# Patent

## Title
Method for increasing seed size by increasing gibberellin content in the seeds

## Abstract

A method is provided for increasing the weight of plant seed, which comprises preparing a genetically modified plant in which gibberellin content has been manipulated in the seed of the plant.

## Background
The present invention relates to a method for increasing the size of plant seed which may be characterised as an increase in seed weight or seed volume.
Techniques of plant breeding have developed from traditional methods to the use of recombinant DNA technology to introduce desirable genetic characteristics into plants, particularly agricultural crop plant species, of interest.
Previous studies in this area have discovered that plant growth can be regulated by the expression of enzymes important in the biosynthesis of gibberellins (GAs). The gibberellins (GAs) are a large group of diterpenoid carboxylic acids that are present in all higher plants and some fungi. Certain members of the group function as plant hormones and are involved in many developmental processes, including seed germination, stem extension, leaf expansion, flower initiation and development, and growth of the seeds and fruit. The biologically active GAs are usually C19 compounds containing a 19-10 lactone, a C-7 carboxylic acid and a 3β-hydroxyl group. The later stages of their biosynthesis involve the oxidative removal of C-20 and hydroxylation at C-3. Hydroxylation at the 2β position results in the production of biologically inactive products. This reaction is the most important route for GA metabolism in plants and ensures that the active hormones do not accumulate in plant tissues. The GA biosynthetic enzymes 7-oxidase, 20-oxidase, 3β-hydroxylase and 2β-hydroxylase are all 2-oxoglutarate-dependent dioxygenases. These are a large group of enzymes for which 2-oxoglutarate is a co-substrate that is decarboxylated to succinate as part of the reaction (see review by Hedden, P. and Kamiya, Y., in Annu. Rev. Plant Physiol. Plant Mol. Biol. 48 431-460 (1997)).
Chemical regulators of plant growth have been used in horticulture and agriculture for many years. Many of these compounds function by changing the GA concentration in plant tissues. For example, growth retardants inhibit the activity of enzymes involved in GA biosynthesis and thereby reduce the GA content. Such chemicals are used commonly, for example, to prevent lodging in cereals and to control the growth of ornamental and horticultural plants. Conversely, GAs may be applied to plants, such as in the application of GA3 to seedless grapes to improve the size and shape of the berry, and to barley grain to improve malt production. Mixtures of GA4 and GA7 are applied to apples to improve fruit quality and to certain conifers to stimulate cone production. There are several problems associated with the use of growth regulators. Some of the growth retardants are highly persistent in the soil making it difficult to grow other crops following a treated crop. Others require repeated applications to maintain the required effect. It is difficult to restrict application to the target plant organs without it spreading to other organs or plants and having undesirable effects. Precise targeting of the growth-regulator application can be very labour intensive. A non-chemical option for controlling plant morphology is, thus, highly desirable.
Gibberellin biosynthesis has been modified in transgenic plants. See for example WO 94/28141 which reports the cloning and expression of a gibberellin (GA) 20-oxidase gene which catalyses the penultimate step in GA biosynthesis, or WO 99/66029 which reports the cloning and expression of nucleic acid sequences encoding a gibberellin 2β-hydroxylase enzyme (GA 2-oxidase) which is a gibberellin inactivation enzyme. It catalyses the 2β-oxidation (2-oxidation) of a gibberellin molecule to introduce a hydroxyl group at C-2 and further catalyses the oxidation of the hydroxyl group introduced at C-2 to yield the ketone derivative.
A system of nomenclature for the GA-biosynthesis genes has now been introduced (Coles et al The Plant Journal 17(5) 547-556 (1999). References to “gibberellin” include all bioactive gibberellin molecules, unless the context specifies otherwise.
It has long been an aim in agriculture to be able to increase the size of seeds produced by plant species of interest. For many plants, the seeds are the main product to be harvested and an increase in size would be beneficial in improving overall crop yields. For all agricultural plant species, an increase in seed size may assist in the success of planting out a crop by providing the developing plant with a larger resource for germination of the seed.
It has now been surprisingly found that by manipulation of the expression of gibberellin enzymes that an increase in the size of seeds can be achieved.
According to a first aspect of the invention, there is provided a method of increasing the weight of plant seed, the method comprising preparing a genetically modified plant in which gibberellin content has been manipulated in the seed of the plant.
The plant seeds produced according to a method of the invention have an increased size in comparison to plant seeds from control plants. An increase in the size of seed of a plant can be characterised by an increase in volume of the seed as well as by its weight.
Plant seeds are the ripened plant ovule containing an embryo of a gymnosperm or an angiosperm plant.
Preferred species of plants include but are not limited to monocotyledonous plants including seed and the progeny or propagules thereof, for example Lolium, Zea, Triticum, Sorghum, Triticale, Saccharum, Bromus, Oryzae, Avena, Hordeum, Secale and Setaria. Especially useful transgenic plants are maize, wheat, barley plants and seed thereof. Suitably the monocotyledonous is selected from the group consisting of wheat, maize, rye, rice, oat, barley, sorghum and millet.
Dicotyledenous plants are also within the scope of the present invention and preferred transgenic plants include but are not limited to the species Fabaceae, Solanum, Brassicaceae, especially potatoes, beans, cabbages, forest trees, roses, clematis, oilseed rape, sunflower, chrysanthemum, poinsettia and antirrhinum (snapdragon). Alternatively, the transgenic plant may be a dicotyledonous plant. Suitably, the dicotyledonous is selected from the group consisting of soybean, canola, and sunflower.
Genetically modified plants according to a method of the invention may be prepared by any convenient procedure, examples of which are described below. The plants are modified to express a nucleic acid sequence encoding an enzyme of gibberellin biosynthesis, or an enzyme which renders gibberellins resistant to inactivation, or to express nucleic acid molecules to inhibit the expression of enzymes of gibberellin inactivation, or to otherwise manipulate the content of gibberellin in the seed, such as for example by mutagenesis, conveniently by chemical mutagenesis. Expression of a nucleic acid sequence includes overexpression above basal or endogenous levels, which can be defined with respect to levels of expression in an unaltered or control plant of the same species. Expression of such nucleic acid sequences therefore includes expression of an exogenous gene introduced into the plant, as well as introduction of promoter sequences to drive expression of the endogenous gene, such as, for example, homologous recombination.
The nucleic acid sequence may be as shown in any one of FIG. 4, 6, 8, or 10, or its complementary strand or a homologous sequence thereto. Alternatively, the nucleic acid may be a nucleic acid sequence that encodes an amino acid sequence or a protein sequence as shown in any one of FIG. 5, 7, 9, or 11, or its complementary strand or a homologous sequence thereto.
In the context of the present invention, the degree of identity between amino acid sequences may be at least 40%, suitably 50% or higher, e.g. 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%. At the nucleotide level, the degree of identity may be at least 50%, suitably 60% or higher, e.g. 65%, 70%, 75%, 80%, 85%, 90% or 95%. A homologous sequence according to the present invention may therefore have a sequence identity as described above. Sequence homology may be determined using any conveniently available protocol, for example using Clustal XTM from the University of Strasbourg and the tables of identities produced using GenedocTM (Karl B. Nicholas).
Also included within the scope of the present invention are nucleic acid sequences which hybridises to a sequence in accordance with the first aspect of the invention under stringent conditions, or a nucleic acid sequence which is homologous to or would hybridise under stringent conditions to such a sequence but for the degeneracy of the genetic code, or an oligonucleotide sequence specific for any such sequence.
Stringent conditions of hybridisation may be characterised by low salt concentrations or high temperature conditions. For example, highly stringent conditions can be defined as being hybridisation to DNA bound to a solid support in 0.5 M NaHPO4, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., and washing in 0.1×SSC/0.1% SDS at 68° C. (Ausubel et al eds. “Current Protocols in Molecular Biology” 1, page 2.10.3, published by Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York, (1989)). In some circumstances less stringent conditions may be required. As used in the present application, moderately stringent conditions can be defined as comprising washing in 0.2×SSC/0.1% SDS at 42° C. (Ausubel et al (1989) supra). Hybridisation can also be made more stringent by the addition of increasing amounts of formamide to destabilise the hybrid nucleic acid duplex. Thus particular hybridisation conditions can readily be manipulated, and will generally be selected according to the desired results. In general, convenient hybridisation temperatures in the presence of 50% formamide are 42° C. for a probe which is 95 to 100% homologous to the target DNA, 37° C. for 90 to 95% homology, and 32° C. for 70 to 90% homology.
Expression of a nucleic acid sequence encoding an enzyme of gibberellin metabolism (i.e. an enzyme of gibberellin biosynthesis or catabolism) may comprise expression of (i) the enzyme gibberellin 20-oxidase (GA20ox), that catalyses the penultimate steps in GA biosynthesis, as in the example included (FIGS. 4 & 5); or (ii) the enzyme gibberellin 3β-hydroxylase (GA 3-oxidase, GA3ox, eg. AtGA3ox1, FIGS. 6 & 7) that catalyses the last step in GA biosynthesis, eg. AtGA3ox1, Genbank No. L37126, Chiang H H et al., Plant Cell 7:195-201; 1995). Expression includes overexpression above basal or endogenous levels.
Expression of a nucleic acid sequence encoding an enzyme which renders gibberellins resistant to inactivation may comprise production of the enzyme gibberellin 1,2-desaturase (eg. FIGS. 8 & 9) which renders GAs resistant to inactivation (GAdes; Genbank No. AJ417493, Tudzynski B, et al., J. Biol. Chem. 278:28635-28643; 2003). Expression includes overexpression above basal or endogenous levels, for example overexpression of the nucleic acid may comprise overproduction of the enzyme with respect to basal or endogenous levels of expression.
Expression of nucleic acid molecules to inhibit the expression of enzymes of gibberellin inactivation (i.e. by reduction or abolition of gene expression) may comprise reduction in activity or concentration of the endogenous gibberellin inactivation enzyme gibberellin 2β-hydroxylase (GA 2-oxidase; GA2ox, eg. FIGS. 10 & 11: TaGA2ox2).
Mutagenesis of the plant may be conveniently achieved by any technique of chemical or radionucleide-induced mutagenesis, or for example by TILLING (McCallum C M, et al., Plant Physiol. 123:439-442; 2000).
Other nucleic acid sequences in accordance with this aspect of the present invention may also comprise a nucleic acid sequence as previously defined in which the coding sequence is operatively linked to a promoter. The promoter may be constitutive and/or specific for expression in a particular plant cell or tissue, preferably in seeds.
Preferably, the nucleic acid sequence comprises a promoter which drives expression of a nucleic acid sequence described above. Such promoter sequences include promoters which occur naturally 5′ to the coding sequence of the sequences shown in FIG. 4, 6, 8, or 10. Promoters may also be selected to constitutively express the nucleic acid coding for the preferred gene sequences defined herein. Expression of the nucleic acid includes overexpression above basal or endogenous levels. Promoters that are induced by internal or external factors, such as chemicals, plant hormones, light or stress could also be used. Examples are the pathogenesis related genes inducible by salicylic acid, copper-controllable gene expression (Mett et al Proc. Nat'l. Acad. Sci. USA 90 4567-4571 (1993)) and tetracycline-regulated gene expression (Gatz et al Plant Journal 2 397-404 (1992)). Examples of gibberellin-inducible genes are γ-TIP (Phillips, A. L., & Huttly, A. K., Plant Mol. Biol. 24 603-615 (1994)) and GAST (Jacobsen, S. E., & Olszewski, N. E., Planta 198 78-86 (1996).
Suitable promoters for driving transgene expression in developing seeds include:

    
    
        (i) High Molecular Weight Glutenin-1-D1 promoter from Wheat (FIG. 3; Lamacchia et al., 2001);
        (ii) End-1 promoter from barley (Clarke B C, et al., Aust. J. Agric. Res. 52:1181-1193; 2001).
        (iii) MAC1 promoter from maize (Sheridan et al., Genetics 142:1009-1020, 1996);
        (iv) Cat3 promoter from maize (GenBank No. L05934, Abler et al., Plant Mol. Biol. 22:10131-1038, 1993);
        (v) Atimyc1 from Arabidopsis (Urao et al., Plant J. Mol. Biol. 32:571-57, 1996; Conceicao et al., Plant 5:493-505, 1994);
        (vi) napA from Brassica napus (GenBank No. J02798);
        (vii) Napin gene family from Brassica napus (Sjodahl et al., Planta 197:264-271, 1995);
        (viii) 2S storage protein promoter from Brassica napus (Dasgupta et al., Gene 133:301-302, 1993);
        (ix) 2S seed storage protein gene family promoter from Arabidopsis; 
        (x) Oleosin 20 kD from Brassica napus (GenBank No. M63985);
        (xi) Oleosin A promoter (GenBank No. U09118) or Oleosin B promoter (GenBank No. U09119) from soybean;
        (xii) Oleosin promoter from Arabidopsis (GenBank No. Z17657);
        (xiii) Oleosin 18 kD promoter from maize (GenBank No. J05212, Lee, Plant Mol. Biol. 26:1981-1987, 1994);
        (xiv) Low molecular weight sulphur rich protein promoter from soybean (Choi et al., Mol. Gen. Genet. 246:266-268, 1995);
        (xv) Promoters derived from zein-encoding genes (including the 15 kD, 16 kD, 19 kD, 22 kD, 27 kD, and gamma-zein genes, Pedersen et al., Cell 29:1015-1026, 1982).
    
    


The rice actin-1 intron may also be used to increase transgene mRNA accumulation, but this is not necessary for the invention.
The nucleic acid sequences of the present invention may also code for RNA which is antisense to the RNA normally found in a plant cell or may code for RNA which is capable of cleavage of RNA normally found in a plant cell. In such an approach, the whole cDNA or smaller fragments (>200 bp) may be amplified by PCR and inserted into an appropriate expression vector in reverse orientation to the primer. Accordingly, the present invention also provides a nucleic acid sequence encoding a ribozyme capable of specific cleavage of RNA encoded by a gene for a gibberellin inactivation enzyme, such as the gibberellin 2-oxidase gene. Such ribozyme-encoding DNA would generally be useful in inhibiting the deactivation of gibberellins. Alternatively, the RNA may encode a short interfering RNA sequence capable of activating the RNAi cellular process for degrading a target RNA species of interest, such as RNA coding a gibberellin deactivation enzyme.
RNAi can involve intron-spliced hairpin (ihpRNA) constructs (Smith, N. A., et al. (2000) Nature, 407:319-320), using 300-600 bp of the transcribed region of the target inserted in sense and antisense orientation flanking the intron of an ihpRNA vector such as pHELLSGATE (Wesley, S. V., et al. (2001) Plant J., 27:581-590.). Design of hammerhead ribozymes against target sequences, for example, GA2ox, may follow guidelines, for example Fritz, J. J., et al. (Methods (2002), 28:276-285). The ribozyme would be produced from synthetic oligonucleotides, annealed and inserted into an appropriate vector. It is preferable to use tissue-specific promoters for expression of antisense/RNAi/ribozymes in transgenic plants to avoid pleiotropic effects in other tissues. The promoters listed in the application are suitable. The constructs or RNAi fragments are introduced into the target species by routine methods in the art as described herein.
Nucleic acid sequences in accordance with the present invention may further comprise 5′ signal sequences to direct expression of the expressed protein product.
Such signal sequences may also include protein targeting sequences which can direct an expressed protein to a particular location inside or outside of a host cell expressing such a nucleic acid sequence. Alternatively, the nucleic acid sequence may also comprise a 3′ signal such as a polyadenylation signal or other regulatory signal.
Preparation of transgenic plants according to the present invention which have increased seed weight may therefore be prepared by modification of a plant cell to contain a nucleic acid sequence as described above which provides for expression of a nucleic acid sequence encoding an enzyme of gibberellin biosynthesis or an enzyme which renders gibberellins resistant to inactivation, or for expression of nucleic acid molecules to inhibit the expression of enzymes of gibberellin inactivation. Such nucleic acid sequences as herein defined can be introduced into plant cells by any suitable means. Expression of a nucleic acid includes overexpression above basal or endogenous levels.
Preferably, nucleic acid sequences of the present invention are introduced into plant cells by transformation using an appropriate vector, e.g. pMON57004 as shown in FIG. 1. Alternatively, a binary vector, for example a modified version of pGPTV-Kan (Becker et al Plant Mol. Biol. 20 1195-1197 (1992)) in which the β-glucuronidase reporter gene is replaced by the HMWGlu-GA20ox1 expression cassette. Such plasmids may be then introduced into Agrobacterium tumefaciens by electroporation and can then be transferred into the host cell via a vacuum filtration procedure. Alternatively, transformation may be achieved using a disarmed Ti-plasmid vector and carried by Agrobacterium by procedures known in the art, for example as described in EP-A-0116718 and EP-A-0270822. Where Agrobacterium is ineffective, the foreign DNA could be introduced directly into plant cells using an electrical discharge apparatus alone, such as for example in the transformation of monocotyledonous plants. Any other method that provides for the stable incorporation of the nucleic acid sequence within the nuclear DNA or mitochondrial DNA of any plant cell would also be suitable. This includes species of plant which are not yet capable of genetic transformation.
Preferably, nucleic acid sequences as described herein for introduction into host cells also contain a second chimeric gene (or “marker” gene) that enables a transformed plant containing the foreign DNA to be easily distinguished from other plants that do not contain the foreign DNA. Examples of such a marker gene include antibiotic resistance (Herrera-Estrella et al EMBO J. 2 987-995 (1983)), herbicide resistance (EP-A-0242246) and glucuronidase (GUS) expression (EP-A-0344029). Expression of the marker gene is preferably controlled by a second promoter which allows expression in cells at all stages of development so that the presence of the marker gene can be determined at all stages of regeneration of the plant.
A whole plant can be regenerated from a single transformed plant cell, and the invention therefore provides transgenic plants (or parts of them, such as propagating material, i.e. protoplasts, cells, calli, tissues, organs, seeds, embryos, ovules, zygotes, tubers, roots, etc.) including nucleic acid sequences as described above.
In the context of the present invention, it should be noted that the term “Genetically modified” should not be taken to be limited in referring to an organism as defined above containing in their germ line one or more genes from another species, although many such organisms will contain such a gene or genes, i.e. a “transgenic” plant. Rather, the term “genetically modified” refers more broadly to any organism whose germ line has been the subject of technical intervention, for example by recombinant DNA technology or chemical mutagenesis. So, for example, an organism in whose germ line an endogenous gene has been deleted, duplicated, activated or modified is a genetically modified organism for the purposes of this invention as much as an organism to whose germ line an exogenous DNA sequence has been added.
Screening of plant cells, tissue and plants for the presence of specific DNA sequences may be performed by Southern analysis as described in Sambrook et al (Molecular Cloning: A Laboratory Manual, Second edition (1989)). This screening may also be performed using the Polymerase Chain Reaction (PCR) by techniques well known in the art.
Transformation of plant cells includes separating transformed cells from those that have not been transformed. One convenient method for such separation or selection is to incorporate into the material to be inserted into the transformed cell a gene for a selection marker. As a result only those cells which have been successfully transformed will contain the marker gene. The translation product of the marker gene will then confer a phenotypic trait that will make selection possible. Usually, the phenotypic trait is the ability to survive in the presence of some chemical agent, such as an antibiotic, e.g. kanamycin, G418, paromomycin, etc, which is placed in a selection media. Some examples of genes that confer antibiotic resistance, include for example, those coding for neomycin phosphotransferase kanamycin resistance (Velten et al EMBO J. 3 2723-2730 (1984)), hygromycin resistance (van den Elzen et al Plant Mol. Biol. 5 299-392 (1985)), the kanamycin resistance (NPT II) gene derived from Tn5 (Bevan et al Nature 304 184-187 (1983); McBride et al Plant Mol. Biol. 14 (1990)) and chloramphenicol acetyltransferase. The PAT gene described in Thompson et al (EMBO J. 6 2519-2523 (1987)) may be used to confer herbicide resistance.
An example of a gene useful primarily as a screenable marker in tissue culture for identification of plant cells containing genetically engineered vectors is a gene that encodes an enzyme producing a chromogenic product. One example is the gene coding for production of β-glucuronidase (GUS). This enzyme is widely used and its preparation and use is described in Jefferson (Plant Mol. Biol. Reporter 5 387-405 (1987)).
Once the transformed plant cells have been cultured on the selection media, surviving cells are selected for further study and manipulation. Selection methods and materials are well known to those of skill in the art, allowing one to choose surviving cells with a high degree of predictability that the chosen cells will have been successfully transformed with exogenous DNA.
After transformation of the plant cell or plant using, for example, the Agrobacterium Ti-plasmid, those plant cells or plants transformed by the Ti-plasmid so that the enzyme is expressed, can be selected by an appropriate phenotypic marker. These phenotypic markers include, but are not limited to, antibiotic resistance. Other phenotypic markers are known in the art and may be used in this invention.
Positive clones are regenerated following procedures well-known in the art. Subsequently transformed plants are evaluated for the presence of the desired properties and/or the extent to which the desired properties are expressed. A first evaluation may include, for example, the level of bacterial/fungal resistance of the transformed plants, stable heritability of the desired properties, field trials and the like.
The methods of present invention extend to methods for the preparation of transgenic plants and the sexual and/or asexual progeny thereof, which have been transformed with a recombinant DNA sequence as defined herein. The regeneration of the plant can proceed by any known convenient method from suitable propagating material either prepared as described above or derived from such material.
The expression “asexual or sexual progeny of transgenic plants” includes by definition according to the invention all mutants and variants obtainable by means of known process, such as for example cell fusion or mutant selection and which still exhibit the characteristic properties of the initial transformed plant, together with all crossing and fusion products of the transformed plant material.
The methods of the invention also concern the proliferation material of transgenic plants. The proliferation material of transgenic plants is defined relative to the invention as any plant material that may be propagated sexually in vivo or in vitro. Particularly preferred within the scope of the present invention are protoplasts, cells, calli, tissues, organs, seeds, embryos, egg cells, zygotes, together with any other propagating material obtained from transgenic plants.
Gibberellin metabolism is the term used to describe the enzyme pathway for the biosynthesis in the cell of the active gibberellin molecules and for the regulation of the biosynthetic pathway through the activity of inhibitory or degradatory enzymes or other mechanisms, which includes the catabolism of gibberellins.
Manipulation of gibberellin content, preferably bioactive gibberellin content as described herein comprises the expression of a nucleic acid sequence encoding an enzyme of gibberellin metabolism (i.e. gibberellin biosynthesis or catabolism) or an enzyme which renders gibberellins resistant to inactivation, or the expression of nucleic acid molecules to inhibit the expression of enzymes of gibberellin inactivation. Expression includes overexpression above basal or endogenous levels. Preferably, the manipulation of gibberellin metabolism is caused by the expression of nucleic acid sequences as herein defined in the seeds of the plant, or by the ablation of the expression of genes that control inactivation of gibberellins in the seeds of the plant. The result of such manipulations is an increase in the level of gibberellin in the seed compared to normal levels of gibberellins in a control plant seed of the same species.
Gibberellin molecules known to occur as biologically active molecules in plant tissues, including seeds are GA1, GA3, GA4 and GA7. However, use of the term “gibberellins” also includes other bioactive gibberellins. In fact, the method of the invention relates to increasing abundance of bioactive GAs as a whole.
Overproduction of enzymes involved in gibberellin biosynthesis coded for by these genes referred to above can be achieved by expression including overexpression of the gene under a suitable promoter active in developing seeds as described above. Expression includes overexpression above basal or endogenous levels. For example, overexpression of the gene may comprise overproduction of the enzymes involved in gibberellin biosynthesis coded for by the gene with respect to basal or endogenous levels of expression.
Reduction or abolition of gene expression as described above can be achieved using antisense or sense suppression, RNAi or the identification of mutants with reduced expression. Reduction or abolition of enzyme activity can be achieved through the identification of mutagen-induced or existing lines with altered properties, for example by TILLING (McCallum C M, et al., Plant Physiol. 123:439-442; 2000).
Increasing GA concentration through manipulation of GA biosynthesis or turnover in during seed development according to the present invention leads to increased seed volume and weight. Weight-per-grain increases of at least 5%, suitably in the range of from 5% to 40%, preferably from 10% to 40%, most preferably from 20% to 30%, can be achieved, compared to plant seed from control plants grown under normal conditions which have not been subject to genetic modification. An increase in weight of at least 5% is statistically significant and represents a measurable and real improvement in crop yield.
As noted above an increase in the size of seed of a plant can be characterised by an increase in volume of the seed as well as by its weight. The present invention therefore also extends to a method of increasing the volume per unit plant seed, the method comprising preparing a transgenic plant in which gibberellin content has been manipulated in the seed of the plant.
According to a further aspect of the invention, there is provided the use of a nucleic acid sequence encoding an enzyme of gibberellin metabolism (i.e. gibberellin biosynthesis or catabolism) in the preparation of plant seed with an increased weight.
According to a further aspect of the invention, there is provided the use of a nucleic acid sequence encoding an enzyme which renders gibberellins resistant to inactivation in the preparation of plant seed with an increased weight.
According to a further aspect of the invention, there is provided the use of a nucleic acid sequence encoding a nucleic acid molecule which inhibits the expression of enzymes of gibberellin inactivation in the preparation of plant seed with an increased weight.
According to a further aspect of the invention, there is provided method of increasing the drought resistance of a plant seed, which comprises preparing a genetically modified plant in which gibberellin content has been manipulated in the seed of the plant, thereby increasing the weight of the plant seed.
By way of illustration and summary, the following scheme sets out a typical process by which genetically modified plant material, including whole plants, may be prepared according to a method of the present invention for increasing seed size. The process can be regarded as involving five steps:

    
    
        (1) first isolating from a suitable source (or DNA library) or synthesising by means of known processes a DNA sequence encoding a protein exhibiting gibberellin enzyme activity, or a DNA sequence which upon expression yields a nucleic acid sequence capable of inhibiting expression of such enzymes;
        (2) operably linking the said DNA sequence in a 5′ to 3′ direction to plant expression sequences as defined herein;
        (3) transforming the construct of step (2) into plant material by means of known processes and expressing it therein;
        (4) screening of the plant material treated according to step (3) for the presence of a DNA sequence encoding a protein exhibiting gibberellin synthetic activity or for a nucleic acid sequence having gibberellin inhibitory activity: and
        (5) optionally regenerating the plant material transformed according to step (3) to a whole plant.
    
    


Alternatively, genetically modified plants in which the gibberellin content has been increased in the seed of the plant through reduced inactivation, resulting in increased weight of plant seed, may be generated as follows:

    
    
        (1) Identify a suitable source of DNA encoding an enzyme or enzymes involved in gibberellin biosynthesis, for example GA 2-oxidases (GA2ox);
        (2) Identify the gene family members that encode the enzyme(s) which is/are expressed in the seeds of the plant, for example, by transcript analysis by Northern blotting, RT-PCR or microarrays;
        (3) Design a single antisense RNA, RNAi or ribozyme construct that will target all the genes encoding enzymes involved in gibberellin biosynthesis which are expressed in the plant seed, if these genes have high sequence identity. Alternatively, design separate antisense RNA, RNAi or ribozyme constructs if sequence homology is lower;
        
            (3a) For antisense RNA, amplify the whole cDNA or smaller fragments (>200 bp) by PCR and insert in an appropriate expression vector in reverse orientation to the promoter; or
            (3b) Design an intron-spliced hairpin (ihpRNA) construct; or
            (3c) Design a hammerhead ribozyme and insert into an appropriate vector;
        
        
        (4) Antisense/RNAi/ribozyme constructs can be introduced into the target species by Agrobacterium-mediated transformation or microprojectile bombardment as appropriate, followed by assessment of effects on GA2ox expression and seed development.
    
    


Another example of an approach for identifying a suitable DNA source is to identify loss-of-function or reduced-function variants of the target genes using TILLING or other sequence variant detection methods. TILLING can identify sequence variants in the target gene(s) in natural or induced populations of crop species (McCallum, C. M., et al. (2000) Plant Physiol., 123:439-442; Comai, L., et al. (2004) Plant J., 37:778-786; Slade, A. J., et al. (2005) Nature Biotechnology, 23:75-81). A simplified protocol could be:

    
    
        (1) Determine gene expression patterns (for example GA2ox) to identify target genes as above;
        (2) Design sequence specific primers to amplify conserved exon-rich regions from genomic DNA (homoeologue-specific primers for allopolyploid species);
        (3) Carry out TILLING method, involving PCR, heteroduplex annealing, cell cleavage and product detection, to identify sequence variants and confirm this by DNA sequencing; and
        (4) Back-cross to remove unwanted mutations and assess the effects.
    
    


A DNA sequence encoding a protein exhibiting gibberellin enzyme activity as used herein includes nucleic acid sequences encoding an enzyme of gibberellin metabolism, which may be gibberellin biosynthesis or gibberellin catabolism, or an enzyme which renders gibberellins resistant to inactivation. A DNA sequence encoding a nucleic acid molecule that is capable of inhibiting the expression of enzymes of gibberellin inactivation may be an antisense DNA sequence or short interfering RNA sequence capable of activating a process of RNAi in the plant cell leading to the inhibition of gene expression of the affected gene of gibberellin biosynthesis.
Alternatively, as described above, the genetically modified plant may be produced through the action of chemically induced mutagenesis of a subject plant or plant tissue, followed by screening to identify plants or plant tissue with the desired genetic characteristics.
In its broadest aspect, the invention can be described as a means of increasing yield of plant crop per unit area of the crop. Such methods of preparing transgenic plants having increased seed size offer several advantages, not least of which is an overall increase in yield. In addition, substantial advantages are associated with the greater storage capacity of increased seed size. These include seedling vigour, whereby larger seeds would nourish the growing seedling for a longer period before it becomes photosynthetically competent and self-sustaining. This would allow deeper planting and thus improve drought resistance.
Preferred features for the second and subsequent aspects of the invention are as for the first aspect mutatis mutandis.



The invention will now be further described by way of reference to the following Examples and Figures which are provided for the purposes of illustration only and are not to be construed as being limiting on the invention. Reference is made to a number of Figures in which:
FIG. 1 shows a map of plasmid pMON57004. Plasmid pMON57004 used as the basis for construct pDE45.
FIG. 2 shows photographs of T3 seeds from DE45 transgenic lines (T2) and a null (azygous) line.
FIG. 3 (SEQ ID No: 1) shows the nucleotide sequence of the promoter of the High Molecular Weight Glutenin-1-D1 gene from wheat (Triticum aestivum cv Cheyenne; EMBL accession AJ301618; Lamacchia, et al., 2001) with added PacI and StuI restriction sites at the 5′ and 3′ ends, respectively.
FIG. 4 (SEQ ID No: 2) shows the nucleotide sequence of the coding region of the gibberellin 20-oxidase-1 gene from Arabidopsis thaliana (accession Landsberg erecta); EMBL accession X83379; Phillips, et al., 1995) with added XhoI and SacI sites at the 5′ and 3′ ends, respectively.
FIG. 5 (SEQ ID No: 3) shows the amino acid sequence of the gibberellin 20-oxidase isozyme-1 from Arabidopsis thaliana as encoded by FIG. 4 (sequence 2).
FIG. 6 (SEQ ID No: 4) shows the nucleotide sequence of the gibberellin 3β-hydroxylase-1 gene from Arabidopsis (AtGA3ox1, Genbank No. L37126, Chiang H H et al., Plant Cell 7: 195-201; 1995).
FIG. 7 (SEQ ID No: 6) shows the protein sequence of the gibberellin 3β-hydroxylase-1 gene from Arabidopsis (AtGA3ox1, Genbank No. L37126, Chiang H H et al., Plant Cell 7: 195-201; 1995).
FIG. 8 (SEQ ID No: 5) shows the nucleotide sequence of the gibberellin 1,2-desaturase from Gibberella fujikuroi which renders GAs resistant to inactivation (GAdes; Genbank No. AJ417493, Tudzynski B, et al., J. Biol. Chem. 278: 28635-28643; 2003).
FIG. 9 (SEQ ID No: 7) shows the protein sequence of the gibberellin 1,2-desaturase from Gibberella fujikuroi which renders GAs resistant to inactivation (Sequences 5 & 6: GAdes; Genbank No. AJ417493, Tudzynski B, et al., J. Biol. Chem. 278:28635-28643; 2003).
FIG. 10 (SEQ ID No: 8) shows the nucleotide sequence of the gibberellin inactivation enzyme from wheat, gibberellin 2β-hydroxylase-2 (GA 2-oxidase-2; TaGA2ox2; Phillips A L., et al., unpublished).
FIG. 11 (SEQ ID No: 9) shows the protein sequence of the gibberellin inactivation enzyme from wheat, gibberellin 2β-hydroxylase-2 (GA 2-oxidase-2; TaGA2ox2; Phillips A L., et al, unpublished).
FIG. 12 shows the main pathways of gibberellin (GA) biosynthesis in plants. The labelled enzymes are: (1) ent-copalyldiphosphate synthase, (2) ent-kaurene synthase, (3) ent-kaurene oxidase, (4,5,6), ent-kaurenoic acid oxidase, (7) GA 13-hydroxylase, (8) GA 20-oxidase, (9,a,b) GA 3β-hydroxylase, (10) GA 2-oxidase.



EXAMPLE 1
Construction of an HMWGlu::AtGA20ox1 Expression Cassette
The promoter of the High Molecular Weight Glutenin-1-D1 gene from wheat (Triticum aestivum cv Cheyenne; EMBL accession AJ301618; Lamacchia, et al., 2001) was amplified by PCR from plasmid pHMWGlu-1-D1 using the primers HMW-L (5′-AAATTAATTAAAAATATGCAACATAATTTCC-3′) (SEQ ID No: 10) and HMW-R (5′-AAAAGGCCTGGTGGACTATCAGTAATTGA-3′) (SEQ ID No: 11) to create PacI and StuI restriction sites at the 5′ and 3′ ends, respectively. This HMWGlu-1-D1 promoter fragment (Sequence 1) was inserted into the PacI-StuI sites of plasmid pMON57004 (FIG. 1) upstream of the rice Actin-1 intron to yield plasmid pMON57004-HMWG. The coding region of the gibberellin 20-oxidase-1 gene from Arabidopsis thaliana (accession Landsberg erecta); EMBL accession X83379; Phillips, et al., 1995) was PCR-amplified from plasmid pAtGA20ox1 using primers 20ox1-L (5′-AAACTCGAGATGGCCGTAAGTTTCGTAAC-3′) (SEQ ID No: 12) and 20ox1-R (5′-AAAGAGCTCTTAGATGGGTTTGGTGAGCC-3′) (SEQ ID No: 13) to create XhoI and SacI sites at the 5′ and 3′ ends, respectively. This AtGA20ox1 fragment (Sequence 2), encoding gibberellin 20-oxidase isozyme 1 (Sequence 3) was inserted into the XhoI-SacI sites of pMON57004-HMWG, between the rice Actin-1 intron and the NOS terminator.
EXAMPLE 2
Wheat Transformation
Bread wheat (Triticum aestivum cv Cadenza) was transformed with plasmid pDE45 by particle bombardment of immature embryo tissue. Wheat plants were grown, 5 per 20 cm diameter pot, in an environmentally-controlled room with air temperatures of 18° C./15° C. (day/night), a relative humidity of 50-70% under ca. 350 μmol/m2/s irradiance with a photoperiod of 16 hours. Immature scutella isolated from seeds at approximately 14-16 days post anthesis were co-transformed using the PDS1000/He micro-projectile bombardment device (BioRad, Hemel Hempstead, UK) with pDE45 and pAHC25 (containing the selectable marker gene bar; Christensen & Quail P 1996) at a 1:1 molar ratio. Plants were recovered via in vitro tissue culture using Bialaphos selection following protocols developed by Barcelo and Lazzeri (1995) and modified by Pastori et al. (2001), Rasco-Gaunt et al. (2001) and Sparks & Jones (2004). Four transgenic T1 lines (T45-2 through T45-5) that survived selection were transferred to soil and grown to maturity in an environmentally-contained glasshouse to produce T2 seed. Twenty (20) T2 seeds of each line were re-sown and leaf material tested by PCR to identify segregants containing the DE45 transgene. All lines were grown through to maturity and T3 grain collected. T3 seedlings were produced and tested for the presence of the DE45 construct by PCR in order to identify lines homozygous for the transgene.
EXAMPLE 3
Seed Analysis
Seeds of the four primary transgenic lines, T45-2 through T45-5, had larger seed than control (non-transformed) lines. Average seed weights (Table 1) indicated that plants containing pDE45 had seeds that were between 10% and 40% heavier than the control.
Larger seeds were also observed on T2 plants that tested positive for the DE45 transgene—see FIG. 2. Mean Grain weights of these T3 seeds were also increased by 19-32% over seeds from null (azygous, non-transgenic) lines (Table 2).







TABLE 1







Grain weights and yields of primary


transgenic (T1) lines (T2 seeds)













Mean grain
Mean Grain
Mean grain


Plant
Construct
no./Ear
weight in g
weight g/plant





T45.2
DE45
60.08
0.049
17.79


T45.3
DE45
63.22
0.056
21.09


T45.4
DE45
54.38
0.063
20.49


T45.5
DE45
56.76
0.061
20.74


Control
n/a
58.45
0.044
15.56



















Grain weights from T2 plants (T3 seeds) and Null (azygous) lines






















Line
45.2
45.3
45.4
45.5
Nulls



Mean grain
61.6
60
61.6
56.8
51.2



weights
62.4
58.4
67.2
66
53



of sub-lines
64.4
58.4
58.4
66.8
50



(g/grain)
50.8
66.8
66
58.8
38.8




50.4
55.6
69.2
60.4
44




66.4
58
60
73.2
46.8




60.8
58.8
68.8
61.6
47.2




52.8
58.8
75.6
63.6
47.6




50.4
59.6
61.2
58.8
54.8




57.6
58.8
62.8
55.6
48.8




59.6
60.4
58.8
49.6
52.4




56

59.2
56.8
51.2







74.8
48.4







70.0
50.8








42.8








45.2








51.2








46.8








50



Means (g/grain)
57.8
59.4
64.1
62.3
48.5



% increase over
19
23
32
29



Null










EXAMPLE 4
Growth of a Subsequent Generation (T3) of Transgenic Wheat Plants and Analysis of the Seed (T4 seeds)
Three independent transgenic (T3) lines, homozygous for the HMWGlu-GA20ox cassette, together with Control (non-transgenic wheat cv. Cadenza) were grown, 15 plants per line, in controlled environment. Plants were grown under a 16 hr day at 700 micromole per meter squared per second of white light, at a temperature of 20° C. (day), 18° C. (night) and 80% relative humidity. A randomized block pattern was used to avoid position effects within the cabinet. At the boot stage, each plant was reduced to three main tillers; after seed filling and maturation, plants were allowed to desiccate completely and the ears harvested. Two seeds were harvested from each of two central spikelets of each ear and the four seeds weighed individually. All harvested seed from each line was then pooled and the volume of 50 seeds measured by ethanol displacement using a 10 ml glass density bottle (Table 3).







TABLE 3







Grain weights and yields of transgenic (T3) lines (T4 seeds)













Line
Line
Line



Control
45.2.3
45.3.11
45.5.8















Mean seed vol (ul)
39.1
46.9
49.6
45.7


Increase over control

20%
27%
17%


Mean seed weight (mg)
51.4
59.8
60.9
57.2


Increase over control

16%
19%
11%









REFERENCES


    Barcelo P, & Lazzeri P. (1995). In Methods in Molecular Biology: Plant Gene Transfer and Expression Protocols, p. 113-123. Eds H. Jones. Humana Press: Totowa N.J.
    Christensen A H, & Quail P H. (1996). Transgenic Research. 5:213-218.
    Lamacchia et al., (2001) J. Exp. Bot. 52:243-250.
    Pastori et al., (2001) Journal of Experimental Botany. 52:857-863.
    Phillips et al., (1995) Plant Physiol. 108:1049-1057.
    Rasco-Gaunt et al., (2001) Journal of Experimental Botany. 52:865-874.
    Sparks C A, & Jones H D. (2004). Transformation of wheat by biolistics, In Transgenic Crops of the World—Essential Protocols. Ed I. S. Curtis. Kluwer: Dordrecht: Netherlands.

## Claims

1. A method of increasing the weight of plant seed, the method comprising preparing a genetically modified plant in which the gibberellin content has been increased in the seed of the plant.

2. The method as claimed in claim 1, in which the plant is monocotyledonous.

3. The method as claimed 2, in which monocotyledonous plant is selected from the group consisting of wheat, maize, rye, rice, oat, barley, sorghum and millet.

4. The method as claimed in claim 1, in which the plant is dicotyledenous.

5. The method as claimed in claim 4, in which the dicotyledonous plant is selected from the group consisting of soybean, canola, and sunflower.

6. A method as claimed in claim 1, in which the gibberellin content has been increased by (i) expression of a nucleic acid sequence encoding an enzyme of gibberellin biosynthesis or of an enzyme which renders gibberellins resistant to inactivation, (ii) by expression of a nucleic acid molecule to inhibit the expression of enzymes of gibberellin inactivation, or (iii) by mutagenesis of the plant.

7. The method as claimed in claim 6, in which the gibberellin content has been increased by expression of a nucleic acid sequence encoding an enzyme of gibberellin metabolism comprising a nucleic acid sequence as shown in SEQ ID NO:2 or SEQ ID NO:4, or its complementary strand or a homologous sequence thereto.

8. The method as claimed in claim 6, in which the enzyme of gibberellin biosynthesis is gibberellin 20-oxidase (GA20ox) or gibberellin 3β-hydroxylase (GA 3-oxidase).

9. The method as claimed in claim 6, in which the gibberellin content has been increased by expression of a nucleic acid sequence encoding an enzyme which renders gibberellins resistant to inactivation comprising a nucleic acid sequence as shown in SEQ ID NO:5.

10. The method as claimed in claim 6, in which the enzyme that renders gibberellins resistant to inactivation is gibberellin 1,2-desaturase (GAdes).

11. The method as claimed in claim 6, in which the gibberellin content has been increased by expression of a nucleic acid molecule that inhibits the expression of gibberellin-2β-hydroxylase (GA 2-oxidase).

12. The method as claimed in claim 1, in which the increase in seed weight is of at least 5%.

13. The method as claimed in claim 12, in which the increase in seed weight is in the range of from 5% to 40%.

14. A plant seed in which the weight has been increased according to the method of claim 1.

15. A genetically modified plant comprising the plant seed of claim 14.

16. A method of increasing the volume per unit plant seed, the method comprising preparing a transgenic plant in which gibberellin content has been increased in the seed of the plant.

17. A method for the preparation of plant seed with an increased weight, said method comprising genetically modifying a plant with a nucleic acid sequence encoding an enzyme of gibberellin metabolism and expressing said enzyme, thereby increasing said plant's gibberellin metabolism and increasing the weight of the seed produced by said plant.

18. A method for the preparation of plant seed with an increased weight, said method comprising genetically modifying a plant with a nucleic acid sequence encoding an enzyme which renders gibberellins resistant to inactivation and expressing said enzyme, thereby rendering said plant's gibberellins resistant to inactivation and increasing the weight of the seed produced by said plant.

19. A method for the preparation of plant seed with an increased weight, said method comprising genetically modifying a plant with a nucleic acid sequence encoding a nucleic acid molecule which inhibits the expression of enzymes of gibberellin inactivation and expressing said nucleic acid molecule, thereby inhibiting said plant's expression of enzymes of gibberellin inactivation and increasing the weight of the seed produced by said plant.

20. A genetically modified plant comprising plant seed having an increased gibberellin content and an increased weight.

21. A genetically modified plant seed with an increased weight comprising an increased gibberellin content.