAs in stem/leaf tissue from internodes 7 and 8 (see text for details)


of transgenic hybrid aspen expressing the AtGA20oxl gene. Means of three independent measurements.
















Genotype
GA12
GA53
GA19
GA9
GA20
GA4
GA1
GA34
GA8





Control
5.78/1.15
3.22/0.41
3.15/1.21
n.d./n.d.
0.45/0.43
0.84/0.88
0.63/0.63
1.63/1.23
15.0/9.4 


Line 1
1.16/0.79
0.29/0.28
0.85/0.55
3.20/0.64
5.36/1.44
9.71/4.03
20.2/1.29
5.00/3.78
58.6/40.7


Line 2
3.02/0.90
1.04/1.25
3.55/0.59
4.34/1.24
3.69/2.84
9.04/6.60
10.2/2.47
8.37/5.94
77.4/43.8


Line 4
1.06/0.71
0.27/0.24
0.81/0.63
5.23/0.86
5.03/2.84
11.3/4.76
11.7/1.58
7.84/4.78
53.2/52.9


Line 7
1.00/0.73
0.29/0.28
1.21/0.60
5.38/0.78
4.74/2.36
20.7/6.29
14.0/1.35
6.03/4.69
68.4/32.7


Line 9
2.49/0.73
0.62/0.26
1.19/0.56
5.67/0.73
6.00/2.16
9.70/5.13
5.42/2.35
10.46/4.85 
70.0/49.6


Line 11
1.67/0.62
0.57/0.28
2.04/0.60
4.31/1.42
5.77/3.31
10.7/5.30
14.2/1.81
9.43/4.28
72.6/42.6


Line 13
0.91/0.78
0.26/0.25
0.90/0.64
6.58/0.64
16.4/3.45
19.8/6.46
29.3/3.22
7.98/3.85
75.9/55.1


Line 14
1.26/0.64
0.27/0.25
0.62/0.55
 3.2/0.41
4.01/1.48
6.99/2.36
11.6/1.38
5.52/4.69
78.6/37.5


Line 15
1.82/0.60
—
5.57/0.74
—
2.53/2.57
3.14/2.58
2.58/1.62
7.53/5.02
57.6/48.2





n.d. = not detectable.






Studies involving the application of plant hormones have shown that GAs can increase cambial activity in trees, especially in conjunction with IAA. Here, the engineered increase in GA levels resulted in faster stem diameter growth (FIG. 4). An effect of elevated GA levels on secondary growth was also observed with the increase in number of xylem fibre cells and xylem fibre length (FIG. 5C, D). The length increase measured in the experiments is about 8% which must be held to be a significant increase, having consequences for the fibre properties and thus the potential uses for the fibrous raw material.
This is consistent with application studies with both GAs and inhibitors of GA biosynthesis. In Populus, fibre and vessel lengths increase in the transition zone between juvenile and mature wood. Therefore, the time it takes for a cell to mature within the cambial region, i.e. the time spent in the different differentiation zones, will ultimately determine its size and cell wall thickness. If the action of GAs like GA1and/or GA4 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 was 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 constitutive GA signal mutants, like sln mutants of barley and spy mutants of Arabidopsis, also indicate that GAs promote leaf elongation. However, 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 polarised processes: length and width development, with ROTUNDIFOLIA and ANGUSTIFOLIA genes playing specific roles. It is not known whether GAs modulate the cellular responses to these genes. However, it is noteworthy that leaf phenotypes in the transgenic GA 20-oxidase aspen change during development, suggesting that the two developmentally separate processes of leaf length and width growth are both affected by GAs, but at different stages.







TABLE 2





Morphological characterisation of wild type and transgenic hybrid aspen


expressing the AtGA20oxl 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 determination the numbers of plants sampled


were 7, 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.

















Genotype











Control
Line 2
Line 11





Internode length
2.19 ± 0.05
3.06 ± 0.09*
3.07 ± 0.09*


(cm ± SE)







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


Leaf length 18
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*





Statistically significant difference are indicated at the 1%* and 5%** probability levels (Fisher's PLSD).






Biomass measurements in transgenic and wild type plants revealed that an increase in GA 20-oxidase activity leads to a general increase in growth. This effect was especially pronounced in the stem, indicating that GAs have a strong effect on stem growth. Spraying experiments with various GAs have previously shown that these hormones tend to increase shoot biomass in Populus at the expense of root growth. Reductions in root formation due to increased GA levels in roots have earlier been shown in trees. In this study, poor root initiation was 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 development was still affected, but to a lesser degree (Table 2). It has also been shown that the effect of GAs on rooting varies with 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 surprising 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 surprising that the inventive method can be applied to a higher species, here represented by hybrid 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 levels. 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
Example 1
Experimental Protocol:
Plant Vector Construction
The Arabidopsis GA 20-oxidase cDNA construct cloned in bluescript pAt2301 [Phillips. A. L. et al. Isolation and expression of three gibberellin 20-oxidase cDNA clones from Arabidopsis. Plant Physiol. 108. 1049-1057 (1995)], recently renamed AtGA20ox1 [Coles. J. P. et al. Modification of gibberellin production and plant development in Arabidopsis by sense and antisense expression of gibberellin 20-oxidase genes. Plant J. 17. 547-556 (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 in vitro mutagenesis. A forward primer 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 primer spanning an internal Hind III site in the GA 20 oxidase gene, were used in a PCR reaction. Using AtGA20ox1 as a template, a PCR product was obtained and subcloned into the pOK12 vector, generating plasmid pOK12.GA20ox5′. An entire GA 20-oxidase cDNA lacking the extra ATG was then re-constituted, by digesting AtGA20ox1, isolating a fragment containing the 3′-end of the GA 20-oxidase gene, and ligation of this fragment into pOK12.GA20ox5′, giving plasmid pOK12/AtGA20ox1. The GA 20-oxidase cDNA was then isolated from pOK12/AtGA20ox1, by digestion with Bam HI, and ligated in sense orientation into the Bam HI site of the binary vector pPCV702.kana [Walden, R., Koncz, C. & Schell, J. Methods Mol. Cell. Biol. 1. 175-194 (1990). The use of gene vectors in plant molecular biology, Methods Mol. Cell Biol. 1, 175-194 (1990)], subjecting it to the control of the CaMV 35S promoter.
Plant Transformation and Growth Conditions
Hybrid aspen Populus tremula L.×P. tremuloides Michx. clone T89 was transformed and regenerated essentially as previously described [Nilsson, O. et al. Spatial pattern of cauliflower mosaic virus 35S promoter-luceferase expression in transgenic hybrid aspen trees monitored by enzymatic assay and non-destructive imaging. Transgenic Research 1. 209-220 (1992)]. Out of 14 independent lines. 10 were selected and multiplied by in vitro shoot culture on half-strength MS-medium containing minerals and vitamins [Murashige. T. & Skoog. F. A revised medium for rapid growth and bio-assay with tobacco tissue cultures. Physiologia Plantarum 15. 473-479 (1962)].
Following root initiation, the plants were dipped in Weibufix rooting powder (Svalof Weibull A B. Hammenhög. Sweden), and potted in a fertilised peat: perlite mixture (5:1) and kept in a growth chamber at 18° C. under a photoperiod of 18 h and a relative humidity of 90%. The photon flux density of the main light period (10 h) was 175 μmol m−2 s−1 at 400-750 nm (Osram Power Staw HQI-TS 400 W/D lamps. Osram. Germany) and daylength extensions were given using low-intensity light (20 μmol−2 s−1). Plants were watered daily, and repotted and fertilised with a complete nutrient solution (SuperbaS. Supra Hydro A B. Landskrona. Sweden) when needed. After 108 days some plants were transferred to short photoperiod 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 were taken for fibre length measurements.
Growth Measurements and Sampling
At seven weeks of age, the plants were marked at an actively growing internode at about the same position in all plants. This was used as a reference point for diameter growth measurements and for counting the internodes. The diameter, height and number of internodes of the plants were measured every 3rd to 4th day. The number of internodes was counted from the top to the reference point: the first internode being defined as the one below the uppermost leaf at least 1 cm long. Plants grown under long days were 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, were sampled for GA analysis and leaves from nodes 9 and 10 for northern analysis. All tissues were frozen in liquid N2 immediately after sampling. For anatomical studies, the length of intemodes 15 and 16 were measured, then excised and fixed immediately in FAA. All parts remaining after sampling were separated into leaf, stem and root fractions and used for fresh weight biomass determinations. Dry weight was determined after drying the samples at 55° C. for 5 days. Dormant plants consisting of three controls and five transgenes (representing lines 2. 11 and 14) were sampled at internodes 1, 10 and 20, counted from the reference point. These samples were used for maceration and subsequent fibre length measurements.
Anatomical Characterisation
Following conventional chemical fixation samples were embedded in L R White [Regan, S., Bourquin, V., Tuominen, H. & Sundberg, B. Accurate and high resolution in situ hybridization analysis of gene expression in secondary stem tissues, Plant J. 19, 363-369 (1999)]. Longitudinal sections for cell counts (internode 15 and 16) and transverse sections for fibre count (internode 30), 2 μm thick, were obtained and stained with toluidine blue. Numbers of epidermal and pith cells in 1 mm of the longitudinal sections were determined for 3 sections per internode. Cell length and number of cells per internode were calculated as the averages found in intemodes 15 and 16. The number of xylem fibres was counted in three separate radial files (from pith to cambium) per individual and the average for each genotype (line 4, 11, 13 and control) was calculated.
Fibre Length Measurements
For fibre length determination, trimmed pieces of outer xylem from the selected internodes were prepared. The samples were macerated by boiling in a solution of 10% hydrogen peroxide and 50% glacial acetic acid for 4-6 h. When totally bleached, they were rinsed with distilled water three times, neutralised with sodium carbonate and washed again in water. Finally, the fibres were separated from each other by shaking in water and measured using a fibre analyser (KAJAANI FiberLab, Valmet Automation Kajaani Ltd. Kajaani, 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 hexadecyltrimethylamonium bromide based method according to Chang et al. [Chang, S., Puryear, J. & Carney, J. A simple and efficient method for isolating RNA from pine trees. Plant Mol. Biol. Rep. 11. 113-116 (1993)]. About 18 μg of total RNA per sample was separated in a formaldehyde agarose gel according to Sambrook et al. [Sambrook, J., Fritsch, E. & Maniatis, T. Molecular Cloning: A Laboratory Manual, Cold Spring 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 HI/Xho I fragment recovered from pOK12/AtGA20ox1 and an ubiquitin-like Expressed Sequence Tag (EST A046p57) obtained in the Populus sequencing project [Sterky, F. et al. Gene discovery in the wood-forming tissues of poplar: Analysis of 5.692 expressed sequence tags. Proc. Natl. Acad. Sci. USA 95. 13330-13335 (1998)] were used as probes for analysis of ectopic AtGA20ox1 and reference gene expression, respectively. Hybridisation was performed in Church buffer [Church, G. M. & Gilbert, W., Genomic sequencing, Proc. Natl. Acad. Sci. USA 81. 1991-1995 (1984)] at 65° C. overnight. The membrane was washed at 65° C. two times three minutes in each of solution: 2×SSPE. 0.1% SDS; 1×SSPE. 0.1% SDS; 0.5×SSPE. 0.1% SDS and 0.1×SSPE. 0.1% SDS. To visualise the expression patterns, the membrane was exposed to a phosphor imager (Molecular Imager GS-525, Bio-Rad Laboratories, Hercules, Calif., 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 described by Peng et al. [Peng, J. R., Richards, D. E., Moritz, T., CanoDelgado, A. & Harberd, N. P., Extragenic suppressors of the Arabidopsis gai mutation alter the dose-response relationship of diverse gibberellin responses, Plant Physiol. 119, 1199-1207 (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 were methylated prior to the HPLC step, and finally they were analysed by GC/MS-selected reaction monitoring (SRM) using a JEOL SX/SX102A four sector mass spectrometer (JEOL, Tokyo, Japan). [2H2]-GAs were used as internal standards.
Results
Generation of Transgenic GA 20-Oxidase Hybrid Aspen
Out of 14 independently transformed lines, 10 were regenerated and selected for further analysis. Southern analysis showed that all the selected transgenic lines have one to three copies of the AtGA20ox1 gene inserted into their genomes, except for line 6, which has at least 4 copies (data not shown). Northern analysis of RNA isolated from leaf tissue showed that the strongest expression of the AtGA20ox1 gene was in lines 2, 7, 9, 14 and 15. Expression was slightly lower in lines 1, 4, 11 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 of actively growing tissue. Transgenic plants showed high levels of the 13-hydroxylated C19-GAs (GA20. GA1 and GA8) and the non 13-hydroxylated C19-GAs (GA9. GA4 and GA34) in both stem and leaves (Table 1). The increase over wild type levels was more pronounced in stem tissue than in leaf tissue. The levels of the biologically active GA1 and GA4 in stem tissues of lines 7 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 GA12 and GA53 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 characteristic phenotype (FIG. 4). Although levels of GA 20-oxidase mRNA varied between the transgenic lines (FIG. 2) there were no strict correlation between expression levels and growth. A detailed study of two of the transgenic lines (Table 2) revealed that the difference in height growth was primarily due to differences in internode lengths. No statistically significant differences in the number of nodes (leaves) were observed (data not shown). However, leaf development was different in the transgenic lines. Young expanding leaves had a different morphology and a higher leaf length to width ratio than leaves of control plants (Table 2). After node 14, i.e. in the fully expanded leaves, there was no significant difference in this morphological ratio, but there was still a clear difference in leaf size between control and transgenic plants. The transgenics had longer and broader leaves and, consequently, higher mean leaf fresh weights (Table 2). Furthermore, the petioles were longer in the transgenic lines than in 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 weight, on average, than the control plants. This difference was especially pronounced in the stem, where the transgenic plants had 126% higher dry weight 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 AtGA20ox1 gene did not affect the lengths of epidermal and pith cells in presumably fully elongated internodes (see Experimental Protocol for definition) in the actively growing transgenic lines (FIG. 5A). However, epidermal and pith cell numbers in these elongated internodes were approximately 55% higher than in the wild type control (FIG. 5B).
Cambial activity 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 were 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 invention has been described with regard to its preferred embodiments, which constitute the best mode presently known to the inventors, it should be understood that various changes and modifications as would be obvious to one having the ordinary skill in this art may be made without departing from the scope of the invention as set forth in the claims appended hereto.
Example 2
The technique of Example I may be applied to any available polypeptide that exhibits GA 20-oxidase activity. GA 20-oxidases have been expressed in non-woody plants and have been shown to result in increased gibberellin levels in such plants, which demonstrate that the GA 20-oxidases will be function regardless of the source of the enzyme and regardless of the woody plant in which the enzyme is expressed. By way of example, the Arabidopsis GA 20-oxidase gene was overexpressed in Arabidopsis (Coles et al. 1999; cited in the present patent application). In addition, GA 20-oxidases have also been overexpressed in other species and resulted in increased gibberellin levels, e.g. tobacco (Arabidopsis GA20ox: Biemelt et al 2004 Plant Phys: 135: 254-265; Citrus GA20ox: Vidal et al. 2001 Physiol. Plant. 112: 251-260) and potato (potato GA20ox: Carrera et al. Plant J. 22: 247-256).
As shown in the sequence alignment in Table 3 (containing SEQ ID NOs: 1-70), many polypeptides exhibiting GA 20-oxidase are available in the art. Additional GA 20-oxidase polypeptides may be readily identified by the three conserved motifs within the polypeptides as shown on Table 3. Furthermore, the large number of polypeptides provide abundant guidance for generation of derivative polypeptides that will have the desired GA 20-oxidase activity. Finally, polypeptides can readily be expressed and screened for GA 20-oxidase activity using routine methods of molecular biology and enzymology, so one of skill in the art will be able to generate and screen polypeptides having GA 20-oxidase activity. This Example 2 sets out a representative protocol for generating transgenic plants expressing a GA 20-oxidase.
Experimental Protocol:
Plant Vector Construction
The a GA 20-oxidase cDNA construct encoding any one of the polypeptides selected from Table 3 is cloned into an expression vector such as the binary vector pPCV702.kana such that the GA 20-oxidaes polypeptide is operably linked to a promoter such as the CaMV 35S promoter.
Plant Transformation and Growth Conditions
The expression vector is then transformed into the desired woody plant using standard techniques available in the art. By way of example, a hybrid aspen Populus tremula L.×P. tremuloides Michx. clone is transformed and regenerated essentially as previously described [Nilsson, O. et al. Spatial pattern of cauliflower mosaic virus 35S promoter-luceferase expression in transgenic hybrid aspen trees monitored by enzymatic assay and non-destructive imaging. Transgenic Research 1. 209-220 (1992)]. Out of the independent lines, a reasonable number are selected and multiplied by in vitro shoot culture on half-strength MS-medium containing minerals and vitamins [Murashige. T. & Skoog. F. A revised medium for rapid growth and bio-assay with tobacco tissue cultures. Physiologia Plantarum 15. 473-479 (1962)].
Following root initiation, the plants are dipped in Weibufix rooting powder (Svalöf Weibull A B. Hammenhög. Sweden), and potted in a fertilised peat: perlite mixture (5:1) and kept in a growth chamber at 18° C. under a photoperiod of 18 h and a relative humidity of 90%. The photon flux density of the main light period (10 h) is 175 μmol m−2 s−1 at 400-750 nm (Osram Power Staw HQI-TS 400 W/D lamps. Osram. Germany) and daylength extensions are given using low-intensity light (20 μmol−2 s−1). Plants were watered daily, and repotted and fertilised with a complete nutrient solution (SuperbaS. Supra Hydro A B. Landskrona. Sweden) when needed. After 108 days some plants are transferred to short photoperiod conditions (10 h) to induce growth cessation. These plants are then cold acclimated and kept dormant at 8° C. for 4 weeks, at which point samples are taken for fibre length measurements.
Growth Measurements and Sampling
At seven weeks of age, the plants are marked at an actively growing internode at about the same position in all plants. This is used as a reference point for diameter growth measurements and for counting the internodes. The diameter, height and number of internodes of the plants are measured every 3rd to 4th day. The number of internodes is counted from the top to the reference point: the first internode being defined as the one below the uppermost leaf at least 1 cm long. Plants grown under long days are harvested after 100 days, using seven control plants and 25 plants representing the reasonable number of transgenic individual lines. Internodes 7 and 8, with the upper leaves included, are sampled for GA analysis and leaves from nodes 9 and 10 for northern analysis. All tissues are frozen in liquid N2 immediately after sampling. For anatomical studies, the length of internodes 15 and 16 were measured, then excised and fixed immediately in FAA. All parts remaining after sampling are separated into leaf, stem and root fractions and used for fresh weight biomass determinations. Dry weight is determined after drying the samples at 55° C. for 5 days. Dormant plants consisting of three controls and five transgenes are sampled at internodes 1, 10 and 20, counted from the reference point. These samples are used for maceration and subsequent fibre length measurements.
Anatomical Characterisation
Following conventional chemical fixation samples are embedded in L R White [Regan, S., Bourquin, V., Tuominen, H. & Sundberg, B. Accurate and high resolution in situ hybridization analysis of gene expression in secondary stem tissues, Plant J. 19, 363-369 (1999)]. Longitudinal sections for cell counts (internode 15 and 16) and transverse sections for fibre count (internode 30), 2 μm thick, are obtained and stained with toluidine blue. Numbers of epidermal and pith cells in 1 mm of the longitudinal sections are determined for 3 sections per internode. Cell length and number of cells per internode are calculated as the averages found in internodes 15 and 16. The number of xylem fibres are counted in three separate radial files (from pith to cambium) per individual and the average for each genotype are calculated.
Fibre Length Measurements
For fibre length determination, trimmed pieces of outer xylem from the selected intemodes are prepared. The samples are macerated by boiling in a solution of 10% hydrogen peroxide and 50% glacial acetic acid for 4-6 h. When totally bleached, they are rinsed with distilled water three times, neutralised with sodium carbonate and washed again in water. Finally, the fibres are separated from each other by shaking in water and measured using a fibre analyser (KAJAANI FiberLab, Valmet Automation Kajaani Ltd. Kajaani, Finland). On average, the lengths of 30-800 fibres per sample are measured.
Northern Analysis
Total RNA is extracted from leaves using a chloroform and hexadecyltrimethylamonium 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 Mol. Biol. Rep. 11. 113-116 (1993)]. About 18 μg of total RNA per sample is separated in a formaldehyde agarose gel according to Sambrook et al. [Sambrook, J., Fritsch, E. & Maniatis, T. Molecular Cloning: A Laboratory Manual, Cold Spring 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. An appropriate fragment recovered from expression vector and an ubiquitin-like Expressed Sequence Tag (EST A046p57) obtained in the Populus sequencing project [Sterky, F. et al. Gene discovery in the wood-forming tissues of poplar: Analysis of 5.692 expressed sequence tags. Proc. Natl. Acad. Sci. USA 95. 13330-13335 (1998)] are used as probes for analysis of ectopic GA 20-oxidase polypeptide and reference gene expression, respectively. Hybridisation is performed in Church buffer [Church, G. M. & Gilbert, W., Genomic sequencing, Proc. Natl. Acad. Sci. USA 81. 1991-1995 (1984)] at 65° C. overnight. The membrane is washed at 65° C., two times three minutes in each of solution: 2×SSPE. 0.1% SDS; 1×SSPE. 0.1% SDS; 0.5×SSPE. 0.1% SDS and 0.1×SSPE. 0.1% SDS. To visualise the expression patterns, the membrane is exposed to a phosphor imager (Molecular Imager GS-525, Bio-Rad Laboratories, Hercules, Calif., USA) overnight.
Quantification of Gibberellins
Samples of 200-300 mg are ground in liquid nitrogen to a homogenous powder, and GAs are analysed as earlier described by Peng et al. [Peng, J. R., Richards, D. E., Moritz, T., CanoDelgado, A. & Harberd, N. P., Extragenic suppressors of the Arabidopsis gai mutation alter the dose-response relationship of diverse gibberellin responses, Plant Physiol. 119, 1199-1207 (1999)] with a few modifications. Volumes of the solvents used for extraction and partitioning, and that of the anion-exchange column, are reduced. Furthermore, the samples are methylated prior to the HPLC step, and finally they are analysed by GC/MS-selected reaction monitoring (SRM) using a JEOL SX/SX102A four sector mass spectrometer (JEOL, Tokyo, Japan). [2H2]-GAs are used as internal standards.
Results
The results obtained for introduction of any of the polypeptides selected from Table 3 into the desired woody plant will be similar the results of Example 1.







TABLE 3

## Claims

1. A transgenic Populus plant comprising a heterologous nucleic acid molecule encoding a GA 20-oxidase, the nucleic acid molecule operably linked to a promoter, wherein expression of the nucleic acid molecule in the Populus plant results in increased xylem fibre length, wherein the GA 20-oxidase exhibits at least 70% amino acid identity to SEQ ID NO:39.

2. The transgenic Populus plant according to claim 1, wherein the transgenic Populus plant is a Populus tremula×P. tremuloides species.

3. A transgenic Populus plant comprising a heterologous nucleic acid molecule encoding a GA 20-oxidase, the nucleic acid molecule operably linked to a promoter, wherein expression of the nucleic acid molecule in the Populus plant results in increased xylem fibre length, wherein the GA 20-oxidase exhibits at least 70% amino acid identity to SEQ ID NO:39, and wherein the polypeptide comprises three motifs in the following order from N-terminal to C-terminal: LPWKET (SEQ ID NO: 71), NYYP (SEQ ID NO: 72), and HXD (SEQ ID NO: 73).

4. The transgenic Populus plant according to claim 3, wherein the transgenic Populus plant is a Populus tremula×P. tremuloides species.

5. The transgenic Populus plant according to claim 3, wherein the GA 20-oxidase has at least 80% amino acid identity to SEQ ID NO: 39.

6. The transgenic Populus plant according to claim 3, wherein the GA 20-oxidase has at least 90% amino acid identity to SEQ ID NO: 39.

7. The transgenic Populus plant according to claim 3, wherein the GA 20-oxidase has at least 95% amino acid identity to SEQ ID NO: 39.

8. The transgenic Populus plant according to claim 3, wherein the GA 20-oxidase has at least 99% amino acid identity to SEQ ID NO: 39.

9. A transgenic seed that produces a Populus plant comprising a heterologous nucleic acid molecule encoding a GA 20-oxidase, the nucleic acid molecule operably linked to a promoter, wherein expression of the nucleic acid molecule in the Populus plant results in increased xylem fibre length, wherein the GA 20-oxidase exhibits at least 70% amino acid identity to SEQ ID NO:39.

10. A transgenic cell derived from the transgenic Populus plant of claim 1, wherein the cell comprises the nucleic acid molecule.

11. A transgenic cell derived from the transgenic Populus plant of claim 3, wherein the cell comprises the nucleic acid molecule.

12. A transgenic, propagating material derived from the transgenic Populus plant of claim 1, wherein the propagating material comprises the nucleic acid molecule.

13. A transgenic, propagating material derived from the transgenic Populus plant of claim 3, wherein the propagating material comprises the nucleic acid molecule.

14. A method of increasing xylem fibre length in a Populus tree comprising introducing a heterologous nucleic acid molecule encoding a GA 20-oxidase into the Populus tree's genome, wherein the nucleic acid molecule is operably linked to a promoter, wherein expression of the nucleic acid molecule in the Populus tree results in increased xylem fibre length, and wherein the GA 20-oxidase exhibits at least 70% amino acid identity to SEQ ID NO:39.

15. The transgenic Populus plant according to claim 1, wherein the nucleic acid molecule is DNA.

16. The transgenic Populus plant according to claim 1, wherein the GA 20-oxidase has at least 80% amino acid identity to SEQ ID NO: 39.

17. The transgenic Populus plant according to claim 1, wherein the GA 20-oxidase has at least 90% amino acid identity to SEQ ID NO: 39.

18. The transgenic Populus plant according to claim 1, wherein the GA 20-oxidase has at least 95% amino acid identity to SEQ ID NO: 39.

19. The transgenic Populus plant according to claim 1, wherein the GA 20-oxidase has at least 99% amino acid identity to SEQ ID NO: 39.

20. The transgenic Populus plant according to claim 1, wherein the GA 20-oxidase is SEQ ID NO: 39.

21. The transgenic Populus plant according to claim 1, wherein the GA 20-oxidase comprises three motifs in the following order from N-terminal to C-terminal: LPWKET (SEQ ID NO: 71), NYYP (SEQ ID NO: 72), and HXD (SEQ ID NO: 73).

22. The transgenic Populus plant according to claim 3, wherein the nucleic acid molecule is DNA.

23. The transgenic Populus plant according to claim 3, wherein the GA 20-oxidase is SEQ ID NO: 39.