US20080295197A1

US20080295197A1 - Method for Modifying Plant Morphology, Biochemistry and Physiology Comprising Expression of Cytokinin Oxydase in the Seeds

Info

Publication number: US20080295197A1
Application number: US11/630,012
Authority: US
Inventors: Thomas Schmulling; Tomas Werner
Original assignee: Individual
Current assignee: Individual
Priority date: 2000-06-16
Filing date: 2005-06-20
Publication date: 2008-11-27
Also published as: JP2009159967A; CN102174541B; CN102174540B; US8859854B2; ES2333102T3; US20130007922A1; US20080222755A1; US7259296B2; CN1474872A; JP2004535753A; CN102174540A; AU8575301A; ATE440142T1; US20110023186A1; EP1290181A2; ES2468315T3; US20080307547A1; EP2103688A2; ZA200210057B; EP2192176A1

Abstract

The present invention relates to methods and compositions for increasing seed yield of a plant. The methods comprise expression of a cytokinin oxidase in the aleurone and/or embryo of a seed. The invention also relates to vectors comprising a nucleic acid encoding a cytokinin oxidase that is operably linked to a promoter capable of driving expression in the aleurone and/or embryo of a seed, and to host cells, transgenic cells and plants comprising such sequences. The use of these sequences for increasing yield is also provided.

Description

The present invention generally relates to methods for modifying plant morphological properties or characteristics, such as developmental processes, and in particular seed development and/or seed yield. The methods comprise expressing a cytokinin degradation control protein, in particular cytokinin oxidase, in the plant, operably under the control of a regulatable promoter sequence, wherein the regulatable promoter is an aleurone and/or embryo-specific promoter sequence. The present invention extends to genetic constructs which are useful in performing the method of the invention and to plants transformed therewith, the plants having altered morphological properties compared to their otherwise isogenic counterparts.
Seeds are the reproduction unit of higher plants. Seeds contain reserve compounds to ensure nutrition of the embryo after germination. These storage compounds contribute significantly to human nutrition as well as to cattle feeding. Seeds consist of three major parts, namely the embryo, the endosperm and the seed coat. Reserve compounds are deposited in storage tissue, which is either the endosperm (resulting from double fertilisation; e.g. in all cereals), the so-called perisperm (derived from the nucellus tissue) or the cotyledons (e.g. bean varieties). The endosperm is covered by a layer of dense cytoplasmic cells, known as the aleurone. Storage compounds include lipids (oil seed rape), proteins (e.g. in the aleuron of cereals) or carbohydrates (starch, oligosaccharides like raffinose).
Starch is the storage compound in the seeds of cereals. The most important cereal species are maize (yearly production ca. 570 million tonnes), rice (540 million tonnes per annum) and wheat (530 million tonnes per annum). Protein-rich seeds include different kinds of beans (Phaseolus spec., Vicia faba, Vigna spec.; ca. 20 million tonnes per annum), pea (Pisum sativum; million tonnes per annum) and soybean (Glycine max; 136 million tonnes per annum). Soybean seeds are also an important source of lipids, as are the seeds of different Brassica species (app. 30 million tonnes per annum), cotton, oriental sesame, flax, poppy, castor bean, sunflower, peanut, coconut, oilpalm and some other plants of lesser economic importance.
After fertilization, the developing seed becomes a sink organ that attracts nutritional compounds from source organs of the plant and uses them to produce the reserve compounds in storage organs, such as seeds, bulbs or tubers. The common concept predicts that cytokinins are a positive regulator of sink strength.
Numerous reports ascribe a stimulatory or inhibitory function to cytokinins in different developmental processes such as root growth and branching, control of apical dominance in the shoot, chloroplast development, and leaf senescence (Mok M. C. (1994) in Cytokines: Chemistry, Activity and Function, eds., Mok, D. W. S. & Mok, M. C. (CRC Boca Raton, Fla.), pp. 155-166). Conclusions about the biological functions of cytokinins have mainly been derived from studies on the consequences of exogenous cytokinin application or endogenously enhanced cytokinin levels (Klee, H. J. & Lanehon, M. B. (1995) in Plant Hormones: Physiology, Biochemisry and Molecular Biology, ed. Davies, P. J. (Kluwer, Dordrecht, the Netherlands), pp. 340-353, Schmülling, T., Rupp, H. M. Frank, M. & Schafer, S. (1999) in Advances in Regulation of Plant Growth and Development, eds. Sumad, M. Pac P. & Beck, E. (Peres, Prague), pp. 85-96).
The cloning of cytokinin oxidase allowed the study of the relevance of iP- and Z-type cytokinins during the whole life cycle of higher plants. The catabolic enzyme cytokinin oxidase (CKX) plays a principal role in controlling cytokinin levels in plant tissues. CKX activity has been found in a great number of higher plants and in different plant tissues. The enzyme is a FAD-containing oxidoreductase that catalyzes the degradation of cytokinins bearing unsaturated isoprenoid side chains and is sometimes referred to as a cytokinin dehydrogenase (Frébortova et al., Biochem. J. 380, 121-130, 2004). The free bases iP and Z, and their respective ribosides are the preferred substrates. The reaction products of iP catabolism are adenine and the unsaturated aldehyde 3-methyl-2-butonal (Armstrong, D. J. (1994) in Cytokinins: Chemistry, Activity and Functions, eds. Mok. D. W. S & Mok, M. C. (CRC Boca Raton, Fla.), pp. 139-154).
Cytokinin oxidase genes from various plant species have been isolated: Zea mays (Morris, R. O., Bilyeu, K. D., Laskey, J. G. & Chemch, N. N. (1999) Biochem. Biophys. Res. Commun. 255, 328-333, Houba-Herin, N., Pethe, C., d'Alayer, J. & Laloue, M. (1999) Plant J. 17, 615-626), Arabidopsis thaliana (WO 01/96580, WO 03/050287), and Dendrobium sp. (Yang et al., J. Exp. Bot. 53, 1899-1907). The manipulation of CKX gene expression is used as a powerful tool to study the relevance of iP- and Z-type cytokinins during the life cycle of higher plants; in particular the manipulation of CKX expression allowed the influence of cytokinin on root growth and shoot development and morphology, on seed size, and on leaf senescence to be determined (WO 01/96580, WO 03/050287, Werner et al. Plant Cell 15, 2632-2550, 2003). Transformants showing constitutive AtCKX mRNA expression and increased cytokinin oxidase activity manifested enhanced formation and growth of roots. Negative effects on shoot growth were also observed.
It was postulated that cytokinins play a fundamental role in maize for establishing seed size, decreasing tip kernel abortion and increased seed setting during unfavourable environmental conditions (WO 00/63401). Smigocki et al. (Proc Natl Acad Sci USA 85, 5131-5135, 1988) observed increased shoot organogenesis upon infection of stems, leaf pieces and seedling with Agrabacterium tumefaciens overexppessing isopentenyltransferase (ipt). It was therefore also postulated that enhanced levels of cytokinins in the seed would provide for improved seed size and increased seed set (WO 00/63401).
It is known in the art that constitutive CKX expression in a plant leads to increased root growth but decreased shoot growth (WO 01/96580). It has furthermore been demonstrated that expression of CKX under the control of a strong constitutive promoter in a plant results in increased seed size, increased embryo size and increased cotyledon size (WO 03/050287). However, it was not known which parts of the seeds would be best suited for CKX expression so as to achieve increased seed yield. The inventors have now shown for the first time that increased seed yield may be obtained relative to control plants when CKX is overexpressed in certain parts of the seed, namely the aleurone and/or embryo. The inventors have also shown that aleurone- and/or embryo-specific overexpression of CKX leads to a larger increase in seed yield compared to endosperm-specific CKX overexpression.
In accordance with the present invention, it has been surprisingly discovered that transgenic plants overexpressing a cytokinin oxidase gene in a confined part of the seed, such confined part being the aleurone and/or embryo, have an increased seed yield compared to corresponding wild type plants. These results are surprising, as a reduced cytokinin content would have been expected to be associated with a reduced organ growth and hence with reduced yield.
The present invention provides a method for modifying plant morphological properties, in particular for increasing seed yield of a plant, comprising expressing a cytokinin oxidase under the control of a regulatable promoter capable of driving expression in the aleurone and/or embryo of a seed. The invention also provides compositions for modifying plant morphological properties; in particular for increasing seed yield of a plant.
“Plant morphology” or “plant morphological characteristic” or similar term will, when used herein, be understood by those skilled in the art to refer to the external appearance of a plant. More particularly the plant morphological characteristic that is improved by using the methods according to the present invention is seed yield.
As used herein, the term “control plant” refers to plants that are, except for the modified or improved morphological characterisitics, as similar as possible or identical to the modified plant. Such control plants are also known as “wild type plants/lines” or “otherwise isogenic plants/lines”. The inventors have furthermore shown that the increase in seed yield is higher for plants wherein CKX is overexpressed in the aleurone and/or embryo, compared to plants wherein CKX is overexpressed in the endosperm. The term “control plants” therefore also includes plants wherein CKX is overexpressed in the endosperm.
There are several well known parameters which may be used for measuring increased seed yield which include but are not limited to: total weight of seeds, total number of seeds, total number of filled seeds, harvest index, and Thousand Kernel Weight. As described in the examples herein, the total weight of seeds may be measured by weighing all filled seeds harvested from a plant. The total number of seeds may be measured by counting the number of seeds harvested from a plant. The total number of filled seeds may be measured by counting the number of filled seeds harvested from a plant. The term “harvest index” as used herein is defined as the ratio between the total seed weight and the above ground area (mm²), multiplied by a factor 10⁶. Thousand Kernel Weight may be derived from the number of filled seeds counted and their total weight.
Advantageously, performance of the methods according to the present invention results in plants having increased yield or biomass, relative to corresponding control plants.
By “yield” is meant the amount of harvested material per area of production. The term “increased yield” encompasses an increase in biomass in one or more parts of a plant relative to the biomass of corresponding control plants. Depending on the crop, the harvested part of the plant may be different, for example it may be seed (e.g. rice, sorghum or corn when grown for seed); total above-ground biomass (e.g. corn, when used as silage, sugarcane), root (e.g. sugar beet), fruit (e.g. tomato), cotton fibres, or any other part of the plant which is of economic value. For example, the methods of the present invention are used to increase seed yield in rice and in corn. The increase in yield encompasses an increase in seed yield, which includes an increase in the total biomass of the seed (total seed weight), total number of seeds and/or an increase in the number of (filled) seeds. The increase in yield is also reflected as an increase in the Harvest Index, which is expressed as a ratio of the yield of harvestable parts, such as seeds, over the total biomass and is also reflected as an increased Thousand Kernel Weight (derived from the number of filled seeds counted and their total weight).
Thus, there is provided a method for increasing seed yield of a plant, increasing the level and/or activity of a cytokinin oxidase (CKX) in the embryo and/or aleurone of a plant seed, wherein said embryo and/or aleurone have increased levels and/or activity of cytokinin oxidase relative to other parts of said seed, and wherein the increase of seed yield comprises at least one of increased total weight of seeds, increased total number of seeds, increased number of filled seeds, increased harvest index or increased thousand kernel weight, each relative to corresponding control plants.
Yield is by its nature a complex parameter where total yield depends on a number of yield components. The parameters for increased yield of a crop are well known by a person skilled in the art. By way of example, key yield components for corn include number of plants per hectare or acre, number of ears per plant, number of rows (of seeds) per ear, number of kernels per row, and Thousand Kernel Weight. The improvement in yield as obtained in accordance with the methods of the invention, may be obtained as a result of one or more of these yield components. By way of example, key yield components for rice include number of plants per hectare or acre, number of panicles per plant, number of spikelets per panicle, seed filling rate and thousand kernel weight. The improvement in yield as obtained in accordance with the methods of the invention may be obtained as a result in one or more of these yield components, preferentially the improvement in yield is obtained primarily on the basis of an increased number of flowers per panicle and an increased seed filling rate.
Performance of the methods according to the present invention result in plants having modified seed yield. The modified yield includes at least an increase in any one or more of total weight of seeds, total seed number, number of filled seeds, thousand kernel weight and harvest index, each relative to control plants. Therefore, according to the present invention, there is provided a method for increasing one or more of: total seed number, total weight of seeds, number of filled seeds, thousand kernel weight and harvest index of plants, which method comprises modulating expression of a nucleic acid molecule encoding a CKX protein and/or modulating activity of the CKX itself in a plant in a aleurone- and/or embryo-preferred way, preferably wherein the CKX protein is encoded by a nucleic acid sequence represented by SEQ ID NO: 26, SEQ ID NO: 42, or a portion thereof or by sequences capable of hybridising therewith or wherein the CKX is represented by SEQ ID NO: 4, SEQ ID NO:36, or a homologue, derivative or active fragment thereof. Alternatively, the CKX may be encoded by a nucleic acid sequence represented by SEQ ID NO: 37, or by a portion thereof or by sequences capable of hybridising therewith, or wherein the CKX is represented by SEQ ID NO: 38, or a homologue, derivative or active fragment of any thereof.
The present invention is applicable to any plant, in particular a monocotyledonous plants and dicotyledonous plants including a fodder or forage legume, ornamental plant, food crop, tree, or shrub selected from the list comprising Acacia spp., Acer spp., Actinidia spp., Aesculus spp., Agathis australis, Albizia amara, Alsophila tricolor, Andropogon spp., Arachis spp, Areca catechu, Astelia fragrans, Astragalus cicer, Avena sativa, Baikiaea plurijuga, Betula spp., Brassica spp., Bruguiera gymnorrhiza, Burkea africana, Butea frondosa, Cadaba fannosa, Calliandra spp, Camellia sinensis, Canna indica, Capsicum spp., Cassia spp., Centroema pubescens, Chaenomeles spp., Cinnamomum cassia, Coffea arabica, Colophospermum mopane, Coronillia vana, Cotoneaster serotina, Crataegus spp., Cucumis spp., Cupressus spp., Cyathea dealbata, Cydonia oblonga, Clyptomeria japonica, Cymbopogon spp., Cynthea dealbata, Cydonia ob/onga, Dalbergia monetaria, Davallia divaricata, Desmodium spp., Dicksonia squarosa, Diheteropogon amplectens, Dioclea spp, Dolichos spp., Dorycnium rectum, Echinochloa pyramidalis, Ehrartia spp., Eleusine coracana, Eragrestis spp., Erythrina spp., Eucalyptus spp., Euclea schimperi, Eulalia villosa, Fagopyrum spp., Feijoa sellowiana, Fragaria spp., Flemingia spp, Freycinetia banksii, Geranium thunbergii, Ginkgo biloba, Glycinejavanica, Gliricidia spp, Gossypium hirsutum, Grevillea spp., Guibourtia coleosperna, Hedysarum spp., Hemarthia altissima, Heteropogon contortus, Hordeum vulgare, Hyparrhenia rufa, Hypericum erectum, Hyperthelia dissoluta, Indigo incamata, Iris spp., Leptarrhena pyrolifolia, Lespediza spp., Lettuca spp., Leucaena leucocephala, Loudetia simplex, Lotonus bainesii, Lotus spp., Macrotyloma axillare, Malus spp., Manihot esculenta, Medicago sativa, Metasequoia glyptostroboides, Musa sapientum, Nicotianum spp., Onobrychis spp., Ornithopus spp., Oryza spp., Peltophorum africanum, Pennisetum spp., Persea gratissima, Petunia spp., Phaseolus spp., Phoenix canariensis, Phornium cookianum, Photinia spp., Picea glauca, Pinus spp., Pisum sativum, Podocarpus totara, Pogonarthria fleckii, Pogonarthria squarrosa, Populus spp., Prosopis cineraria, Pseudotsuga menziesii, Pterolobium stellatum, Pyrus communis, Quercus spp., Rhaphiolepsis umbellata, Rhopalostylis sapida, Rhus natalensis, Ribes grossularia, Ribes spp., Robinia pseudoacacia, Rosa spp., Rubus spp., Salix spp., Schyzachyrium sanguineum, Sciadopitys verticillata, Sequoia sempervirens, Sequoiadendron giganteum, Sorghum bicolor, Spinacia spp., Sporobolus fimbriatus, Stiburus alopecuroides, Stylosanthos humilis, Tadehagi spp, Taxodium distichum, Themeda triandra, Trifolium spp., Triticum spp., Tsuga heterophylla, Vaccinium spp., Vicia spp. Vitis vinifera, Watsonia pyramidata, Zantedeschia aethiopica, Zea mays, amaranth, artichoke, asparagus, broccoli, brussel sprout, cabbage, canola, carrot, cauliflower, celery, collard greens, flax, kale, lentil, oilseed rape, okra, onion, potato, rice, soybean, straw, sugarbeet, sugar cane, sunflower, tomato, squash, and tea, amongst others, or the seeds of any plant specifically named above or a tissue, cell or organ culture of any of the above species.
According to a preferred embodiment of the present invention, the plant is a crop plant such as soybean, sunflower, canola, alfalfa, rapeseed, cotton, tomato, potato or tobacco. Further preferably, the plant is a monocotyledonous plant, such as sugarcane. More preferably the plant is a cereal, such as rice, maize, wheat, barley, millet, rye, sorghum or oats.
The term “plant” as used herein encompasses whole plants, ancestors and progeny of the plants and plant parts, including seeds, shoots, stems, leaves, roots (including tubers), flowers, and tissues and organs, wherein each of the aforementioned comprise the gene/nucleic acid of interest. The term “plant” also encompasses suspension cultures, callus tissue, embryos, meristematic regions, gametophytes, sporophytes, pollen and microspores, again wherein each of the aforementioned comprise the gene/nucleic acid of interest. The terms “plant” and “plant part” are used interchangeably with the terms “plants” and “plant parts”.
It should be clear that, although the invention is supported in the examples section by AtCKX genes and proteins, the inventive concept also relates to the use of cytokinin oxidases isolated from other plants and expressed in the aleurone and/or embryo of a seed of these other plants to obtain similar effects in plants as described in the examples section.
The cytokinin oxidase gene family contains at least six members from Arabidopsis. It is anticipated that functional homologs of the described Arabidopsis cytokinin oxidases can be isolated from other organisms, given the evidence for the presence of cytokinin oxidase activity in many green plants (Hare and van Staden, Physiol Plant 91:128-136, 1994; Jones and Schreiber, Plant Growth Reg 23:123-134, 1997), as well as in other organisms (Armstrong, in Cytokinins: Chemistry, Activity and Function. Eds Mok and Mok, CRC Press, pp 139-154, 1994). Therefore, the sequence of the cytokinin oxidase, functional in the invention, need not to be identical to those described herein. This invention is particularly useful for cereal crops and monocot crops in general and cytokinin oxidase genes from for example wheat or maize may be used as well (Morris et al., 1999; Rinaldi and Comandini, 1999). It is envisaged that other genes with cytokinin oxidase activity or with any other cytokinin metabolizing activity (see Za{hacek over (z)}imalová et al., Biochemistry and Molecular Biology of Plant Hormones, Hooykaas, Hall and Libbenga (Eds.), Elsevier Science, pp 141-160, 1997) can also be used for the purpose of this invention. Similarly, genes encoding proteins that would increase endogenous cytokinin-metabolizing activity can also be used for the purpose of this invention. In principle, similar phenotypes could also be obtained by interfering with genes that function downstream of cytokinin such as receptors or proteins involved in signal transduction pathways of cytokinin.
Any nucleotide sequence encoding a polypeptide with cytokinin oxidase activity may be used in the methods of the invention. For example, any of the various sequences provided herein encoding a polypeptide with cytokinin oxidase activity may be used in the methods of increasing seed yield.
The terms “protein(s)”, “peptide(s)”, “polypeptide(s)” or “oligopeptide(s)” when used herein refer to amino acids in a polymeric form of any length. These terms also include known amino acid modifications as well as non-naturally occurring amino acid residues, L-amino acid residues and D-amino acid residues.
The CKX proteins useful in the methods according to the invention are defined herein as having cytokinin oxidase/dehydrogenase activity and comprising at least 2 sequences of 17 and 19 consecutive amino acid residues respectively with a consensus sequence as shown below:
Consensus sequence 1 (17 amino acids): hTDYLhhoIGGTLSssG, (SEQ ID NO: 44)
Consensus sequence 2 (19 amino acids): cLFxushGsLGQFGIIstA, (SEQ ID NO: 45) wherein the capital letters are the standard single letter IUPAC codes for the various amino acids and the other letters symbolise the nature of the amino acids, as shown in Table 1. The right column lists for each class the particular amino acids that are allowed in the consensus sequences. This classification is based on the amino acid grouping as defined in the SMART database (Schultz et al. (1998) Proc. Natl. Acad. Sci. USA 95, 5857-5864; Letunic et al. (2002) Nucleic Acids Res 30, 242-244).

TABLE 1

Class	Key	Allowed amino acids

h	hydrophobic	G, H, L, R, T, W, Y
o	alcohol	S, T
l	aliphatic	V, I
s	small	A, D, G, N, T, V
c	charged	D, E, R
x	any	Y, R, N, F, D, H
u	tiny	A, G, S
t	turnlike	R, N

The crystal structure of maize CKX was elucidated by Malito et al. (J. Mol. Biol. 341, 1237-1249, 2004), which allowed a structure-function analysis and the identification of amino acids that were important for the catalytic activity of maize CKX. These results may be extrapolated to other CKX proteins by comparison of protein sequences.
Methods for measuring cytokinin oxidase/dehydrogenase activity are well known in the art. Suitable methods are based on the conversion of [2⁻³H]iP to adenine (Motyka et al., Plant Physiology 112, 1035-1043, 1996), on calorimetric assays (Libreros-Minotta and Tipton, Anal. Biochem. 231, 339-341, 1995) or on the measurement of reduced electron acceptors (Bilyeu et al., Plant Physiol. 125, 378-386, 2001).
The present invention relates to methods for increasing seed yield. In particular, the methods comprise expression in the aleurone and/or embryo of a seed, of a nucleic acid encoding a cytokinin oxidase selected from the group consisting of:

- (a) nucleic acids comprising a DNA sequence as given in any of SEQ ID NOs: 42, 37, 27, 1, 3, 5, 7, 9, 11, 25, 26, 28 to 31, 33 or 34, or the complement thereof,
- (b) nucleic acids comprising the RNA sequences corresponding to any of SEQ ID NOs: 42, 37, 27, 1, 3, 5, 7, 9, 11, 25, 26, 28 to 31, 33 or 34, or the complement thereof,
- (c) nucleic acids specifically hybridizing to any of SEQ ID NOs: 42, 37, 27, 1, 3, 5, 7, 9, 11, 25, 26, 28 to 31, 33 or 34, or to the complement thereof,
- (d) nucleic acids encoding a protein comprising the amino acid sequence as given in any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 32, 35, 36 or 38, or the complement thereof,
- (e) nucleic acids as defined in any of (a) to (d) characterized in that said nucleic acid is DNA, genomic DNA, cDNA, synthetic DNA or RNA wherein T is replaced by U,
- (f) nucleic acid which is degenerate compared to a nucleic acid as given in any of SEQ ID NOs: 42, 37, 27, 1, 3, 5, 7, 9, 11, 25, 26, 28 to 31, 33 or 34, or which is degenerate compared to a nucleic acid as defined in any of (a) to (e) as a result of the genetic code,
- (g) nucleic acids which are divergent from a nucleic acid encoding a protein as given in any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 35, 36 or 38, or which is divergent from a nucleic acid as defined in any of (a) to (e), due to the differences in codon usage between the organisms,
- (h) nucleic acids encoding a protein as given in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 35, 36 or 38, or nucleic acids as defined in (a) to (e) which are divergent due to the differences between alleles,
- (i) nucleic acids encoding a protein as given in any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 35, 36 or 38,
- (j) functional fragments of nucleic acids as defined in any of (a) to (i) having the biological activity of a cytokinin oxidase, and
- (k) nucleic acids encoding a plant cytokinin oxidase, comprising the consensus sequence hTDYLhhoIGGTLSssG and cLFxushGsLGQFGIIstA or comprising expression in seeds of a nucleic acid encoding a protein that reduces the level of active cytokinins in plants or plant parts.

The expression of a nucleic acid encoding a CKX polypeptide or a homologue thereof may also be increased by introducing a genetic modification (preferably in the locus of a CKX gene).
In addition to the cytokinin oxidase genes and corresponding proteins described above, a cytokinin oxidase 2 gene (CKX2) is particularly suited for use in increasing seed yield in a plant. In addition to the Arabidopsis thaliana CKX2 set forth in SEQ ID NO:36, other CKX proteins from Arabidopsis thaliana, such as the ones represented in GenBank Accessions NP_—181682, NP_—200507, NP_—849470, NP_—194703, NP_—850863 or AAG30909 may be used. CKX proteins from other species like Zea mays (for example GenBank Accessions CAE55202, CAE55200 or AAC27500), Dendrobium (GenBank CAC17752), Hordeum vulgare (GenBank AAN16383, M050082, AAM08400), or rice (GenBank NP_—913145, NP_—916348, NP_—922039) are also available for use in the methods and compositions of the present invention. A prokaryotic homologue of SEQ ID NO: 36 is represented by GenBank Accession P46377.
Therefore, the present invention more generally relates to method for increasing seed yield in a plant, said method comprising increasing the level and/or activity of a cytokinin oxidase (CKX) in the embryo and/or aleurone of a plant seed, wherein said embryo and/or aleurone have increased levels and/or activity of cytokinin oxidase relative to other parts of said seed. Preferred cytokinin oxidases to be used are encoded by the nucleic acids encoding the cytokinin oxidases as defined above. More preferably, the cyokinin oxidases to be used are from Arabidopsis thaliana, most preferably the cyokinin oxidase to be used is AtCKX2 encoded by one of SEQ ID NO: 3, 26, 37 or 42.
The terms “gene(s)”, “nucleic acid(s)”, “nucleic acid sequence(s)”, “nucleotide sequence(s)”, or “nucleic acid molecule(s)”, when used herein refer to nucleotides, either ribonucleotides or deoxyribonucleotides or a combination of both, in a polymeric form of any length. The terms furthermore include double-stranded and single-stranded DNA and RNA. The terms also include known nucleotide modifications such as methylation, cyclization and ‘caps’ and substitution of one or more of the naturally occurring nucleotides with an analog such as inosine. The terms also encompass peptide nucleic acids (PNAs), a DNA analogue in which the backbone is a pseudopeptide consisting of N-(2-aminoethyl)-glycine units rather than a sugar.
A “coding sequence” or “open reading frame” or “ORF” is defined as a nucleotide sequence that can be transcribed into mRNA and/or translated into a polypeptide when placed under the control of appropriate control sequences or regulatory sequences, i.e. when the coding sequence or ORF is present in an expressible format. This coding sequence or ORF is bound by a 5′ translation start codon and a 3′ translation stop codon. A coding sequence or ORF can include, but is not limited to RNA, mRNA, cDNA, recombinant nucleotide sequences, synthetically manufactured nucleotide sequences or genomic DNA. Such coding sequence or ORF can be interrupted by intervening nucleic acid sequences.
Genes and coding sequences essentially encoding the same protein but isolated from different sources can consist of substantially divergent nucleic acid sequences. Reciprocally, substantially divergent nucleic acid sequences can be designed to effect expression of essentially the same protein. These nucleic acid sequences are the result of e.g. the existence of different alleles of a given gene, of the degeneracy of the genetic code or of differences in codon usage. Thus, amino acids such as methionine and tryptophan are encoded by a single codon whereas other amino acids such as arginine, leucine and serine can each be translated from up to six different codons. Differences in preferred codon usage are known in the art. For example, the codon GGC (for glycine) is the most frequently used codon in Agrobacterium tumefaciens (36.2‰), is the second most frequently used codon in Oryza sativa but is used at much lower frequencies in A. thaliana and Medicago sativa (9‰ and 8.4‰, respectively). Of the four possible codons encoding glycine, this GGC codon is most preferably used in A. tumefaciens and O. sativa. However, in A. thaliana this is the GGA (and GGU) codon whereas in M. sativa this is the GGU (and GGA) codon.
Alleles exist in nature, and encompassed within the methods of the present invention is the use of these natural allelic variants. Allelic variants encompass Single Nucleotide Polymorphisms (SNPs), as well as Small Insertion/Deletion Polymorphisms (INDELs). The size of INDELs is usually less than 100 bp. SNPs and INDELs form the largest set of sequence variants in naturally occurring polymorphic strains of most organisms.
DNA sequences as defined in the current invention can be interrupted by intervening sequences. With “intervening sequences” is meant any nucleic acid sequence which disrupts a coding sequence comprising such inventive DNA sequence or which disrupts the expressible format of a DNA sequence comprising the inventive DNA sequence. Removal of the intervening sequence restores the coding sequence or the expressible format. Examples of intervening sequences include introns and mobilizable DNA sequences such as transposons. With “mobilizable DNA sequence” is meant any DNA sequence that can be mobilized as the result of a recombination event.
The methods according to the present invention may also be practised using an alternative splice variant of a nucleic acid molecule encoding a CKX protein. The term “alternative splice variant” as used herein encompasses variants of a nucleic acid molecule in which selected introns and/or exons have been excised, replaced or added. Such variants will be ones in which the biological activity of the protein remains unaffected, which can be achieved by selectively retaining functional segments of the protein. Such splice variants may be found in nature or can be manmade. Methods for making such splice variants are well known in the art. Therefore according to another aspect of the present invention, there is provided a method for modifying the morphological characteristics of plants, and in particular seed yield, comprising expression in the aleurone and/or embryo of a seed of an alternative splice variant of a nucleic acid molecule encoding a CKX. Preferably, the splice variant is a splice variant of a sequence represented by SEQ ID NO: 1, 3, 5, 7, 9, 11 or 33. A preferred splice variant of AtCKX2 is as represented by SEQ ID NO: 38.
Also useful in the methods of in the present invention are nucleic acids capable of hybridising under reduced stringency conditions, preferably under stringent conditions, with a CKX encoding nucleic acid. Preferred is a nucleic acid capable of hybridising to a nucleic acid represented by SEQ ID NO: 42 or 26.
“Hybridization” is the process wherein substantially homologous complementary nucleotide sequences anneal to each other. The hybridization process can occur entirely in solution, i.e. both complementary nucleic acids are in solution. Tools in molecular biology relying on such a process include PCR, subtractive hybridization and DNA sequence determination. The hybridization process can also occur with one of the complementary nucleic acids immobilized to a matrix such as magnetic beads, Sepharose beads or any other resin. Tools in molecular biology relying on such a process include the isolation of poly (A+) mRNA. The hybridization process can furthermore occur with one of the complementary nucleic acids immobilized to a solid support such as a nitrocellulose or nylon membrane or immobilized by e.g. photolithography to e.g. a silicious glass support (the latter known as nucleic acid arrays or microarrays or as nucleic acid chips). Tools in molecular biology relying on such a process include RNA and DNA gel blot analysis, colony hybridization, plaque hybridization and microarray hybridization. In order to allow hybridization to occur, the nucleic acid molecules are generally thermally or chemically (e.g. by NaOH) denatured to melt a double strand into two single strands and/or to remove hairpins or other secondary structures from single stranded nucleic acids. The stringency of hybridization is influenced by conditions such as temperature, salt concentration and hybridization buffer composition. High stringency conditions for hybridization include high temperature and/or low salt concentration (salts include NaCl and Na₃-citrate) and/or the inclusion of formamide in the hybridization buffer and/or lowering the concentration of compounds such as SDS (detergent) in the hybridization buffer and/or exclusion of compounds such as dextran sulfate or polyethylene glycol (promoting molecular crowding) from the hybridization buffer. Conventional hybridization conditions are described in e.g. Sambrook et al. (1989) but the skilled craftsman will appreciate that numerous different hybridization conditions can be designed in function of the known or the expected homology and/or length of the nucleic acid sequence. Sufficiently low stringency hybridization conditions are particularly preferred to isolate nucleic acids heterologous to the DNA sequences of the invention defined supra. Elements contributing to heterology include allelism, degeneration of the genetic code and differences in preferred codon usage as discussed supra.
The term “specifically hybridizing” or “hybridizing specifically” refers to the binding, duplexing, or hybridizing of a molecule to a particular nucleotide sequence under medium to stringent conditions when that sequence is presented in a complex mixture e.g., total cellular DNA or RNA.
“Stringent hybridization conditions” and “stringent hybridization wash conditions” in the context of nucleic acid hybridization experiments such as Southern and Northern hybridizations are sequence dependent and are different under different environmental parameters. For example, longer sequences hybridize specifically at higher temperatures. The T_mis the temperature under defined ionic strength and pH, at which 50% of the target sequence hybridizes to a perfectly matched probe. Specificity is typically the function of post-hybridization washes. Critical factors of such washes include the ionic strength and temperature of the final wash solution.
Generally, stringent conditions are selected to be about 50° C. lower than the thermal melting point (T_m) for the specific sequence at a defined ionic strength and pH. The T_mis the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. The T_mis dependent upon the solution conditions and the base composition of the probe, and may be calculated using the following equation:
$T_{m} = 79.8 ° C . + (18.5 \times Log [Na +]) + (58.4 ° C . \times % [G + C]) - (820 / # bp in duplex) - (0.5 \times % formamide)$
More preferred stringent conditions are when the temperature is 20° C. below T_m, and the most preferred stringent conditions are when the temperature is 10° C. below T_m. Nonspecific binding may also be controlled using any one of a number of known techniques such as, for example, blocking the membrane with protein-containing solutions, addition of heterologous RNA, DNA, and SDS to the hybridization buffer, and treatment with RNase.
Wash conditions are typically performed at or below stringency. Generally, suitable stringent conditions for nucleic acid hybridization assays or gene amplification detection procedures are as set forth above. More or less stringent conditions may also be selected.
For the purposes of defining the level of stringency, reference can conveniently be made to Sambrook, J., E. F. Fritsch, et al. 1989 “Molecular Cloning: a Laboratory Manual, 2^ndEdition, Cold Spring Harbor, N.Y., Cold Spring Harbor Laboratory Press. An example of low stringency conditions is 4-6×SSC/0.1-0.5% w/v SDS at 37°-45° C. for 2-3 hours. Depending on the source and concentration of the nucleic acid involved in the hybridization, alternative conditions of stringency may be employed such as medium stringent conditions. Examples of medium stringent conditions include 14×SSC/0.25% w/v SDS at ≧45° C. for 2-3 hours. An example of high stringency conditions includes 0.1-1×SSC/0.1% w/v SDS at 60 C for 1-3 hours. The skilled artisan is aware of various parameters which may be altered during hybridization and washing and which will either maintain or change the stringency conditions. For example, another stringent hibridization condition is hybridization at 4×SSC at 65° C., followed by a washing in 0.1×SSC at 65° C. for about one hour. Alternatively, an exemplary stringent hybridization condition is in 50% formamide, 4×SSC, at 42° C. Still another example of stringent conditions include hybridization at 62° C. in 6×SSC, 0.05× BLOTTO, and washing at 2×SSC, 0.1% SDS at 62° C.
Clearly, the current invention embodies the use of DNA sequences hybridising to DNA sequences encoding a cytokinin oxidase, homologue, derivative or immunologically active and/or functional fragment thereof as defined below in methods for increasing seed yield comprising expression of these DNA sequences in the aleurone and/or embryo of a seed. Preferably the cytokinin oxidase is a plant cytokinin oxidase, more specifically the Arabidopsis thaliana (At)CKX.
“Homologues” of a CKX polypeptide may also be useful in the present invention.
“Homologues” of a CKX useful in the methods of the invention are those peptides, oligopeptides, polypeptides, proteins and enzymes which contain amino acid substitutions, deletions and/or additions relative to the protein with respect to which they are a homologue, without altering one or more of its functional properties, in particular without reducing the activity of the resulting protein. For example, a homologue of a CKX will consist of a bioactive amino acid sequence variant of this CKX.
Two special forms of homology, orthologous and paralogous homology, are evolutionary concepts used to describe ancestral relationships of genes. The term “paralogous” relates to homologous genes that result from one or more gene duplications within the genome of a species. The term “orthologous” relates to homologous genes in different organisms due to ancestral relationship of these genes. The term “homologues” as used herein also encompasses paralogues and orthologues of the proteins useful in the methods according to the invention. Orthologous genes can be identified by querying one or more gene databases with a query gene of interest, using for example, the BLAST program. The highest-ranking subject genes that result from the search are then again subjected to a BLAST analysis, and only those subject genes that match again with the query gene are retained as true orthologous genes. For example, to find a rice orthologue of an Arabidopsis thaliana gene, one may perform a BLASTN or TBLASTX analysis on a rice database (such as (but not limited to) the Oryza sativa Nipponbare database available at the NCBI (http://www.ncbi.nim.nih.gov) or the genomic sequences of rice (cultivars indica or japonica)). In a next step, the obtained rice sequences are used in a reverse BLAST analysis using an Arabidopsis database. The results may be further refined when the resulting sequences are analysed with ClustalW and visualised in a neighbour joining tree. The method can be used to identify orthologues from many different species.
BLAST (Basic Local Alignment Search Tool) is a family of programs (http://www.ncbi.nlm.nih.gov/BLAST/) aiming to identify regions of optimal local alignment, i.e. the alignment of some portion of two nucleic acid or protein sequences, and to detect relationships among sequences which share only isolated regions of similarity (Altschul et al., Nucleic Acids Res. 25: 3389-3402 (1997)).
To produce such homologues, amino acids present in the protein can be replaced by other amino acids having similar properties, for example hydrophobicity, hydrophilicity, hydrophobic moment, antigenicity, propensity to form or break α-helical structures or β-sheet structures, and so on. An overview of physical and chemical properties of amino acids is given in Table 2.

TABLE 2

Properties of naturally occurring amino acids.

Charge properties/
hydrophobicity	Side group	Amino Acid

Nonpolar	Aliphatic	ala, ile, leu, val
hydrophobic	aliphatic, S-containing	met
	aromatic	phe, trp
	imino	pro
polar uncharged	Aliphatic	gly
	Amide	asn, gln
	Aromatic	tyr
	Hydroxyl	ser, thr
	Sulfhydryl	cys
Positively charged	Basic	arg, his, lys
Negatively charged	Acidic	asp, glu

The homologues useful in the methods according to the invention preferably have cytokinin oxidase/dehydrogenase activity and comprise at least 2 sequences of 17 and 19 consecutive amino acid residues respectively with a consensus sequence as shown below:
Consensus sequence 1 (17 amino acids): hTDYLhhoIGGTLSssG, (SEQ ID NO: 44)
Consensus sequence 2 (19 amino acids): cLFxushGsLGQFGIIstA, (SEQ ID NO: 45) wherein the capital letters are the standard single letter IUPAC codes for the various amino acids and the other letters symbolise the nature of the amino acids, as shown in table 2 above. The right column lists for each class the particular amino acids that are allowed in the consensus sequences.
Substitutional variants of a protein of the invention are those in which at least one residue in the protein or amino acid sequence has been removed and a different residue inserted in its place. Amino acid substitutions are typically of single residues, but may be clustered depending upon functional constraints placed upon the polypeptide; insertions will usually be of the order of about 1-10 amino acid residues and deletions will range from about 1-20 residues. Preferably, amino acid substitutions will comprise conservative amino acid substitutions, such as those described supra.
Insertional amino acid sequence variants of a protein of the invention are those in which one or more amino acid residues are introduced into a predetermined site in the protein. Insertions can comprise amino-terminal and/or carboxy-terminal fusions as well as intra-sequence insertions of single or multiple amino acids. Generally, insertions within the amino acid sequence will be smaller than amino or carboxyl terminal fusions, of the order of about 1 to 10 residues. Examples of amino- or carboxy-terminal fusion proteins or peptides include the binding domain or activation domain of a transcriptional activator as used in a two-hybrid system, phage coat proteins, (histidine)₆-tag, glutathione S-transferase, protein A, maltose-binding protein, dihydrofolate reductase, Tag100 epitope (EETARFQPGYRS), c-myc epitope (EQKLISEEDL), FLAG®-epitope (DYKDDDK), lacZ, CMP (calmodulin-binding peptide), HA epitope (YPYDVPDYA), protein C epitope (EDQVDPRLIDGK) and VSV epitope (YTDIEMNRLGK).
Deletional variants of a protein of the invention are characterized by the removal of one or more amino acids from the amino acid sequence of the protein.
Amino acid variants of a protein of the invention may readily be made using peptide synthetic techniques well known in the art, such as solid phase peptide synthesis and the like, or by recombinant DNA manipulations. The manipulation of DNA sequences to produce variant proteins which manifest as substitutional, insertional or deletional variants are well known in the art. For example, techniques for making substitution mutations at predetermined sites in DNA having known sequence are well known to those skilled in the art, such as by M13 mutagenesis, T7-Gen in vitro mutagenesis kit (USB, Cleveland, Ohio), QuickChange Site Directed mutagenesis kit (Stratagene, San Diego, Calif.), PCR-mediated site-directed mutagenesis or other site-directed mutagenesis protocols.
Derivatives of a CKX protein may also be useful in the methods of the present invention. “Derivatives” of a protein of the invention are those peptides, oligopeptides, polypeptides, proteins and enzymes which comprise at least about five contiguous amino acid residues of the polypeptide but which retain the biological activity of this protein. A “derivative” may further comprise additional naturally-occurring, altered glycosylated, acylated or non-naturally occurring amino acid residues compared to the amino acid sequence of a naturally-occurring form of the polypeptide. Alternatively or in addition, a derivative may comprise one or more non-amino acid substituents compared to the amino acid sequence of a naturally-occurring form of the polypeptide, for example a reporter molecule or other ligand, covalently or non-covalently bound to the amino acid sequence such as, for example, a reporter molecule which is bound thereto to facilitate its detection.
With “immunologically active” is meant that a molecule or specific fragments thereof such as specific epitopes or haptens are recognized by, i.e. bind to antibodies. Specific epitopes may be determined using, for example, peptide scanning techniques as described in Geysen et al. (1996) (Geysen, H. M., Rodda, S. J. and Mason, T. J. (1986). A priori delineation of a peptide which mimics a discontinuous antigenic determinant. Mol. Immunol. 23, 709-715.).
The term “fragment of a sequence” or “part of a sequence” means a truncated sequence of the original sequence referred to. The truncated sequence (nucleic acid or protein sequence) can vary widely in length; the minimum size being a sequence of sufficient size to provide a sequence with at least a comparable function and/or activity or the original sequence referred to (e.g. “functional fragment”), while the maximum size is not critical. In some applications, the maximum size usually is not substantially greater than that required to provide the desired activity and/or function(s) of the original sequence. Typically, the truncated amino acid sequence will range from about 5 to about 60 amino acids in length. More typically, however, the sequence will be a maximum of about 50 amino acids in length, preferably a maximum of about 60 amino acids. It is usually desirable to select sequences of at least about 10, 12 or 15 amino acids, up to a maximum of about 20 or 25 amino acids. Functional fragments comprise at least 50 amino acids, include an FAD binding domain (as defined in Pfam (version 14.0, june 2004) accession number 1565, Bateman et al., Nucleic Acids Research Database Issue 32, D138-D141, 2004) and exhibit cytokinin oxidase/dehydrogenase activity. Most preferably, functional fragments exhibit cytokinin oxidase/dehydrogenase activity and comprise an amino acid sequence corresponding to the sequence spanning from Leu 94 to Leu 461 of SEQ ID NO: 26. Functional fragments can also include those comprising an epitope which is specific for the CKX proteins.
It should thus be understood that functional fragments may also be immunologically active fragments.
In the methods of the invention use of homologues, derivatives and/or immunologically active and/or functional fragments of the cytokinin oxidases as defined supra is contemplated. Particularly preferred homologues, derivatives and/or immunologically active and/or functional fragments of the cytokinin oxidase proteins which are contemplated for use in the methods of the current invention are derived from plants, more specifically from Arabidopsis thaliana, even more specifically the cytokinin oxidases are the Arabidopsis thaliana (At)CKX, or are capable of being expressed in the aleurone and/or embryo of a seed of cereals, preferably of rice or corn. The present invention clearly contemplates the use of functional homologues or derivatives and/or immunologically active fragments of the AtCKX proteins and is not to be limited in application to the use of a nucleotide sequence encoding one of these AtCKX proteins in the methods of the present invention.
To effect expression of a CKX protein in a cell, tissue or organ, preferably of plant origin, either the protein may be introduced directly to the cell, such as by microinjection or ballistic means or alternatively, an isolated nucleic acid molecule encoding the CKX protein may be introduced into the cell, tissue or organ in an expressible format.
In the context of the present invention it should be understood that the term “expression” and/or ‘overexpression’ are used interchangeably and both relate to an “enhanced and/or ectopic expression” of a plant cytokinin oxidase. It should be clear that herewith an enhanced expression of the plant cytokinin oxidase as well as “de novo” expression of plant cytokinin oxidases is meant. Methods for increasing expression of genes or proteins are well documented in the art and include, for example, overexpression driven by appropriate promoters, the use of transcription enhancers or translation enhancers. Isolated nucleic acids which serve as promoter or enhancer elements may be introduced in an appropriate position (typically upstream) of a non-heterologous form of a polynucleotide so as to upregulate expression of a CKX-encoding nucleic acid or variant thereof. For example, endogenous promoters may be altered in vivo by mutation, deletion, and/or substitution (see, Kmiec, U.S. Pat. No. 5,565,350; Zarling et al., PCT/US93/03868), or isolated promoters may be introduced into a plant cell in the proper orientation and distance from a gene of the present invention so as to control the expression of the gene. Methods for reducing the expression of genes or gene products are well documented in the art.
The expression of a nucleic acid encoding a CKX polypeptide or a homologue thereof may also be increased in the aleurone and/or embryo of a seed by introducing a genetic modification (preferably in the locus of a CKX gene). The locus of a gene as defined herein is taken to mean a genomic region, which includes the gene of interest and 10 kb up- or down stream of the coding region.
The genetic modification may be introduced, for example, by any one (or more) of the following methods: T-DNA activation, TILLING, site-directed mutagenesis, directed evolution and homologous recombination or by introducing and expressing in a plant a nucleic acid encoding a CKX polypeptide or a homologue thereof. Following introduction of the genetic modification, there follows a step of selecting for modified expression of a nucleic acid encoding a CKX polypeptide or a homologue thereof, which modification in expression gives plants having increased yield.
T-DNA activation tagging (Hayashi et al. Science (1992) 1350-1353) involves insertion of T-DNA, usually containing a promoter (may also be a translation enhancer or an intron), in the genomic region of the gene of interest or 10 kb up- or down stream of the coding region of a gene in a configuration such that the promoter directs expression of the targeted gene. Typically, regulation of expression of the targeted gene by its natural promoter is disrupted and the gene falls under the control of the newly introduced promoter. The promoter is typically embedded in a T-DNA. This T-DNA is randomly inserted into the plant genome, for example, through Agrobacterium infection and leads to overexpression of genes near the inserted T-DNA. The resulting transgenic plants show dominant phenotypes due to overexpression of genes close to the introduced promoter. According to the present invention, the promoter to be introduced is a promoter capable of driving expression in the aleurone and/or embryo of a seed.
A genetic modification may also be introduced in the locus of a CKX gene using the technique of TILLING (Targeted Induced Local Lesions In Genomes). This is a mutagenesis technology useful to generate and/or identify, and to eventually isolate mutagenised variants of a CKX nucleic acid capable of exhibiting CKX activity. TILLING also allows selection of plants carrying such mutant variants. These mutant variants may even exhibit higher CKX activity than that exhibited by the gene in its natural form. TILLNG combines high-density mutagenesis with high-throughput screening methods. The steps typically followed in TILLING are: (a) EMS mutagenesis (Redei G P and Koncz C (1992) In Methods in Arabidopsis Research, Koncz C, Chua N H, Schell J, eds. Singapore, World Scientific Publishing Co, pp. 16-82; Feldmann et al., (1994) In Meyerowitz E M, Somerville C R, eds, Arabidopsis. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp 137-172; Lightner J and Caspar T (1998) In J Martinez-Zapater, J Salinas, eds, Methods on Molecular Biology, Vol. 82. Humana Press, Totowa, N.J., pp 91-104); (b) DNA preparation and pooling of individuals; (c) PCR amplification of a region of interest; (d) denaturation and annealing to allow formation of heteroduplexes; (e) DHPLC, where the presence of a heteroduplex in a pool is detected as an extra peak in the chromatogram; (e) identification of the mutant individual; and (g) sequencing of the mutant PCR product. Methods for TILLING are well known in the art (McCallum et al., (2000) Nat Biotechnol 18: 455-457; reviewed by Stemple (2004) Nat Rev Genet. 5(2): 145-50).
Site-directed mutagenesis may be used to generate variants of CKX nucleic acids. Several methods are available to achieve site-directed mutagenesis, the most common being PCR based methods (current protocols in molecular biology. Wiley Eds. http://www.4ulr.com/products/currentprotocols/index.html).
Directed evolution may also be used to generate variants of CKK nucleic acids. This consists of iterations of DNA shuffling followed by appropriate screening and/or selection to generate variants of CKX nucleic acids or portions thereof encoding CKX polypeptides or homologues or portions thereof having an modified biological activity (Castle et al., (2004) Science 304(5674): 1151-4; U.S. Pat. Nos. 5,811,238 and 6,395,547).
T-DNA activation, TILLING, site-directed mutagenesis and directed evolution are examples of technologies that enable the generation of novel alleles and CKX variants.
Homologous recombination allows introduction in a genome of a selected nucleic acid at a defined selected position. Homologous recombination is a standard technology used routinely in biological sciences for lower organisms such as yeast or the moss Physcomitrella. Methods for performing homologous recombination in plants have been described not only for model plants (Offring a et al. (1990) EMBO J. 9(10): 3077-84) but also for crop plants, for example rice (Terada et al. (2002) Nat Biotech 20(10): 1030-4; Iida and Terada (2004) Curr opin Biotech 15(2):132-8). The nucleic acid to be targeted (which may be a CKX nucleic acid or variant thereof as hereinbefore defined) need not be targeted to the locus of a CKX gene, but may be introduced in, for example, regions of high expression. The nucleic acid to be targeted may be an improved allele used to replace the endogenous gene or may be introduced in addition to the endogenous gene.
The invention also relates to a method for the production of modified plants having increased seed yield, comprising the introduction of a genetic modification, which genetic modification results in increased expression of cytokinin oxidase in the aleurone and/or embryo of a seed.
The invention furthermore relates to a method for increasing seed yield in a plant comprising the introduction of a nucleic acid molecule encoding a CKX as defined above, operably linked to one or more control sequences or a vector stably integrated into the genome of a plant cell capable of driving expression in the aleurone and/or embryo of a seed.
Therefore, the invention also relates to a vector comprising a nucleic acid encoding a CKX as defined above, wherein the vector is an expression vector and wherein the nucleic acid encoding a CKX is operably linked to one or more control sequences allowing the expression in the aleurone and/or embryo of a seed.
In the present invention, the inventors have surprisingly shown that the expression of cytokinin oxidases in the aleurone and/or embryo of a seed resulted in the above-mentioned seed-related features. Examples of seed-specific promoters include but are not limited to those listed in Table 3.

TABLE 3

Examples of plant-expressible promoters capable
of driving expression in the seed

	EXPRESSION
GENE SOURCE	PATTERN	REFERENCE

α-amylase (Amy32b)	aleurone	Lanahan, M. B., et al., Plant Cell
		4: 203-211, 1992; Skriver, K., et al.
		Proc. Natl. Acad. Sci. (USA) 88:
		7266-7270, 1991
cathepsin β-like gene	aleurone	Cejudo, F. J., et al. Plant Molecular
		Biology 20: 849-856, 1992.
seed-specific genes	seed	Simon, et al., Plant Mol. Biol. 5: 191,
		1985; Scofield, et al., J. Biol. Chem.
		262: 12202, 1987.; Baszczynski, et
		al., Plant Mol. Biol. 14: 633, 1990.
Brazil Nut albumin	seed	Pearson, et al., Plant Mol. Biol. 18:
		235-245, 1992.
Legumin	seed	Ellis, et al., Plant Mol. Biol. 10: 203-
		214, 1988.
glutelin (rice)	seed	Takaiwa, et al., Mol. Gen. Genet. 208:
		15-22, 1986; Takaiwa, et al., FEBS
		Letts. 221: 43-47, 1987.
Zein	seed	Matzke et al Plant Mol Biol,
		14(3): 323-32 1990
NapA	seed	Stalberg, et al, Planta 199: 515-519,
		1996.
wheat LMW and HMW	endosperm	Mol Gen Genet 216: 81-90, 1989;
glutenin-1		NAR 17: 461-2, 1989
wheat SPA	seed	Albani et al, Plant Cell, 9: 171-184,
		1997
wheat α, β, γ-gliadins	endosperm	EMBO 3: 1409-15, 1984
barley ltr1 promoter	endosperm
barley B1, C, D,	endosperm	Theor Appl Gen 98: 1253-62, 1999;
hordein		Plant J 4: 343-55, 1993; Mol Gen
		Genet 250: 750-60, 1996
barley DOF	endosperm	Mena et al, The Plant Journal, 116(1):
		53-62, 1998
blz2	endosperm	EP99106056.7
synthetic promoter	endosperm	Vicente-Carbajosa et al., Plant J. 13:
		629-640, 1998.
rice prolamin NRP33	endosperm	Wu et al, Plant Cell Physiology 39(8)
		885-889, 1998
rice α-globulin Glb-1	endosperm	Wu et al, Plant Cell Physiology 39(8)
		885-889, 1998
rice OSH1	embryo	Sato et al, Proc. Natl. Acad. Sci. USA,
		93: 8117-8122, 1996
rice α-globulin	endosperm	Nakase et al. Plant Mol. Biol. 33: 513-
REB/OHP-1		522, 1997
rice ADP-glucose PP	endosperm	Trans Res 6: 157-68, 1997
maize ESR gene family	endosperm	Plant J 12: 235-46, 1997
sorgum γ-kafirin	endosperm	PMB 32: 1029-35, 1996
KNOX	embryo	Postma-Haarsma et al, Plant Mol.
		Biol. 39: 257-71, 1999
rice oleosin	embryo and aleuron	Wu et at, J. Biochem., 123: 386, 1998
sunflower oleosin	seed (embryo and	Cummins, et al., Plant Mol. Biol. 19:
	dry seed)	873-876, 1992

Further examples of promoters suitable for seed specific expression in a plant may be found in the examples section, Table 4.
In accordance with the present invention, there are provided methods and compositions for increasing seed yield in a plant. Seed yield may be increased by increasing the expression of a cytokinin oxidase gene in the embryo and/or aleurone of a plant seed. Thus, an embryo and/or aleurone preferred promoter may be utilized to drive expression of a cytokinin oxidase in these particular components of a seed.
In accordance with the present invention, a cytokinin oxidase gene may be placed in a genetic construct such as a vector, under control of a embryo and/or aleurone-preferred promoter. An example of such a promoter capable of driving expression in the embryo and/or aleurone is the sequence as represented in GenBank under accession number AF019212 (sequence from nucleotide 1 to 1256, hereafter named PRO0218).
For example, the AtCKX2 gene (SEQ ID NO: 42) may be placed under control of a promoter capable of driving expression in the seed, more particularly a promoter capable of driving expression in the embryo and/or aleurone, for example PRO0218.
Therefore, there is provided a vector comprising the isolated nucleic acid molecule having the sequence set forth in SEQ ID NO: 41 or 43.
These constructs may then be used to transform plants, either dicotyledonous or monocotyledonous. In accordance with the present invention, plants transformed with a vector according to the present invention have an increased seed yield, when compared to nullizygous control plants.
Preferably, the vector of the invention comprises a coding sequence or open reading frame (ORF) encoding a cytokinin oxidase protein or a homologue or derivative thereof or an immunologically active and/or functional fragment thereof as defined supra. Preferably the cytokinin oxidase is a plant cytokinin oxidase and more specifically an Arabidopsis thaliana (At)CKX. Most preferably the CKX is as represented by one of SEQ ID NO: 4, 36 or 38.
With “vector” or “vector sequence” or “genetic construct” is meant a DNA sequence which can be introduced in an organism by transformation and can be stably maintained in this organism. Vector maintenance is possible in e.g. cultures of Escherichia coli, A. tumefaciens, Saccharomyces cerevisiae or Schizosaccharomyces pombe. Other vectors such as phagemids and cosmid vectors can be maintained and multiplied in bacteria and/or viruses. Vector sequences generally comprise a set of unique sites recognized by restriction enzymes, the multiple cloning site (MCS), wherein one or more non-vector sequence(s) can be inserted. As an alternative to multiple cloning sites, the vector may also comprise recombination sites. Gene cloning through recombination is well known in the art.
With “non-vector sequence” is accordingly meant a DNA sequence which is integrated in one or more of the sites of the MCS comprised within a vector.
“Expression vectors” form a subset of vectors which, by virtue of comprising the appropriate regulatory or control sequences enable the creation of an expressible format for the inserted non-vector sequence(s), thus allowing expression of the protein encoded by this non-vector sequence(s). Expression vectors are known in the art enabling protein expression in organisms including bacteria (e.g. E. coli), fungi (e.g. S. cerevisiae, S. pombe, Pichia pastoris), insect cells (e.g. baculoviral expression vectors), animal cells (e.g. COS or CHO cells) and plant cells (e.g. potato virus X-based expression vectors).
By “expressible format” is meant that the isolated nucleic acid molecule is in a form suitable for being transcribed into mRNA and/or translated to produce a protein, either constitutively or following induction by an intracellular or extracellular signal, such as an environmental stimulus or stress (mitogens, anoxia, hypoxia, temperature, salt, light, dehydration, etc) or a chemical compound such as IPTG (isopropyl-β-D-thiogalactopyranoside) or such as an antibiotic (tetracycline, ampicillin, rifampicin, kanamycin), hormone (e.g. gibberellin, auxin, cytokinin, glucocorticoid, brassinosteroid, ethylene, abscisic acid etc), hormone analogue (indoleacetic acid (IAA), 2,4-D, etc), metal (zinc, copper, iron, etc), or dexamethasone, amongst others. As will be known to those skilled in the art, expression of a functional protein may also require one or more post-translational modifications, such as glycosylation, phosphorylation, dephosphorylation, or one or more protein-protein interactions, amongst others. All such processes are included within the scope of the term “expressible format”.
It should be understood that for expression in monocots of the cytokinin oxidase genes according to the methods of the invention, a nucleic acid sequence corresponding to the cDNA sequence should be used to avoid mis-splicing of introns in monocots. Preferred cDNA sequences to be expressed in monocots have a nucleic acid sequence as represented in any of SEQ ID NOs: 25 to 30, SEQ ID NO: 34, SEQ ID NO: 37, or SEQ ID NO: 42.
Constitutive expression of the cytokinin oxidase gene in plants resulted in increased root growth and decreased shoot growth, illustrating the importance of confined expression of the cytokinin oxidase gene for general plant growth properties. Containment of cytokinin oxidase activity can be achieved by using cell-, tissue- or organ-specific promoters, since cytokinin degradation is a process limited to the tissues or cells that express the CKX protein, this in contrast to approaches relying on hormone synthesis.
Preferably, expression of a protein in a specific cell, tissue, or organ, preferably of plant origin, is effected by introducing and expressing an isolated nucleic acid molecule encoding the protein, such as a cDNA molecule, genomic gene, synthetic oligonucleotide molecule, mRNA molecule or open reading frame, into this cell, tissue or organ, wherein the nucleic acid molecule is placed operably in connection with suitable regulatory or control sequences including a promoter, preferably a plant-expressible promoter, and a terminator sequence. In particular, and according to the methods of the present invention, a nucleic acid sequence encoding a CKX is operably linked to a promoter capable of driving expression in the aleurone and/or embryo of a seed.
Reference herein to a “promoter” is to be taken in its broadest context and includes the transcriptional regulatory sequences derived from a classical eukaryotic genomic gene, including the TATA box which is required for accurate transcription initiation, with or without a CCAAT box sequence and additional regulatory or control elements (i.e. upstream activating sequences, enhancers and silencers) which alter gene expression in response to developmental and/or external stimuli, or in a tissue-specific manner.
The term “promoter” also includes the transcriptional regulatory sequences of a classical prokaryotic gene, in which case it may include a −35 box sequence and/or a −10 box transcriptional regulatory sequences.
The term “promoter” is also used to describe a synthetic or fusion molecule, or derivative which confers, activates or enhances expression of a nucleic acid molecule in a cell, tissue or organ.
Promoters may contain additional copies of one or more specific regulatory elements, to further enhance expression and/or to alter the spatial expression and/or temporal expression of a nucleic acid molecule to which it is operably linked. Such regulatory elements may be placed adjacent to a heterologous promoter sequence to drive expression of a nucleic acid molecule in response to e.g. copper, glucocorticoids, dexamethasone, tetracycline, gibberellin, cAMP, abscisic acid, auxin, wounding, ethylene, jasmonate or salicylic acid or to confer expression of a nucleic acid molecule to specific cells, tissues or organs such as meristems, leaves, roots, embryo, flowers, seeds or fruits.
In the context of the present invention, the promoter is a plant-expressible promoter sequence, capable of driving expression in the aleurone and/or embryo of a seed. By “plant-expressible” is meant that the promoter sequence, including any additional regulatory elements added thereto or contained therein, is at least capable of inducing, conferring, activating or enhancing expression in a plant cell, tissue or organ, preferably a monocotyledonous or dicotyledonous plant cell, tissue, or organ, in casu the aleurone and/or embryo.
The terms “plant-operable” and “operable in a plant” when used herein, in respect of a promoter sequence, shall be taken to be equivalent to a plant-expressible promoter sequence.
Regulatable promoters as part of a binary viral plant expression system are also known to the skilled artisan (Yadav 1999-WO9922003; Yadav 2000-WO0017365).
In the present context, a “regulatable promoter sequence” is a promoter that is capable of conferring expression on a structural gene in a particular cell, tissue, or organ or group of cells, tissues or organs of a plant, optionally under specific conditions, however does generally not confer expression throughout the plant under all conditions. Accordingly, a regulatable promoter sequence may be a promoter sequence that confers expression on a gene to which it is operably linked in a particular location within the plant or alternatively, throughout the plant under a specific set of conditions, such as following induction of gene expression by a chemical compound or other elicitor.
Preferably, the regulatable promoter used in the methods of the present invention confers expression in a specific location within the plant, either constitutively or following induction, however not in the whole plant under any circumstances. In particular, the regulatable promoter for use in the present invention is a promoter capable of driving expression in the aleurone and/or embryo of a seed. Other types of such promoters are cell-specific promoter sequences, tissue-specific promoter sequences, organ-specific promoter sequences, cell cycle specific gene promoter sequences, inducible promoter sequences and constitutive promoter sequences that have been modified to confer expression in a particular part of the plant at any one time, such as by integration of such constitutive promoter within a transposable genetic element (Ac, Ds, Spm, En, or other transposon).
The term “cell-specific” shall be taken to indicate that expression is predominantly in a particular cell or cell-type, preferably of plant origin, albeit not necessarily exclusively in this cell or cell-type. The term “tissue-specific” shall be taken to indicate that expression is predominantly in a particular tissue or tissue-type, preferably of plant origin, albeit not necessarily exclusively in this tissue or tissue-type. Similarly, the term “organ-specific” shall be taken to indicate that expression is predominantly in a particular organ, preferably of plant origin, albeit not necessarily exclusively in this organ. Similarly, the term “cell cycle specific” shall be taken to indicate that expression is predominantly cyclic and occurring in one or more, not necessarily consecutive phases of the cell cycle albeit not necessarily exclusively in cycling cells, preferably of plant origin.
Those skilled in the art will be aware that an “inducible promoter” is a promoter the transcriptional activity of which is increased or induced in response to a developmental, chemical, environmental, or physical stimulus. Similarly, the skilled craftsman will understand that a “constitutive promoter” is a promoter that is transcriptionally active throughout most, but not necessarily all parts of an organism, preferably a plant, during most, but not necessarily all phases of its growth and development.
Those skilled in the art will readily be capable of selecting appropriate promoter sequences for use according to the present invention from publicly-available or readily-available sources, without undue experimentation.
Placing a nucleic acid molecule under the regulatory control of a promoter sequence, or operably linking with a promoter sequence, means positioning the nucleic acid molecule such that expression is controlled by the promoter sequence. A promoter is usually, but not necessarily, positioned upstream, or at the 5′-end, and within 2 kb of the start site of transcription, of the nucleic acid molecule which it regulates. In the construction of heterologous promoter/structural gene combinations it is generally preferred to position the promoter at a distance from the gene transcription start site that is approximately the same as the distance between that promoter and the gene it controls in its natural setting (i.e., the gene from which the promoter is derived). As is known in the art, some variation in this distance can be accommodated without loss of promoter function. Similarly, the preferred positioning of a regulatory sequence element with respect to a heterologous gene to be placed under its control is defined by the positioning of the element in its natural setting (i.e., the gene from which it is derived). Again, as is known in the art, some variation in this distance can also occur.
The term “terminator” refers to a DNA sequence at the end of a transcriptional unit which signals termination of transcription. Terminators are 3′-non-translated DNA sequences containing a polyadenylation signal, which facilitates the addition of polyadenylate sequences to the 3′-end of a primary transcript. Terminators active in cells derived from viruses, yeasts, molds, bacteria, insects, birds, mammals and plants are known and described in the literature. They may be isolated from bacteria, fungi, viruses, animals and/or plants.
Examples of terminators particularly suitable for use in the gene constructs of the present invention include the Agrobacterium tumefaciens nopaline synthase (NOS) gene terminator, the Agrobacterium tumefaciens octopine synthase (OCS) gene terminator sequence, the Cauliflower mosaic virus (CaMV) 35S gene terminator sequence, the Oryza sativa ADP-glucose pyrophosphorylase terminator sequence (t3′Bt2), the Zea mays zein gene terminator sequence, the rbcs-1A gene terminator, and the rbcs-3A gene terminator sequences, amongst others.
Those skilled in the art will be aware of additional promoter sequences and terminator sequences which may be suitable for use in performing the invention. Such sequences may readily be used without any undue experimentation.
In the context of the current invention, the terms “ectopic expression” or “ectopic overexpression” of a gene or a protein are conferring to expression patterns and/or expression levels of this gene or protein normally not occurring under natural conditions, more specifically is meant increased expression and/or increased expression levels in one or more of: aleurone and/or embryo of a seed.
Preferably, the promoter sequence used in the context of the present invention is operably linked to a coding sequence or open reading frame (ORF) encoding a cytokinin oxidase protein or a homologue, derivative or an immunologically active and/or functional fragment thereof as defined supra.
The vector may optionally comprise a selectable marker gene. As used herein, the term “selectable marker gene” or “selectable marker” or “marker for selection” or “screenable marker” includes any gene which confers a phenotype on a cell in which it is expressed to facilitate the identification and/or selection of cells which are transfected or transformed with a gene construct of the invention or a derivative thereof. Suitable selectable marker genes contemplated herein include the ampicillin resistance (Amp^r), tetracycline resistance gene (Tc^r), bacterial kanamycin resistance gene (Kan^r), phosphinothricin resistance gene, neomycin phosphotransferase gene (nptII), hygromycin resistance gene, β-glucuronidase (GUS) gene, chloramphenicol acetyltransferase (CAT) gene, green fluorescent protein (gfp) gene (Haseloff et al, 1997), and luciferase gene, amongst others.
The invention also relates to a host cell containing any of the nucleic acid molecules or vectors of the invention. This host cell is chosen from the group comprising bacterial, insect, fungal, plant or animal cells.
The invention further relates to a method for the production of transgenic plants, plant cells or plant tissues comprising the introduction of a nucleic acid molecule of the invention in an expressible format or a vector of the invention in this plant, plant cell or plant tissue.
Therefore, there is provided a method for producing a plant having increased seed yield, such method comprising:

- (a) introducing into a plant cell a genetic construct comprising an isolated nucleic acid molecule encoding a cytokinin oxidase wherein the isolated nucleic acid molecule is operably linked to a promoter capable of driving expression in the aleurone and/or embryo, endosperm of a seed;
- (b) regenerating a plant therefrom;
- (c) growing the regenerated plant to seed set; and
- (d) selecting a plant with increased seed yield compared to a corresponding control plant.

Generally after transformation, plant cells or cell groupings are selected for the presence of one or more markers which are encoded by plant-expressible genes co-transferred with the gene of interest, following which the transformed material is regenerated into a whole plant.
Following DNA transfer and regeneration, putatively transformed plants may be evaluated, for instance using Southern analysis, for the presence of the gene of interest, copy number and/or genomic organisation. Alternatively or additionally, expression levels of the newly introduced DNA may be monitored using Northern and/or Western analysis, both techniques being well known to persons having ordinary skill in the art.
The generated transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques. For example, a first generation (or T1) transformed plant may be selfed to give homozygous second generation (or T2) transformants, and the T2 plants further propagated through classical breeding techniques.
The generated transformed organisms may take a variety of forms. For example, they may be chimeras of transformed cells and non-transformed cells; clonal transformants (e.g., all cells transformed to contain the expression cassette); grafts of transformed and untransformed tissues (e.g., in plants, a transformed rootstock grafted to an untransformed scion).
The invention also relates to a transgenic plant cell comprising a nucleic acid sequence of the invention which is operably linked to regulatory elements allowing transcription and/or expression of the nucleic acid in the aleurone and/or embryo of a seed.
According to another preferred embodiment, the invention relates to a transgenic plant cell as described hereinabove wherein the nucleic acid of the invention is stably integrated into the genome of this plant cell.
The invention further relates to a transgenic plant or plant tissue comprising plant cells as herein described and also to a harvestable part of such transgenic plant, preferably selected from the group consisting of seeds, leaves, fruits, stem cultures, roots, tubers, rhizomes and bulbs. The present invention furthermore relates to products directly derived from a harvestable part of a transgenic plant according to the invention, such as dry pellets or powders, oil, fat and fatty acids, starch, or proteins. The invention also relates to the progeny derived from any of the transgenic plants according to the invention.
Preferably, transgenic plants are produced which express in the aleurone and/or embryo of a seed a nucleic acid encoding a CKX, preferably an Arabidopsis CKX, most preferably a CKX encoded by a nucleic acid as set forth in any of SEQ ID NOs: 3, 26, 37, or 42 or an ortholog of such nucleic acid. Preferably, the ortholog is derived from a related species of the transgenic plant. Even more preferably, the ortholog is specific (native or endogenous) to the species of the transgenic plant.
Means for introducing recombinant DNA into plant tissue or cells include, but are not limited to, transformation using CaCl₂and variations thereof, in particular the method described by Hanahan (1983), direct DNA uptake into protoplasts (Krens et al., 1982; Paszkowski et al, 1984), PEG-mediated uptake to protoplasts (Armstrong et al, 1990) microparticle bombardment, electroporation (Fromm et al., 1985), microinjection of DNA (Crossway et al., 1986), microparticle bombardment of tissue explants or cells (Christou et al, 1988; Sanford, 1988), vacuum-infiltration of tissue with nucleic acid, or in the case of plants, T-DNA-mediated transfer from Agrobacterium to the plant tissue as described essentially by An et al. (1985), Dodds et al., (1985), Herrera-Estrella et al. (1983a, 1983b, 1985). Methods for transformation of monocotyledonous plants are well known in the art and include Agrobacterium-mediated transformation (Cheng et al., 1997-WO9748814; Hansen 1998-WO9854961; Hiei et al., 1994-WO9400977; Hiei et al., 1998-WO9817813; Rikiishi et al., 1999-WO9904618; Saito et al., 1995-WO9506722), microprojectile bombardment (Adams et al., 1999-U.S. Pat. No. 5,969,213; Bowen et al., 1998-U.S. Pat. No. 5,736,369; Chang et al., 1994-WO9413822; Lundquist et al., 1999-U.S. Pat. No. 5,874,265/U.S. Pat. No. 5,990,390; Vasil and Vasil, 1995-U.S. Pat. No. 5,405,765. Walker et al., 1999-U.S. Pat. No. 5,955,362), DNA uptake (Eyal et al., 1993-WO9318168), microinjection of Agrobacterium cells (von Holt, 1994-DE4309203) and sonication (Finer et al., 1997-U.S. Pat. No. 5,693,512).
A whole plant may be regenerated from the transformed or transfected cell, in accordance with procedures well known in the art. Plant tissue capable of subsequent clonal propagation, whether by organogenesis or embryogenesis, may be transformed with a gene construct of the present invention and a whole plant regenerated therefrom. The particular tissue chosen will vary depending on the clonal propagation systems available for, and best suited to, the particular species being transformed. Exemplary tissue targets include leaf disks, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristematic tissue (e.g., apical meristem, axillary buds, and root meristems), and induced meristem tissue (e.g., cotyledon meristem and hypocotyl meristem).
The term “organogenesis”, as used herein, means a process by which shoots and roots are developed sequentially from meristematic centers.
The term “embryogenesis”, as used herein, means a process by which shoots and roots develop together in a concerted fashion (not sequentially), whether from somatic cells or gametes.
Preferably, the transgenic plant produced according to the inventive method is transfected or transformed with a genetic sequence by any art-recognized means, such as microprojectile bombardment, microinjection, Agrobacterium-mediated transformation (including in planta transformation), protoplast fusion, or electroporation, amongst others. Most preferably such plant is produced by Agrobacterium-mediated transformation.
Agrobacterium-mediated transformation or agrolistic transformation of plants, yeast, molds or filamentous fungi is based on the transfer of part of the transformation vector sequences, called the T-DNA, to the nucleus and on integration of this T-DNA in the genome of the eukaryote.
With “T-DNA”, or transferred DNA, is meant that part of the transformation vector flanked by T-DNA borders which is, after activation of the Agrobacterium vir genes, nicked at the T-DNA borders and is transferred as a single stranded DNA to the nucleus of an eukaryotic cell.
With “agrolistic transformation” is meant a transformation method combining features of Agrobacterium-mediated transformation and of biolistic DNA delivery. As such, a T-DNA containing target plasmid is co-delivered with DNA/RNA enabling in planta production of VirD1 and VirD2 with or without VirE2 (Hansen and Chilton 1996; Hansen et al. 1997; Hansen and Chilton 1997-WO9712046).
With “foreign DNA” is meant any DNA sequence that is introduced in the host's genome by recombinant techniques. The foreign DNA includes e.g. a T-DNA sequence or a part thereof such as the T-DNA sequence comprising the selectable marker in an expressible format. Foreign DNA furthermore include intervening DNA sequences as defined supra.
The present invention also encompasses use of CKX-encoding nucleic acids and use of CKX polypeptides operably linked to a promoter capable of driving expression in the aleurone and/or embryo of a seed. One such use relates to increasing plant, especially seed yield. The seed yield may include one or more of the following: increased number of flowers per panicle, increased total seed weight, increased number of filled seeds, increased thousand kernel weight and increased harvest index, each relative to corresponding wild type plants.
In particular, the present invention provides use of cytokinin oxidase-encoding nucleic acids operably linked to a promoter capable of driving overexpression in the aleurone and/or embryo relative to other parts of the seed for increasing seed yield of a plant. One such use is wherein the cytokinin oxidase-encoding nucleic acid is comprised in a genetic construct that is introduced into a plant cell. In particular, use of cytokinin oxidase-encoding nucleic acids operably linked to a promoter capable of driving overexpression in the aleurone and/or embryo relative to other parts of the seed is envisaged for increasing seed yield of a plant, wherein the cytokinin oxidase-encoding nucleic acid is selected from the group consisting of:

- (a) nucleic acids comprising a DNA sequence as given in any of SEQ ID NOs: 42, 37, 27, 1, 3, 5, 7, 9, 11, 25, 26, 28 to 30, 33 or 34, or the complement thereof,
- (b) nucleic acids comprising the RNA sequences corresponding to any of SEQ ID NOs: 42, 37, 27, 1, 3, 5, 7, 9, 11, 25, 26, 28 to 30, 33 or 34, or the complement thereof,
- (c) nucleic acids specifically hybridizing to any of SEQ ID NOs: 42, 37, 27, 1, 3, 5, 7, 9, 11, 25, 26, 28 to 30, 33 or 34, or to the complement thereof,
- (d) nucleic acids encoding a protein comprising the amino acid sequence as given in any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 35, 36 or 38, or the complement thereof,
- (e) nucleic acids as defined in any of (a) to (d) characterized in that said nucleic acid is DNA, genomic DNA, cDNA, synthetic DNA or RNA wherein T is replaced by U,
- (f) nucleic acid which is degenerate compared to a nucleic acid as given in any of SEQ ID NOs: 42, 37, 27, 1, 3, 5, 7, 9, 11, 25, 26, 28 to 30, 33 or 34, or which is degenerate compared to a nucleic acid as defined in any of (a) to (e) as a result of the genetic code,
- (g) nucleic acids which are divergent from a nucleic acid encoding a protein as given in any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 35, 36 or 38, or which is divergent from a nucleic acid as defined in any of (a) to (e), due to the differences in codon usage between the organisms,
- (h) nucleic acids encoding a protein as given in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 35, 36 or 38 or nucleic acids as defined in (a) to (e) which are divergent due to the differences between alleles,
- (i) nucleic acids encoding a protein as given in any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 35, 36 or 38,
- (j) functional fragments of nucleic acids as defined in any of (a) to (i) having the biological activity of a cytokinin oxidase, and
- (k) nucleic acids encoding a plant cytokinin oxidase, comprising the consensus sequence hTDYLhhoIGGTLSssG and cLFxushGsLGQFGIIstA.

Furthermore, use of SEQ ID NO: 41 and the use of a vector comprising a cytokinin oxidase as defined in (a) to (k) above for increasing seed yield in a plant is provided.
Those skilled in the art will be aware that the invention described herein is subject to variations and modifications other than those specifically described. It is to be understood that the invention described herein includes all such variations and modifications. The invention also includes all such steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any or more of these steps or features.
Throughout this specification, unless the context requires otherwise the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.
As used herein, the term “derived from” shall be taken to indicate that a particular integer or group of integers has originated from the species specified, but has not necessarily been obtained directly from the specified source.
The following examples are given by means of illustration of the present invention and are in no way limiting. The contents of all references included in this application are incorporated by reference herein as if fully set forth.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic representation of plant cytokinin oxidase genes. Shown are the structures of different cytokinin oxidase genes isolated from maize (ZmCKX1, accession number AF044603, Biochem. Biophys. Res. Com. 255:328-333, 1999) and Arabidopsis (AtCKX1 to AtCKX4). Exons are denominated with ‘E’ and represented by shaded boxes. Introns are represented by white boxes. Further indicated are the gene sizes (in kb, on top of each structure), the gene accession numbers (under the names) and a size bar representing 0.5 kb.

FIG. 2. Alignment of plant cytokinin oxidase amino acid sequences. The amino acid sequences from cytokinin oxidases from maize (ZmCKX1) and Arabidopsis (AtCKX1 to AtCKX4) are aligned. Identical amino acid residues are marked by a black box, similar amino acid residues are in a grey box. Amino acid similarity groups: (M,I,L,V), (F,W,Y), (G,A), (S,T), (R,K,H), (E,D), (N,Q),

FIG. 3: Schematic presentation of the entry clone p41, containing CDS0427-2 within the AttL1 and AttL2 sites for Gateway® cloning in the pDONR201 backbone. CDS0427_—2 is the internal code for the Arabidopsis thaliana CKX2 coding sequence (SEQ ID NO: 26). This vector contains also a bacterial kanamycin-resistance cassette and a bacterial origin of replication.

FIG. 4: Binary vector p37 for the expression in Oryza sativa of the Arabidopsis thaliana CKX2 gene under the control of the PRO0218 promoter. This vector contains a T-DNA derived from the Ti plasmid, limited by a left border (LB repeat, LB Ti C58) and a right border (RB repeat, RB Ti C58)). From the left border to the right border, this T-DNA contains: a selectable and a screenable marker for selection of transformed plants, each under control of a constitutive promoter; the PRO0218_CDS0427_—2-zein and rbcS-deltaGA double terminator cassette for expression of the Arabidopsis thaliana CKX2 gene. This vector also contains an origin of replication from pBR322 for bacterial replication and a selectable marker (Spe/SmeR) for bacterial selection with spectinomycin and streptomycin.

FIG. 5: Binary vector p35 for the expression in Oryza sativa of the Arabidopsis thaliana CKX2 gene under the control of the PRO0090 promoter. This vector contains a T-DNA derived from the Ti plasmid, limited by a left border (LB repeat, LB Ti C58) and a right border (RB repeat, RBTi C58)). From the left border to the right border, this T-DNA contains: a selectable and a screenable marker for selection of transformed plants, each under control of a constitutive promoter; the PRO0090—CDS0427_—2-zein and rbcS-deltaGA double terminator cassette for expression of the Arabidopsis thaliana CKX2 gene. This vector also contains an origin of replication from pBR322 for bacterial replication and a selectable marker (Spe/SmeR) for bacterial selection with spectinomycin and streptomycin.

EXAMPLES

Example 1

Brief Description of the Sequences of the Invention and Examples of Seed Specific Promoters


SEQ ID NO:	DESCRIPTION

1	AtCKX1 genomic
2	AtCKX1 protein
3	AtCKX2 genomic
4	AtCKX2 protein
5	AtCKX3 genomic
6	AtCKX3 protein
7	AtCKX4 genomic
8	AtCKX4 protein
9	AtCKX5 genomic (short version)
10	AtCKX5 protein (short version)
11	AtCKX6 genomic
12	AtCKX6 protein
13	5′primer AtCKX1
14	3′primer AtCKX1
15	5′primer AtCKX2
16	3′primer AtCKX2
17	5′primer AtCKX3
18	3′primer AtCKX3
19	5′primer AtCKX4
20	3′primer AtCKX4
21	5′primer AtCKX5
22	3′primer AtCKX5
23	5′primer AtCKX6
24	3′primer AtCKX6
25	AtCKX1 cDNA
26	AtCKX2 cDNA
27	AtCKX3 cDNA
28	AtCKX4 cDNA
29	AtCKX5 cDNA (short version)
30	AtCKX6 cDNA
31	AtCKX2 cDNA fragment
32	AtCKX2 peptide fragment
33	AtCKX5 genomic (long version)
34	AtCKX5 cDNA (long version)
35	AtCKX5 protein (long version)
36	AtCKX2, CDS0427_2 deduced protein sequence
37	AtCKX2 splice variant, DNA sequence
38	AtCKX2 splice variant, deduced protein sequence
39	PRM3769 (sense, start codon at positions 35 to
	37)
40	PRM1526 (reverse, complementary stop codon at
	positions 30-32)
41	Expression cassette with PRO0218 - CDS0427_2 -
	zein and rbcS-deltaGA double terminator
42	AtCKX2, CDS0427_2 cDNA
43	Expression cassette with PRO0090 - CDS0427_2 -
	zein and rbcS-deltaGA double terminator
44	HTDYLhholGGTLSssG signature
45	cLFxushGsLGQFGllstA signature

TABLE 4

Examples of promoters suitable for seed specific
or seedling preferred expression

	Gene name	Expression

	Metallothionein Mte	embryo/scutellum + calli
	putative beta-amylase	embryo/scutellum
	unknown	scutellum
	proteinase inhibitor Rgpi9	seed
	structural protein	young tissues, calli,
		embryo/scutellum
	prolamine
10 Kda	strong in endosperm
	allergen RA2	seed
	prolamine RP7	endosperm
	Metallothioneine-like ML2	embryo/scutellum + calli
	prolamine RM9	strong in endosperm
	prolamine RP5	strong in endosperm
	putative methionine	embryo
	aminopeptidase
	putative 40S ribosomal protein	weak in endosperm
	alpha-globulin	strong in endosperm
	alanine aminotransferase	aleurone, endosperm
	cyclophyllin 2	shoot, embryo/endosperm
	sucrose synthase SS1 (barley)	medium constitutive,
		shoot endosperm/aleurone
	trypsin inhibitor ITR1 (barley)	weak in endosperm
	WSI18	embryo/aleurone
	aquaporine	seedlings
	RAB21	embryo/aleurone
	OSH1	seedling
	Arceline 5A	seed
	Cruciferine	seed
	Albumine 2S3	seed
	Albumine 2S2	seed
	FAE1	embryo
	Phaseolin Beta subunit	seed
	Lec1	embryo
	Gamma zein	seed
	lipid Transfer Protein	seed

Example 2

Seed-Preferred or Seedling-Preferred Expression of a CKX2 Gene Results in Increased Seed Yield

A) DNA Manipulation and Cloning of AtCKX2

Unless otherwise stated, recombinant DNA techniques are performed according to standard protocols described in (Sambrook & Russell (2001) Molecular Cloning: a laboratory manual, 3rd Edition Cold Spring Harbor Laboratory Press, CSH, New York) or in Volumes 1 and 2 of Ausubel et al. (1994), Current Protocols in Molecular Biology, Current Protocols. Standard materials and methods for plant molecular work are described in Plant Molecular Biology Labfax (1993) by R. D. D. Croy, published by BIOS Scientific Publications Ltd (UK) and Blackwell Scientific Publications (UK).
The Arabidopsis CKX2 gene (corresponding to SEQ ID NO: 42) was amplified by PCR using as template an Arabidopsis thaliana seedling cDNA library (Invitrogen, Paisley, UK). After reverse transcription of RNA extracted from seedlings, the cDNAs were cloned into pCMV Sport 6.0. Average insert size of the bank was 1.5 kb, and original number of clones was 1.59×10⁷cfu. The original titer was determined to be 9.6×10⁵cfu/ml, and became after a first amplification 6×10¹¹cfu/ml. After plasmid extraction, 200 ng of template was used in a 50 μl PCR mix. Primers prm3769 (SEQ ID NO: 39) and prm1526 (SEQ ID NO: 40), which include the AttB sites for Gateway recombination, were used for PCR amplification.
PCR was performed using Hifi Taq DNA polymerase in standard conditions. A PCR fragment of 1506 bp was amplified and purified also using standard methods. The first step of the Gateway procedure, the BP reaction, was then performed, during which the PCR fragment recombines in vivo with the pDONR plasmid to produce, according to the Gateway terminology, an “entry clone”, p41 (FIG. 3). pDONR was purchased from Invitrogen, as part of the Gateway technology.

B) Vector Construction

The entry clone p41 was subsequently used in an LR reaction with p831 or p830, both destination vectors according to the Gateway™ terminology, used for rice transformation.
p831 contains as functional elements within the T-DNA borders a plant selectable marker, a screenable marker and a Gateway cassette intended for LR in vivo recombination with the sequence of interest already cloned in the donor vector. The PRO0218 promoter for embryo and aleurone preferred expression is located upstream of this Gateway cassette.
Similarly, p830 contains as functional elements within the T-DNA borders: a plant selectable marker; a screenable marker; and a Gateway cassette intended for LR in vivo recombination with the sequence of interest already cloned in the donor vector. The PRO0090 promoter for endosperm-preferred expression is located upstream of this Gateway cassette.
After the recombination step, the resulting expression vectors p37 (originating from p831, FIG. 4) and p35 (originating from p830, FIG. 5) were transformed into Agrobacterium strain LBA4404 and subsequently into Oryza sativa plants.

C) Transformation of Rice

Mature dry seeds of the rice japonica cultivar Nipponbare were dehusked. Sterilization was done by incubating the seeds for one minute in 70% ethanol, followed by 30 minutes in 0.2% HgCl₂and by 6 washes of 15 minutes with sterile distilled water. The sterile seeds were then germinated on a medium containing 2,4-D (callus induction medium). After a 4-week incubation in the dark, embryogenic, scutellum-derived calli were excised and propagated on the same medium. Two weeks later, the calli were multiplied or propagated by subculture on the same medium for another 2 weeks. 3 days before co-cultivation, embryogenic callus pieces were sub-cultured on fresh medium to boost cell division activity. The Agrobacterium strain LBA4404, harbouring T-DNA vectors comprising a suitable selection marker, was used for co-cultivation. Agrobacterium was cultured for 3 days at 28° C. on AB medium with the appropriate antibiotics. The bacteria were then collected and suspended in liquid co-cultivation medium at an OD6% of about 1. The suspension was transferred to a petri dish and the calli were immersed in the suspension during 15 minutes. Next, the callus tissues were blotted dry on a filter paper, transferred to solidified co-cultivation medium and incubated for 3 days in the dark at 25° C.
Hereafter, co-cultivated callus was grown on 2,4-D-containing medium for 4 weeks in the dark at 28° C. in the presence of a selective agent at a suitable concentration. During this period, rapidly growing resistant callus islands developed. Upon transfer of this material to a regeneration medium and incubation in the light, the embryogenic potential was released and shoots developed in the next four to five weeks. Shoots were excised from the callus and incubated for 2 to 3 weeks on an auxin-containing medium from which they were transferred to soil. Hardened shoots were grown under high humidity and short days in a greenhouse. Finally seeds were harvested three to five months after transplanting. The method yielded single locus transformants at a rate of over 50% (Aldemita and Hodges, 1996, Chan et al., 1993, Hiei et al., 1994).

D) Evaluation of Transformants: Vegetative Growth Measurements

Approximately 15 to 20 independent T0 transformants were generated. The primary transformants were transferred from tissue culture chambers to a greenhouse for growing and harvest of T1 seed. Four events (for p37 transformants, PRO0218 promoter) or five events (for p35 transformants, PRO0090 promoter) of which the T1 progeny segregated 3:1 for presence/absence of the transgene were retained. For each of these events, 10 T1 seedlings containing the transgene (hetero- and homo-zygotes), and 10 T1 seedlings lacking the transgene (nullizygotes), were selected by monitoring visual marker expression. The selected T1 plants were transferred to a greenhouse. Each plant received a unique barcode label to link unambiguously the phenotyping data to the corresponding plant. The selected T1 plants were grown on soil in 10 cm diameter pots under the following environmental settings: photoperiod: 11.5 h, daylight intensity: 30,000 lux or more, daytime temperature: 28° C. or higher, night time temperature: 22° C., relative humidity: 60-70%. Transgenic plants and the corresponding nullizygotes were grown side-by-side at random positions. From the stage of sowing until the stage of maturity the plants were passed several times through a digital imaging cabinet. At each time point digital images (2048×1536 pixels, 16 million colours) were taken of each plant from at least 6 different angles.
In a next step, the mature primary panicles were harvested, bagged, barcode-labelled and then dried for three days in the oven at 37° C. The panicles were then threshed and all the seeds were collected and counted. The filled husks were separated from the empty ones using an air-blowing device. The empty husks were discarded and the remaining fraction was counted again. The filled husks were weighed on an analytical balance and the cross-sectional area of the seeds was measured using digital imaging. This procedure allows deriving a set of seed-related parameters.
The parameters described below were derived in an automated way from the digital images using image analysis software and were analysed statistically.
A two factor ANOVA (ANalysis Of VAriance) corrected for the unbalanced design was used as statistical model for the overall evaluation of plant phenotypic characteristics. An F-test was carried out on all the parameters measured in all the plants and of all the events transformed with that gene. The F-test was carried out to check for an effect of the gene over all the transformation events and to verify for an overall effect of the gene, also named herein “global gene effect”. If the value of the F test showed that the data are significant, than it was concluded that there is a “gene” effect, meaning that not only presence or the position of the gene is causing the effect. The threshold for significance for a true global gene effect was set at 5% probability level for the F test.
To check for an effect of the genes within an event, i.e., for a line-specific effect, a t-test was performed within each event using data sets from the transgenic plants and the corresponding null plants. “Null plants” or “Null segregants” or “Nullizygotes” are the plants treated in the same way as the transgenic plant, but from which the transgene has segregated. Null plants can also be described as the homozygous negative transformed plants. The threshold for significance for the t-test was set at a 10% probability level. The results for some events can be under or below this threshold. This is based on the hypothesis that a gene might only have an effect in certain positions in the genome, and that the occurrence of this position-dependent effect is not uncommon. This kind of gene effect is also named herein a “line effect of the gene”. The p value was obtained by comparing the t value to the t distribution or alternatively, by comparing the F value to the F distribution. The p value gives the probability of the null hypothesis (i.e., that there is no effect of the transgene) is correct. The threshold for significance was set at a 5% p-value for the F test and a 10% p-value for the t-test.
Vegetative growth and seed yield was measured according to the methods as described above. The inventors surprisingly found that the seed yield of transgenic plants was increased for both the endosperm-preferred and embryo and/or aleurone-preferred promoter constructs (expressed as total weight of seeds, number of (filled) seeds and harvest index), when compared to null plants, and that transgenic plants with the embryo and/or aleurone-preferred promoter constructs additionally had an increased Thousand Kernel Weight compared to transgenics with the endosperm-preferred promoter construct. The inventors furthermore observed that the yield increase was higher for plants transformed with the embryo and/or aleurone-preferred promoter constructs than for plants with the endosperm-preferred promoter construct. Details are given in paragraphs E and F.
The data obtained in the experiment with T1 plants were then confirmed in a further experiment with T2 plants. Seed batches from the positive plants (both hetero- and homozygotes) in T1, were screened by monitoring marker expression. For each chosen event, the heterozygote seed batches were then retained for T2 evaluation. Within each seed batch an equal number of positive and negative plants were grown in the greenhouse for evaluation. In particular, four events of p37 T2 transformants and three events of p35 T2 transformants were selected for further analysis. For both p37 T2 transformants and p35 T2 transformants, a total of 120 plants were tested, evenly distributed over each event.

E) Evaluation of P37 Transformants: Measurement of Seed-Related Parameters

Upon analysis of the seeds as described above, the inventors found that plants transformed with the AtCKX2 gene under control of the embryo and/or aleurone-preferred promoter had a higher total weight of seeds, a higher number of filled seeds, a higher harvest index and a higher Thousand Kernel Weight than plants lacking the CKX2 transgene. These findings were consistent over 2 independent experiments with T1 plants as well as in an experiment with T2 plants, as shown in table 5. In addition to these yield parameters, 3 lines in T1 scored also positive for the total number of seeds. This increase in total seed number was confirmed in T2, where the effect was shown to be a significant global gene effect (mean increase +24%, p-value from the F-test 0.0032).

TABLE 5

Analysis of seed related parameters for p37 transformants

	T1 generation, 1^st	T1 generation, 2^nd
	experiment	experiment	T2 generation

	Difference over null	Difference over null	Difference over
Parameter	plants	plants	null plants	p-value

Total weight of	+22%	+22%	+51%	0.0000
seeds
Number of filled	+22%	+17%	+46	0.0000
seeds
Harvest Index	+25%	+20%	+37%	0.0025
Thousand	+1%	+5%	+3%	0.0000
Kernel Weight

The total seed weight was measured by weighing all filled seeds harvested from a transformed rice plant. The number of filled seeds was determined by counting the number of filled seeds harvested from a transformed rice plant. The total seed number was determined by counting the number of seeds harvested from a plant. The harvest index is defined as the ratio between the total seed weight and the above ground area (mm²), multiplied by a factor 106. Thousand Kernel Weight (TKW) was derived from the number of filled seeds that were counted, and their total weight. The figures gave the mean increase (in %) of each parameter calculated from transgenes versus corresponding nullizygotes of 4 independent events in T1 generation, each event comprising 10 plants carrying the transgene and 10 nullizygotes, and of 4 independent events in the T2 generation, each event comprising 20 plants carrying the transgene and 20 nullizygotes. The p-values of the F-test listed for the data of the T2 generation demonstrate that the obtained increases for the various seed yield parameters are all significant and that there is clearly an overall gene effect.

F) Evaluation of p35 Transformants: Measurement of Seed-Related Parameters

Plants transformed with the AtCKX2 gene under control of the endosperm-preferred promoter also had a better yield compared to the control nullizygous plants, in particular for total seed weight, number of filled seeds and harvest index. The total seed weight, number of filled seeds and harvest index are defined as above.
In a first experiment, plants of the T1 generation of five independent events were compared, for each event 10 T1 plants carrying the transgene versus 10 corresponding control T1 plants. For the parameter “total seed weight”, two out of the five events had a significant increase (58% and 67%, with a p-value of the t-test of 0.0551 and 0.0211 respectively). Similar results were obtained for the number of filled seeds, for which these two lines showed in increase of 47% and 68% with a p-value of respectively 0.0846 and 0.0166. The two lines also scored positive for Harvest Index (increases of 41% (p-value of 0.0223) and 31% respectively). Besides these two lines, a third line also scored significantly higher than the corresponding nullizygous control plants (+41%, p-value of 0.035).
The positive data for seed yield observed in the T1 generation were confirmed in the T2 generation. Data are given in Table 6.

TABLE 6

Analysis of seed related parameters for p37 transformants

T2 generation

Parameter	Difference over null plants	p-value

Total seed weight	+24%	0.0484
Number of filled seeds	+26%	0.0254
Harvest index	+19%	0.0277

The figures give the mean increase (in %) of each parameter calculated from transgenes versus corresponding nullizygotes of 3 independent events in the T2 generation, each event comprising 20 plants carrying the transgene and 20 nullizygotes. The p-values of the F-test listed for the data of the T2 generation demonstrate that the obtained increases for the various seed yield parameters are all significant and that there is clearly an overall gene effect.

REFERENCES

Aldemita, R. R. and Hodges, T. K. (1996) Agrobacterium tumefaciens-mediated transformation of japonica and indica rice varieties. Planta 199, 612-617.

Altschul, S. F., Madden, T. L., Schäffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J. (1997) “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.” Nucl. Acids Res. 25, 3389-3402.
Armstrong, D. J. (1994) in Cytokinins: Chemistry, Activity and Functions, eds. Mok. D. W. S & Mok, M. C. (CRC Boca Raton, Fla.), pp. 139-154.
An, G., Watson, B. D., Stachel, S., Gordon, M. P., and Nester, E. W. (1985). New cloning vehicles for transformation of higher plants. EMBO J. 4, 277-284.
Armstrong, C. L., Petersen, W. P., Buchholz, W. G., Bowen, B. A., and Sulc, S. L. (1990). Factors affecting PEG-mediated stable transformation of maize protoplasts. Plant Cell Reports 9, 335-339.

Chan, M. T., Chang, H. H., Ho, S. L., Tong, W. F., and Yu, S. M. (1993) Agrobacterium mediated production of transgenic rice plants expressing a chimeric alpha-amylase promoter/beta-glucuronidase gene. Plant Mol. Biol. 22, 491-506.

Christou, P., McCabe, D. E., and Swain, W. F. (1988). Stable transformation of soybean callus by DNA-coated gold particles. Plant Physiol. 87, 671-674.
Crossway, A., Oakes, J. V., Irvine, J. M., Ward, B., Knauf, V. C., and Shewmaker, C. K. (1986). Integration of foreign DNA following microinjection of tobacco mesophyll protoplasts. Mol. Gen. Genet. 202, 179-185.
Dodds, J. H. (1985). “Plant genetic engineering.” Cambridge University Press.
Ellis, J. G., Llewellyn, D. J., Dennis, E. S., and Peacock, W. J. (1987). Maize Adh-1 promoter sequences control anaerobic regulation: addition of upstream promoter elements from constitutive genes is necessary for expression in tobacco. EMBO J. 6, 11-16.
Hanahan, D. (1983). Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166, 557-580.
Hansen, G. and Chilton, M. D. (1996). “Agrolistic” transformation of plant cells: integration of T-strands generated in planta. Proc. Natl. Acad. Sci. U.S.A 93, 14978-14983.
Hansen, G., Shillito, R. D., and Chilton, M. D. (1997). T-strand integration in maize protoplasts after codelivery of a T-DNA substrate and virulence genes. Proc. Natl. Acad. Sci. U.S.A 94, 11726-11730.

Herrera-Estrella, L., De Block, M., Messens, E. H. J. P., Van Montagu, M., and Schell, J. (1983). Chimeric genes as dominant selectable markers in plant cells. EMBO J. 2, 987-995.

Hiei, Y.; Ohta, S.; Komari, T.; and Kumashiro, T. (1994) Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J. 6, 271-282.
Houba-Herin, N., Pethe, C., d'Alayer, J & Laloue, M. (1999) Plant J. 17:615-626.
Klee, H. J. & Lanehon, M. B. (1995) in Plant Hormones: Physiology, Biochemisry and Molecular Biology, ed. Davies, P. J. (Kluwer, Dordrdrocht, the Netherlands), pp. 340-353.
Krens, F. A., Molendijk, L., Wullems, G. J., and Schilperoort, R. A. (1982). In vitro transformation of plant protoplasts with Ti-plasmid DNA. Nature 296, 72-74.
Mok M. C. (1994) in Cytokines: Chemistry, Activity and Function, eds., Mok, D. W. S. & Mok, M. C. (CRC Boca Raton, Fla.), pp. 155-166.
Morris, R. O. et al. (1999). Isolation of a gene encoding a glycosylated cytokinin oxidase from maize. Biochem. Biophys. Res. Commun. 255, 328-333
Motyka, V., Faiss, M., Strnad, M., Kaminek, M. and Schmuelling, T. (1996). Changes in cytokinin content and cytokinin oxidase activity in response to derepression of ipt gene transcription in transgenic tobacco calli and plants. Plant Physiol. 112, 1035-1043.
Paszkowski, J., Shillito, R. D., Saul, M., Mandak, V., and Hohn, T. H. B. P. I. (1984). Direct gene transfer to plants. EMBO J. 3. 2717-2722.
Rinaldi, A. C. and Comandini, O. (1999). Cytokinin oxidase strikes again. Trends in Plant Sc. 4, 300.
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). “Molecular Cloning: A Laboratory Manual.” Cold Spring Harbor Laboratory Press.
Schmülling, T., Rupp, H. M. Frank, M & Schafer, S. (1999) in Advances in Regulation of Plant Growth and Development, eds. Sumad, M. Pac P. & Beck, E. (Peres, Prague), pp. 85-96.

Claims

1. A method for increasing seed yield in a plant, said method comprising increasing the level and/or activity of a cytokinin oxidase (CKX) in the embryo and/or aleurone of a plant seed, wherein said embryo and/or aleurone have increased levels and/or activity of cytokinin oxidase relative to other parts of said seed.

2. The method according to claim 1, wherein said increased expression in the embryo and/or aleurone is effected by introducing a genetic modification.

3. The method according to claim 2, wherein said genetic modification is effected by one of: T-DNA activation, TILLING, site-directed mutagenesis or directed evolution.

4. A method for increasing seed yield in a plant, said method comprising introducing into a plant cell a genetic construct comprising an isolated nucleic acid molecule encoding a cytokinin oxidase wherein said isolated nucleic acid molecule is operably linked to a promoter capable of driving expression in the embryo and/or aleurone of a seed.

5. A method for producing a plant having increased seed yield and increased expression of a cytokinin oxidase in the embryo and/or aleurone of a seed, said method comprising introducing into a plant cell a genetic construct comprising an isolated nucleic acid molecule encoding a cytokinin oxidase wherein said isolated nucleic acid molecule is operably linked to a promoter capable of driving expression in the embryo and/or aleurone of a seed.

6. A method for producing a plant having increased seed yield and increased expression of a cytokinin oxidase in the embryo and/or aleurone of a seed, said method comprising:

(a) introducing into a plant cell a genetic construct comprising an isolated nucleic acid molecule encoding a cytokinin oxidase, wherein said isolated nucleic acid molecule is operably linked to a promoter capable of driving expression in the embryo and/or aleurone of a seed;

(b) regenerating a plant therefrom;

(c) growing the regenerated plant to seed set; and

(d) selecting a plant with increased seed yield compared to a corresponding wild type plant.

7. The method of any of claim 4 wherein the promoter capable of driving expression in the embryo and/or aleurone comprises nucleotides 1-1256 of SEQ ID NO: 41.

8. The method of claim 2 wherein the nucleic acid molecule encoding a cytokinin oxidase is selected from the group consisting of:

(a) nucleic acids comprising a DNA sequence as given in any of SEQ ID NOs: 42, 37, 27, 1, 3, 5, 7, 9, 11, 25, 26, 28 to 30, 33 or 34, or the complement thereof,

(b) nucleic acids comprising the RNA sequences corresponding to any of SEQ ID NOs: 42, 37, 27, 1, 3, 5, 7, 9, 11, 25, 26, 28 to 30, 33 or 34, or the complement thereof,

(c) nucleic acids specifically hybridizing to the complement of any of SEQ ID NOs: 42, 37, 27, 1, 3, 5, 7, 9, 11, 25, 26, 28 to 30, 33 or 34,

(d) nucleic acids encoding a protein comprising the amino acid sequence as given in any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 35, 36 or 38, or the complement thereof,

(e) nucleic acids as defined in any of (a) to (d) characterized in that said nucleic acid is DNA, genomic DNA, cDNA, synthetic DNA or RNA wherein T is replaced by U,

(f) nucleic acid which is degenerate compared to a nucleic acid as given in any of SEQ ID NOs: 42, 37, 27, 1, 3, 5, 7, 9, 11, 25, 26, 28 to 30, 33 or 34, or which is degenerate compared to a nucleic acid as defined in any of (a) to (e) as a result of the genetic code,

(g) nucleic acids which are divergent from a nucleic acid encoding a protein as given in any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 35, 36 or 38, or which is divergent from a nucleic acid as defined in any of (a) to (e), due to the differences in codon usage between the organisms,

(h) nucleic acids encoding a protein as given in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 35, 36 or 38, or nucleic acids as defined in (a) to (e) which are divergent due to the differences between alleles,

(i) nucleic acids encoding a protein as given in any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 35, 36 or 38,

(j) functional fragments of nucleic acids as defined in any of (a) to (i) having the biological activity of a cytokinin oxidase, and

(k) nucleic acids encoding a plant cytokinin oxidase, comprising the consensus sequence hTDYLhholGGTLSssG and cLFxushGsLGQFGIIstA or comprising expression in seeds of a nucleic acid encoding a protein that reduces the level of active cytokinins in plants or plant parts.

9. The method of claim 4, wherein the isolated nucleic acid molecule operably linked to a promoter capable of driving expression in the embryo and/or aleurone comprises the nucleotide sequence as set forth in SEQ ID NO: 41.

10. The method of claim 1 wherein the increase in seed yield comprises at least one of: total weight of seeds, total number of seeds, total number of filled seeds, harvest index, and thousand kernel weight.

11. An isolated nucleic acid molecule encoding a cytokinin oxidase operably linked to a promoter capable of driving overexpression of said nucleic acid molecule in the embryo and/or aleurone of a seed relative to other parts of said seed.

12. The isolated nucleic acid molecule of claim 11 wherein the nucleic acid molecule encoding a cytokinin oxidase is selected from the group consisting of:

(g) nucleic acids which are divergent from a nucleic acid encoding a protein as given in any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 35, 36 or 38, or which is divergent from a nucleic acid as defined in any of (a) to (e), due to the differences in codon usage,

(h) nucleic acids encoding a protein as given in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 35, 36 or 38 or nucleic acids as defined in (a) to (e) which are divergent due to the differences between alleles,

(k) nucleic acids encoding a plant cytokinin oxidase, comprising the consensus sequence hTDYLhholGGTLSssG and cLFxushGsLGQFGIIstA or comprising expression, preferably in seeds, of a nucleic acid encoding a protein that reduces the level of active cytokinins in plants or plant parts.

13. A vector comprising the isolated nucleic acid molecule of claim 11.

14. A vector comprising the isolated nucleic acid molecule having the sequence set forth in SEQ ID NO: 41.

15. A host cell comprising the isolated nucleic acid molecule of claim 11.

16. A host cell comprising a vector of claims 13.

17. The host cell of claim 16, which is a bacterial, fungal, or plant cell.

18. Plant having increased seed yield, comprising a genetic modification, which genetic modification results in increased expression of cytokinin oxidase in the aleurone and/or embryo of a seed, relative to other parts of said seed.

19. A transgenic plant, plant part, or plant tissue comprising plant cells of claim 17.

20. The transgenic plant of claim 19 which is a dicotyledonous or monocotyledonous plant.

21. Transgenic plant according to claim 20, wherein said monocotyledonous plant is selected from a group consisting of sugarcane, rice, maize, wheat, barley, millet, rye, oats, and sorghum.

22. A transgenic harvestable part, or a product directly derived from a transgenic harvestable part of the transgenic plant of claim 19.

23. A transgenic harvestable part of the transgenic plant of claim 22 selected from the group consisting of seeds, leaves, fruits, stem cultures, rhizomes, roots, tubers and bulbs.

24. Transgenic progeny of the plant or plant part of claim 19.

25. Harvestable parts of the plant according to claim 18, or products directly derived from said harvestable parts.

26-29. (canceled)

30. A method for increasing seed yield in a plant, said method comprises introducing into a plant cell the vector according to claim 13.

31. The method according to claim 1, wherein said increased expression in the embryo and/or aleurone is effected by introducing a genetic modification in the locus of the cytokinin oxidase-encoding gene.