WO2000053746A2 - Method of delaying or inhibiting sprouting in plants - Google Patents

Abstract

The use of a DNA sequence encoding a SNF1-related protein kinase (SnRK) for the inhibition of sprouting in plant tissues is provided. Such sequences can be used in the production of transgenic plants which have sterile progeny.

Description

METHOD OF DELAYING OR INHIBITING SPROUTING IN PLANTS The present invention relates to the use of antisense DNA sequences in the control of plant growth, particularly growth of crop plants.

The sucrose nonfermenting-1 (SNF1) protein kinase currently comprises SNF1 itself in the yeast Saccharomyces cerevisiae, the AMP-activated protein kinase (AMPK) in mammals, the SNFl-related protein kinases (SnRKs) in higher plants. The SNF1 family have roles in the regulation of both metabolism and gene expression. These are currently better understood in yeast and animals, but there is increasing evidence that plant members of the family play very similar roles to their yeast and animal counterparts.

The preferred carbon source for the yeast Saccharomyces cerevisiae is glucose and, if adequate glucose is available, a large number of genes are switched off. This process is known as glucose repression and genes regulated in this way include those required for growth on alternative carbon sources like sucrose, galactose, maltose, glycerol and ethanol (Gancedo, J. M., Microbiol. And Molec. Biol. Reviews 62 334-361 (1998); Ronne, H., Trends in Genetics 11 12-17 (1995)). The SNF1 gene was originally defined via mutations which would not grow on sucrose (SNF = sucrose non- fermenting), but is required for derepression of essentially all glucose-repressed genes (Gancedo, (1998); Ronne, (1995), supra) as well as other functions such as sporulation, glycogen accumulation (Hardy et al., J. Biol. Chem. 269 27907-27913 (1994)) and peroxisome biogenesis (Simon βt al., Yeast 8 303-309 (1992)). It has been cloned by complementation of snfl mutants and shown to encode an M_r 72,000 protein (72kDa) with an N-terminal domain characteristic of protein serine/threonine kinases (Celenza, J. L. and Carlson, M., Science 233 1175-1180 (1986)).

The AMP-activated protein kinase (AMPK) was originally detected in the form of protein fractions which inactivated HMG-CoA reductase (Beg et al, Biochem. Biophys. Res. Comm. 54 1362-1369 (1973)) or acetyl-CoA carboxylase (Carlson, C. A., and Kim, K. H., J. Biol. Chem. 248 378-380 (1973)). It is activated by AMP (Carling et al, FEBS Lett 223 217-222 (1987); Carling et al, Eur. J. Biochem. 186 129-136 (1989)) and by phosphorylation by an upstream protein kinase (Hawley et al, J. Biol. Chem. 271 27879-27887 (1996)), and has been likened to a cellular "fuel gauge" (Hardie, D. G. and Carling, D., Eur. J. Biochem. 246 259-273 (1997)). Its activation has been demonstrated in response to a variety of conditions of stress in mammalian cells, and, once activated, it phosphorylates and inactivates regulatory enzymes of ATP-consuming, anabolic pathways such as acetyl-CoA carboxylase (fatty acid synthesis) (Davies et al, Eur. J. Biochem. 187 183-190 (1990); Davies et al, Eur. J. Biochem. 203 615-623 (1992)) and HMG-CoA reductase (sterol/isoprenoid synthesis) (Clarke, P. R. and Hardie, D. G., EMBO J. 9 2439-2446 (1990); Gillespie, J. G., and Hardie, D. G., EERS lett. 306 59-62 (1992)). It also seems likely, although not yet proven, that AMPK will regulate gene expression.

AMPK was purified in 1994 and shown to be a heterotrimeric complex of three subunits, α,β and γ (Davies et al, Eur. J. Biochem. Ill 351-357 (1994); Mitchelhill et al, J. Biol. Chem. 269 2361-2364 (1994)). Amino acid and DNA sequencing of the catalytic α subunit (Carling et al, J. Biol Chem. 269 11442-11448 (1994); Gao et al, J. Biol. Chem. 271 8675-8681 (1995); Gao et al, Biochim. Biophys. Ada 1266 73-82 (1996); Mitchelhill et al, (1994) supra; Woods et al, J. Biol. Chem. 271 10282-10290 (1996)) showed that it was closely related to SNFl and to the plant SNFl-related (SnRK) family.

The first plant SnRK sequence to be reported was RKINl, a cDNA isolated from a rye endosperm cDNA library (Alderson et al, Proc. Nat'l Acad. Sci. USA 88 8602-8605 (1991)). RKINl encodes an M_r 57,710 (57.71kDa) protein of 502 amino acid residues showing 48% amino acid sequence identity with SNFl and AMPK. The protein kinase catalytic domains, comprising approximately 250 amino acids in the N-terminal half of the three proteins, are the most similar, showing approximately 62-64% amino acid sequence identity. AMPK is slightly larger than RKINl, with a M_r of 63,000 (63kDa), while SNFl is larger still, with an M_r of 72,000 (72kDa). The size variation is due almost entirely to differences in the C-terminal regions which, however, still retain 29-34% amino acid sequence identity. SNFl also has a slightly longer N- terminal region, prior to the catalytic domain.

Genomic clones, cDNAs and PCR products very similar to RKINl have now been cloned from Arabidopsis thaliana (AKIN 10 and ATSKINl), barley (BKIN2 and

BKIN12), oat (ASPK1-3), potato (PKIN1), rice (RSR7) sugar beet (SBKIN154) and tobacco (NPK5). All except RSK1 and NPK5 were cloned at Long Ashton Research

Station. They encode very similar protein kinases, the lowest degree of amino acid sequence identity between any two being 68% and the size varying only from M_r 57,710 (RKLNl) to 58,910 (AKLNIO) (See references from (1) Le Guen et al.Gene

120 249-254 (1992); (2) Huttly, A. K. and Philips A. L., Plant Molec. Biol. 27 1043-

1052 (1995); (3) Halford et al, The Plant Journal 2 791-797 (1992); (4) Hannappel et al, Plant Molec. Biol. 27 1235-1240 (1995); (5) Muranaka et al, Mol. Cell. Biol. 14

2958-2965 (1994); (6) Man et al,Plant Molec. Biol. 34 31-43 (1997); (7) Alderson et al, Proc. Nat'l Acad. Sci. USA 88 8602-8605 (1991); (8) Monger et al.Plant Growth

Regulation 22 181-188 (1997); (9) Lakatos et al, Plant Physiol. 113 1004 (1997).

These genes have been classed as the SnRKl subfamily. The plant SnRK family also comprises two other subfamilies of protein kinases which are closely related to SnRKl s in the catalytic domain, though less so than SNFl or AMPK, with approximately 42-45% amino acid sequence identity with the SnRKl s in this region. One of these comprises M_r approximately 40,000 (40kDa) protein, the first of which to be cloned was PKABA1 from wheat (Anderberg, R. J. and Walker-Simmons, M. K., Proc. Nat'l Acad. Sci. USA 89 10183-10187 (1992)). They have been called the SnRK2 subfamily. They have relatively short C-terminal domains compared with

SnRKl s, characterised by the presence of a short acidic "patch". The other subfamily of plant kinases relates to the SnRKl s currently comprises two members, WPK4 (Sano, H., and Yousseffian, S., Proc. Nat'l Acad. Sci. USA 91 2582-2586 (1991)), which was isolated from wheat, and a hitherto un-named gene from Arabidopsis thaliana (EMBL accession number Z97336). This subfamily has been called SnRK3.

The WPK4 protein kinase has an M_r of 58,000 (58kDa), which means it is the same size as members of the SnRKl subfamily. However, it is clearly a member of a distinct subfamily, with no similarity in the C-terminal domain. The nomenclature relating to the SnRK subfamilies is as published in Halford and Hardie (1998) (Halford, N. G. and Hardie, D. G. Plant Mol Biol. 37 735-748 (1998)).

A plot of the evolutionary distances between the plant SnRKl s divides them into two clusters, SnRKl a and SnRKlb. Barley and oat have representatives in both groups, whereas no dicotyledenous species has been found to contain a SnRKlb. The two groups appear to be expressed in a different manner. The SnRKl a group is expressed in all tissues (Halford et α/.,(1992); Le Guen et al, (1992) supra) while the SnRKlb group, where analysed, has been shown to be expressed seed-specifically (Alderson et al, (1992); Hannappel et α/.,(1995) supra). Most of the divergence between the SnRKl a and SnRKlb proteins is in the C-terminal regions, but the functional significance of this is not known.

In parallel with molecular biological studies, the SnRK family was discovered independently via biochemical approaches. Crucial to this was the development of an assay for SNFl-related protein kinase activity, which used the SAMS peptide (His- Met-Arg-Ser-Ala-Met-Ser-Gly-Leu-His-Leu-Val-Lys-Arg-Arg), a synthetic peptide based on the sequence around the primary phosphorylation site for AMPK on rat acetyl-CoA carboxylase. This was a relatively specific substrate for AMPK in rat liver extracts (Davies et al, Eur. J. Biochem. 186 123-128 (1989)), but could also be used to detect peptide kinase activities in plant extracts (MacKintosh et al, Eur. J. Biochem. 209 923-931 (1992)). Purification of one of these activities from cauliflower showed that its biochemical properties were closely related to those of mammalian AMPK (Ball et al, Eur. J. Biochem. 219 743-750 (1994); MacKintosh et al, (1992) supra). Since it phosphorylated and inactivated a bacterially-expressed HMG-CoA reductase (HMG1 from Arabidopsis thaliana (Dale et al, Eur. J. Biochem. 233 506- 513 (1995)), it was termed HMG-CoA reductase kinase (HRK-A).

Although HRK-A was not activated by AMP, it behaved similar to AMPK during purification, had a similar native molecular mass, and a very similar specificity for protein and peptide substrates (Ball et al, (1994) supra; Dale et al, FEBS Lett 361 191-195 (1995); McKintosh et al, (1992) supra). The catalytic subunit was found to have an M_r of 58,000 (58.0kDa), which is exactly the mass predicted for the catalytic subunits of the SnRKl subfamily. This M_r 58,000 (58.0kDa) polypeptide was found to cross-react with antisera raised to a SnRKl peptide and heterologously-expressed protein (Ball et al, FEBS Lett 311 189-192 (1995)). A SAMS peptide kinase has since been partially purified from barley endosperm as well (Barker et al, Plant Physiol. 112 1141-1149 (1996)), and this preparation also contained a M_r 60,000 (60.0kDa) polypeptide that cross-reacted with the SnRKl antisera. The major SAMS peptide kinase activities in cauliflower and barley are, therefore, almost certainly encoded by genes corresponding to the SnRKl subfamily. Like Cauliflower HRK-A, the barley kinase also phosphorylated bacterially-expressed HMG-CoA reductase from Arabidopsis thaliana. A detailed analysis of SAMS peptide kinase activity has now been performed in potato (Man et al, Plant Mol Biol. 34 31-43 (1997)). The activity (measured as nmoles phosphate incorporated into the peptide per minute per mg protein, in the standard assay described by Davies et al. (1989) supra in tissues from whole plants ranged from 0.21 nmoles/min mg in stolons to 0.033 nmoles/min/mg in leaves. Much higher activities were measured in mini-tubers (0.84 nmoles/min/mg) and callus cultures (1.66 nmoles/min/mg).

One of the functions of SNFl in yeast is the transcriptional regulation of genes encoding enzymes of carbohydrate metabolism and a key question regarding SnRKl is whether or not they play an analogous role in plants. Expression of the rye SnRKl cDNA, RKINl, in a yeast snfl mutant restored SNFl function to the extent that the yeast could utilise non-fermentable carbon sources such as ethanol and glycerol (Alderson et al, Proc. Nat'l Acad. Sci. USA 88 8602-8605 (1991)). A similar experiment has been performed with the tobacco SnRKl, NPK5, using sucrose utilisation as the selectable marker (Muranaka et al, Mol. Cell. Biol. 14 2958-2965 (1994)). The snfl mutant yeast expressing the NPK5 protein kinase was able to grow on the sucrose medium and were shown to contain an invertase activity. These complementation experiments both showed that plant SnRKl s can substitute for SNFl in the sugar sensing signalling pathway in yeast, suggesting that an analogous signalling system might exist in plant cells. Further studies with NPK5 showed that it rescued snfl mutants in a glucose-regulated manner, indicating that the regulation of the yeast and plant systems may be very similar (Jiang, R., and Carlson, M., Genes Dev. 10 3105-3115 (1996)).

This has been investigated further by down-regulating SnRKl activity in potato plants using antisense techniques (Halford et al, Biochem. Soc. Trans. 22 953-957 (1994); Purcell et al, Plant J. - in press (1998)). Two chimaeric genes were constructed and used to transform potato cv. Desiree. One consisted of 1.5 kb of the promoter of a patatin gene (Rocha-Sosa et al, EMBO J. 8 23-29 (1989)), an antisense 503 bp PCR product derived from the potato SnRKl gene, PKIN1 (Man et al, Plant Mol. Biol. 34

31-43 (1997)), and an octopine synthase gene polyadenylation signal (Gielen et al, EMBO J. 3 835-846 (1984)). The other was identical except that the patatin promoter was replaced with 1.6 kb of the promoter of ST-LS1, the gene that encodes the M_r 10,000 (lO.OkDa) protein of photosystem II. The patatin promoter gives expression only in the tubers while the ST-LS1 promoter gives highest levels of expression in the leaves and is expressed to a lesser extent in stems (Eckes et al, Mol. Gen. Genet. 199 216-224 (1985); and Mol. Gen. Genet. 205 14-22 (1986)).

Expression of the antisense transcript in tubers resulted in a reduction of up to 79% in SAMS peptide kinase activity, confirming that PKLN1 is responsible for most if not all of the SAMS peptide kinase activity in potato tubers. The activity of sucrose synthase, one of the enzymes which catalyses the conversation of sucrose to hexoses in plants, had decreased in one of the lines (PAT 1.10) expressing the antisense sequence in the tubers. On further investigation, sucrose synthase gene expression was shown to have decreased to undetectable levels in transgenic tubers from this line and another line,

PAT 1.3, expressing the antisense sequence, and to be uninducible by sucrose in excised leaves from two lines expressing the antisense sequence under the control of the ST-LS1 promoter. In wild-type plants, the sucrose synthase gene, Sus4, is expressed in tubers and is induced in exercised leaves by incubation with sucrose (Fu, H., and Park, W. D., Plant Cell 7 1369-1385 (1995)). From these results, it would appear that SnRKl s play a role in the control of carbohydrate metabolism through the direct regulation of sucrose synthase gene expression. Prevention of pre-harvest losses in agricultural crop plants in the field and post-harvest losses of crops in storage is a significant issue in modern farming practice. Common problems include pre-harvest sprouting of grain crops in the field pre-harvest and post- harvest sprouting of seeds, tubers, such as potatoes and also of root crops. Potatoes are a crop of major agricultural importance because of their food content and ease of storage and transportation. They form a major part of the diet of the population in many countries. Potato plants not only provide a food crop but also the tubers for use as seed potatoes for the next year. However, as with any foodstuff or seed, care must be taken in the storage of the tubers to avoid premature sprouting which can lead to the tuber no longer being useful for human consumption or for use as a seed potato. It is well known that potato tubers will readily sprout under conditions of sufficient light, humidity and temperature. Sprouting of cereals during storage is also a problem for similar reasons.

Tubers in the UK have to be stored from September until July. Two of the major problems which must be avoided during this period are sprouting and the accumulation of sugars. The former can be reduced by storage at low temperature and the use of sprout suppressants but low temperature causes sugars to accumulate. Therefore a genetic means to delay sprouting and/or cold-induced sweetening could be of great commercial value.

The seeds of other crops of agricultural importance are also prone to sprouting pre- harvest in the field and also post-harvest during storage which leads to losses of potential crop yields. For example, the quality of barley malting is particularly affected by premature sprouting. Additionally in cereal crops, there is also a recognised problem in the pre-harvest sprouting of cereal crops in the field which also leads to losses in crop yields at harvest.

SnRK's (SNFl -Related Protein Kinases) as their name indicates, are members of the SNFl family. SNFl was first characterised genetically in yeast while its mammalian counterpart AMPK was characterised biochemically. The results from both the yeast and mammalian work taken together was thought to indicate a role for these protein kinases in metabolic control both at the level of enzyme modification (i.e. inactivation by phosphorylation) and at the level of gene expression. The studies on antisense SnRKl expression in plants also showed a role in gene expression, namely sucrose synthase gene expression. These studies in potatoes also showed that normal growth and development of leaves and tubers was not affected by antisense expression under ideal growth conditions. However, a previously unknown property of SnRK's has now been discovered. It has been surprisingly found that manipulation of SnRK activity can be used to control sprouting in stored tubers from crops reproduced by vegetative propagation and also in seeds from seed-producing crops and to affect pollen viability in seed-producing crops.

According to a first aspect of the present invention, there is provided the use of an antisense DNA sequence encoding a SNFl-related protein kinase (SnRK) for the inhibition of sprouting in plant tissues.

The sucrose non- fermenting- 1 -related protein kinases (SNFl-related protein kinase) or SnRK's comprise a family currently known to include 3 sub-families, i.e. SnRKl (including SnRKl a and SnRKlb), SnRK2 and SnRK3. In a particular embodiment of the present invention, the use relates to SnRK's from the sub-family SnRKl.

In a preferred embodiment the SNFl-related protein kinase can regulate the transcription of genes encoding enzymes of carbohydrate metabolism. For example, the enzyme under transcriptional control can be phosphofructokinase, pyruvate kinase, acid invertase, starch synthase, adenine diphosphoglucose pyrophosphorylase, sucrose synthase, 6-phospho-fructokinase (pyrophosphate) or sucrose phosphate synthetase. A generally suitable enzyme is sucrose synthase.

In a use according to this aspect of the present invention, the inhibition of sprouting can comprise a restriction in starch accumulation in the plant tissues. The restriction may be total or partial, i.e. the plant tissues of plants prepared according to the present invention may not contain starch.

In the present invention the antisense sequence can be derived from members of the SnRK family. The family members known to date and their EMBL, PLRJ or Genbank accession numbers are shown as follows in Tables 1, 2 and 3 (adapted from Halford, N. G. and Hardie, D. G. Plant Mol. Biol. 37 735-748 (1998)).

One preferred antisense sequence is RKINl, the rye SnRKl that was the first plant SNRK1 to be characterised (Alderson et al (1991) supra). A particularly preferred sequence is the sequence from nucleotide 608 to nucleotide 1111 of RKINl as described by Alderson et al (1991), and to corresponding region of other SnRK sequences. The equivalent region, which comprises 503bp, of a potato SnRKl sequence is shown in Figure 2 as PKLN503. The sequence can be prepared from potato (Solanum tuberosum) c.v. Desiree cDNA or cloned SnRKl sequences by PCR amplification using oligonucleotide primers with sequences that are highly conserved in the plant SNFl-related family. The PCR product may also include 3'- and 5'- restriction sites as appropriate to facilitate insertion into vectors or for other purposes.

An antisense DNA sequence in accordance with the present invention may comprise any polynucleotide sequence capable of hybridising to the sense strand of the DNA encoding a SNFl-related protein kinase-1 (SnRKl). Hybridisation of the strands leads to down-regulation of gene expression. The polynucleotide sequence may include modified nucleotide bases or deletions or substitutions from the native sequence provided that functionally it is able to hybridise with the sense strand. Hybridisation may be under highly stringent or less stringent conditions as appropriate. In methods according to the present invention, a reasonable degree of specificity of hybridisation is desired and so relatively stringent conditions may be used to form the duplexes of probe and DNA sequence to be amplified. Such stringent conditions may be characterised by low salt concentration or high temperature conditions. As used in the present application, the term "highly stringent conditions" means hybridisation to DNA bound to a solid support in 0.5M NaHPO₄, 7% sodium dodecyl sulfate (SDS), ImM EDTA at 65°C, and washing in O. lxSSC/0.1 % SDS at 68°C (Ausubel et al eds. " Current Protocols in Molecular Biology" 1, page 2.10.3, published by Green Publishing Associates, Inc. and John Wiley & Sons, Inc. New York (1989)). In some circumstances, less stringent hybridisation conditions may be required. As used in the present application, the term "moderately stringent conditions" means washing in 0.2xSSC/0J % SDS at 42°C (Ausubel et al (1989) supra). Hybridisation conditions can also be rendered more stringent by the addition of increasing amounts of formamide, to destabilise the hybrid duplex. Thus particular hybridisation conditions can be readily be manipulated, and will be generally be selected according to the desired results. In general, convenient hybridisation temperatures in the presence of 50% formamide are: 42°C for a probe which is 95 to 100% homologous to the target DNA, 37°C for 90 to 95% homology, and 32°C for 70 to 90% homology.

In accordance with the present invention, the antisense sequence may be used to transform plants such that inhibition of sprouting is caused by expression of the antisense DNA.

Nucleic acid sequences of the present invention can be introduced into plant cells by transformation using the binary vector pLARS120, a modified version of pGPTV- Kan (Becker et al Plant Mol. Biol. 20 1195-1197 (1992)) in which the β- glucuronidase reporter gene is replaced by the Cauliflower mosaic virus 35S promoter from pBI220 (Jefferson, R. A., Plant Mol Biol. Rep. 5 387-405 (1987)). Such plasmids may be then introduced into Agrobactenum tumefaciens by electroporation and can then be transferred into the host cell via a vacuum filtration procedure. Alternatively, transformation may be achieved using a disarmed Ti- plasmid vector and carried by Agrobactenum by procedures known in the art, for example as described in EP-A-0116718 and EP-A-0270822. Where Agrobactenum is ineffective, the foreign DNA could be introduced directly into plant cells using an electrical discharge apparatus alone, such as for example in the transformation of monocotyledonous plants. Any other method that provides for the stable incorporation of the nucleic acid sequence within the nuclear DNA or mitochondrial DNA of any plant cell would also be suitable. This includes species of plant which are not yet capable of genetic transformation.

Preferably, nucleic acid sequences in accordance with the invention for introduction into host cells also contain a second chimeric gene (or "marker" gene) that enables a transformed plant containing the foreign DNA to be easily distinguished from other plants that do not contain the foreign DNA. Examples of such a marker gene include antibiotic resistance (Herrera-Estrella et al EMBO J. 2 987-995 (1983)), herbicide resistance (EP-A-0242246) and glucuronidase (GUS) expression (EP-A- 0344029). Expression of the marker gene is preferably controlled by a second promoter which allows expression in cells at all stages of development so that the presence of the marker gene can be determined at all stages of regeneration of the plant.

A whole plant can be regenerated from a single transformed plant cell, and the invention therefore provides transgenic plants (or parts of them, such as propagating material, i.e. protoplasts, cells, calli, tissues, organs, seeds, embryos, ovules, zygotes, tubers, roots, etc.) including nucleic acid sequences in accordance with the invention as described above. In the context of the present invention, it should be noted that the term "transgenic" should not be taken to be limited in referring to an organism as defined above containing in their germ line one or more genes from another species, although many such organisms will contain such a gene or genes.

Rather, the term refers more broadly to any organism whose germ line has been the subject of technical intervention by recombinant DNA technology. So, for example, an organism in whose germ line an endogenous gene has been deleted, duplicated, activated or modified is a transgenic organism for the purposes of this invention as much as an organism to whose germ line an exogenous DNA sequence has been added.

Suitably the plant is of the family Leguminosea, Graminaceae, Saccharaceae, Solonaceae, Fabaceae, or Brassicaceae . Preferred plant genera include but are not limited to monocotyledonous plants including seed and the progeny or propagules thereof, for example Lolium, Zea, Triticum, Sorghum, Triticale, Saccharum, Bromus, Oryzae, Avena, Hordeum, Secale and Setaria. Especially useful transgenic plants are maize, wheat, rice, barley plants and seed thereof. Dicotyledenous plants are also within the scope of the present invention and preferred transgenic plants include but are not limited to the families Fabaceae, Solanum, Brassicaceae, e.g. potatoes, beans, cabbages, forest trees, roses, clematis, oilseed rape, sunflower, chrysanthemum, poinsettia and antirrhinum (snapdragon).

The potato plant, Solanum tuberosum, is particularly preferred, including agricultural and non-agricultural varieties, i.e. Desiree, Cara, Maris Bard, Maris Piper, Hermes, Record, Saturna, Erntestoltz, Lady Rosetta, etc. Conveniently, where transformation of potato plants is to be carried out, the transformation of plants can be carried out using Agrobacterium tumefaciens-mQdi∑Λed transformation of leaf discs and a suitable vector is a pBLN-19 based disarmed binary vector (Bevan, M., Nucleic Acids Research 12 8711-8712 (1984)). Potato is readily amenable to Agrobacterium tumefaciens-mcdiated transformation and antisense expression of transgenes has been used extensively in the investigation of metabolic regulation (Nisser et al in "Antisense Nucleic Acids and Proteins", ed.s Mol, J. Ν. M. and van der Krohl, A. R., pages 141-159, Marcel-Dekker, New York (1991); Mϋller-Roer et al EMBO J 11 1229-1238 (1992); Zrenner et alPlanta 190247-252 (1993); Zrenner et al Plant J. I 97-107 (1995)).

A particularly preferred antisense sequence is a construct of an antisense PCR product derived from a potato SNEi-related gene, e.g. PKINl (Man et al (1997) supra), a promoter sequence and optionally further sequences. The promoter sequence may be, for example, a tuber-specific promoter such as the promoter of a patatin gene, up to 1.5kb (Rocha-Sosa et al (1989) supra), or it may be a patatin promoter described by Mignery et al (Gene 62 27-44 (1988)). Alternatively, the promoter may be leaf/stem- specific such as the promoter of ST-LS1, the gene which encodes the 10,000 molecular mass (lO.OkDa) protein of photosystem II, up to 1.6kb (Eckes et al (1985, 1986) supra). Other promoters that could be used include the Cauliflower mosaic virus (CaMV) 35S gene promoter, which may also be present as a twin promoter. Additional sequences may include but are not limited to regulatory or marker sequences, such as for example an octopine synthase gene polyadenylation signal (Gielen et α/ (1984) swprø).

Screeriing of plant cells, tissue and plants for the presence of specific DNA sequences may be performed by Southern analysis as described in Sambrook et al (Molecular Cloning: A Laboratory Manual, Second edition (1989)). This screening may also be performed using the Polymerase Chain Reaction (PCR) by techniques well known in the art.

Transformation of plant cells includes separating transformed cells from those that have not been transformed. One convenient method for such separation or selection is to incorporate into the material to be inserted into the transformed cell a gene for a selection marker. As a result only those cells which have been successfully transformed will contain the marker gene. The translation product of the marker gene will then confer a phenotypic trait that will make selection possible. Usually, the phenotypic trait is the ability to survive in the presence of some chemical agent, such as an antibiotic, e.g. kanamycin, G418, paromomycin, etc, which is placed in a selection media. Some examples of genes that confer antibiotic resistance, include for example, those coding for neomycin phosphotransferase kanamycin resistance (Velten et al EMBO J. 3 2723-2730 (1984)), hygromycin resistance (van den Elzen et al Plant Mol. Biol. 5 299-392 (1985)), the kanamycin resistance (NPT II) gene derived from Tn5 (Bevan et al Nature 304 184-187 (1983); McBride et al Plant Mol. Biol. 14 (1990)) and chloramphenicol acetyltransf erase. The PAT gene described in Thompson et al (EMBO J. 6 2519-2523 (1987)) may be used to confer herbicide resistance.

An example of a gene useful primarily as a screenable marker in tissue culture for identification of plant cells containing genetically engineered vectors is a gene that encodes an enzyme producing a chromogenic product. One example is the gene coding for production of β-glucuromdase (GUS). This enzyme is widely used and its preparation and use is described in Jefferson (Plant Mol. Biol. Reporter 5 387- 405 (1987)).

Once the transformed plant cells have been cultured on the selection media, surviving cells are selected for further study and manipulation. Selection methods and materials are well known to those of skill in the art, allowing one to choose surviving cells with a high degree of predictability that the chosen cells will have been successfully transformed with exogenous DNA.

After transformation of the plant cell or plant using, for example, the Agrobacterium Ti-plasmid, those plant cells or plants transformed by the Ti-plasmid so that the enzyme is expressed, can be selected by an appropriate phenotypic marker. These phenotypic markers include, but are not limited to, antibiotic resistance. Other phenotypic markers are known in the art and may be used in this invention.

Positive clones are regenerated following procedures well-known in the art. Subsequently transformed plants are evaluated for the presence of the desired properties and/or the extent to which the desired properties are expressed. A first evaluation may include, for example, the level of bacterial/fungal resistance of the transformed plants, stable heritability of the desired properties, field trials and the like. By way of illustration and summary, the following scheme sets out a typical process by which transgenic plant material, including whole plants, may be prepared. The process can be regarded as involving up to seven steps:

(1) first isolating from a suitable source or synthesising by means of known processes an antisense DNA sequence derived from a DNA sequence encoding a SNFl-related protein kinase (SnRK);

(2) operably linking the said DNA sequence in a 5a to 3a direction to plant expression sequences as defined hereinbefore;

(3) transforming the construct of step (2) into plant material by means of known processes and expressing it therein;

(4) selecting for the presence of a marker gene where appropriate;

(5) optionally, screen the plant material treated according to step (3) for the presence of an antisense DNA sequence derived from a DNA sequence encoding a SNFl-related protein kinase (SnRK);

(6) optionally regenerating the plant material transformed according to step (3) to a whole plant; and

(7) optionally, screen the plant generated according to step (5) for the presence of an antisense DNA sequence derived from a DNA sequence encoding a SNFl-related protein kinase (SnRK).

The present invention thus also comprises transgenic plants and the sexual and/or asexual progeny thereof, which have been transformed with a recombinant DNA sequence according to the invention. The regeneration of the plant can proceed by any known convenient method from suitable propagating material either prepared as described above or derived from such material.

The expression "asexual or sexual progeny of transgenic plants" includes by definition according to the invention all mutants and variants obtainable by means of known process, such as for example cell fusion or mutant selection and which still exhibit the characteristic properties of the initial transformed plant, together with all crossing and fusion products of the transformed plant material.

Another object of the invention concerns the proliferation material of transgenic plants. The proliferation material of transgenic plants is defined relative to the invention as any plant material that may be propagated sexually in vivo or in vitro. Particularly preferred within the scope of the present invention are protoplasts, cells, calli, tissues, organs, seeds, embryos, egg cells, zygotes, together with any other propagating material obtained from transgenic plants.

References in the present application to plant tissue therefore include, but are not limited to the following: whole plants or parts of them, such as propagating material, i.e. pollen granules, anthers, protoplasts, cells, calli, tissues, organs, seeds, embryos, ovules, zygotes, tubers, roots, etc. , as well as vegetative growth such as leaves, stalks, buds, organs, flowers, fruits, tubers, roots etc.

Plant tissues or plant materials derived from transgenic plants prepared as described above are particularly advantageous as vegetative propagation of the plant is inhibited by the expression of the antisense DNA sequence in accordance with the present invention, i.e. tubers, leaves, stems, seeds, buds etc.

The inhibition of sprouting can occur prior to the harvest of the plant tissue or it can occur post-harvest of the plant tissue. Pre-harvest inhibition includes inhibition while the plant is still in the soil awaiting harvest. Post-harvest inhibition of sprouting includes inhibition during storage of the plant tissue, e.g. in grain storage etc.

In uses according to this aspect of the invention, the inhibition of sprouting may comprise inhibition of pollen development. The inhibition of pollen development can be taken to include any reduction (i.e. full or partial inhibition) in development over that achieved by otherwise similar plant pollen at an equivalent stage in gametogenesis. The inhibition of pollen development may comprise pollen sterility. Inhibition of pollen development or pollen sterility can lead to the production of non- viable pollen. Accordingly, the present invention includes the inhibition of transmission of the antisense DNA sequence through the male or female gametes of the plant. The generation of plants with non-viable pollen provides for progeny of the plant tissue which are sterile.

In the context of the present invention "sprouting" is defined to include all forms of vegetative or non-sexual propagation by plants from plant tissues, i.e. sprouting may occur in tubers, roots, seeds, cuttings, buds or on plant stems on mature plants where a wound has been caused. It includes the production of new leaves or shoots or the initiation of such processes to cause leaf or shoot development to begin, or for shoots to grow or to develop, or the production of a pollen tube from a pollen grain. The inhibition of sprouting in plant tissues is defined as the prevention or (statistically) significant reduction or retardation in these forms of growth or the creation of male sterility by inhibition of pollen tube formation from a pollen grain or sufficient impairment of function to prevent fertilisation.

According to a second aspect of the present invention, there is provided a method for inhibiting sprouting in plant tissues, the method comprising the steps of (a) transforming a plant with an antisense DNA sequence encoding a SNFl-related protein kinase (SnRK); and (b) expressing said DNA sequence in a plant tissue. According to a third aspect of the present invention there is provided the use of a DNA sequence encoding a SNFl-related protein kinase (SnRK) for the inhibition of sprouting in plant tissues. Typically, expression of the transgene results in the inhibition of the expression of the native genes ("switching-off ') by co-suppression. Alternative mechanisms of inhibition are also included within this aspect of the invention through expression of the DNA sequence. This aspect of the present invention also extends to a method of inhibiting sprouting in plant tissues. Such methods include a method for inhibiting sprouting in plant tissues, the method comprising the steps of (a) transforming a plant with a DNA sequence encoding a SNFl-related protein kinase (SnRK); and (b) expressing said DNA sequence in a plant tissue. The inhibition may be caused by down-regulation of gene expression as a result of transformation of the plant with a DNA sequence encoding a SNFl-related protein kinase (SnRK).

According to a fourth aspect of the present invention there is provided the use of a

DNA sequence encoding biologically inactive SNFl-related protein kinase (SnRK) for the inhibition of sprouting in plant tissues. In this aspect of the invention, the biologically inactive SNFl-related protein kinase (SnRK) may be prepared by synthesising a gene construct for the enzyme lacking a crucial amino acid, e.g. at the active site, or by including amino acids which disrupt the normal secondary or tertiary structure of the protein, e.g. proline residues^* or cysteine residues. Overexpression of such a gene construct would compete for substrate with the native enzyme leading to loss of biological function. Overexpression of related enzymes AMPK or SNFl could also be used to compete for substrate. This aspect of the present invention also extends to a method of inhibiting sprouting in plant tissues.

Such variants include polypeptides that are substantially homologous to native SnRK's, but which have an amino acid sequence different from that of a native SnRK because of one or more deletions, insertions or substitutions. The variant DNA or amino acid sequences are at least 80% identical to a native SnRK sequence, preferably 90% and suitably 95% identical. The percent identity may be determined, for example, by comparing sequence information using the GAP computer program version 6.0 described by Devereux et al (Nucleic Acids Res. 12 387 (1984)) and available from the University of Wisconsin Genetics Computer Group (UWGCG). The preferred default parameters for the GAP program include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) for nucleotides, and the weighted comparison matrix of Gribskov and Burgess (Nucleic Acids Res. 14 6745 (1986)), as described by Schwartz and Dayhoff, eds. Atlas of Protein Sequences and Structure, National Biomedical Research Foundation, pages 353-358 (1979)); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps.

An example of a variant of the present invention is a SnRK sequence as defined above, apart from the substitution of one or more amino acids with one or more other amino acids. The skilled person is aware that various amino acids have similar properties. One or more such amino acids of a substance can often be substituted by one or more other such amino acids without eliminating a desired activity of that substance. Thus the amino acids glycine, alanine, valine, leucine and isoleucine can often be substituted for one another (amino acids having aliphatic side chains). Of these possible substitutions it is preferred that glycine and alanine are used to substitute for one another (since they have relatively short side chains) and that valine, leucine and isoleucine are used to substitute for one another (since they have larger aliphatic side chains which are hydrophobic). Other amino acids which can often be substituted for one another include: phenylalanine, tyrosine and tryptophan (amino acids having aromatic side chains); lysine, arginine and histidine (amino acids having basic side chains); aspartate and glutamate (amino acids having acidic side chains); asparagine and glutamine (amino acids having amide side chains); and cysteine and methionine (amino acids having sulphur containing side chains). Substitutions of this nature are often referred to as "conservative" or "semi- conservative" amino acid substitutions.

Amino acid deletions or insertions may also be made relative to the amino acid sequence of a SnRK. Thus, for example, amino acids which do not have a substantial effect on the activity of the polypeptide, or at least which do not eliminate such activity, may be deleted. Amino acid insertions relative to the SnRK sequence above can also be made. Amino acid changes relative to the sequence given in a) above can be made using any suitable technique e.g. by using site-directed mutagenesis.

It should be appreciated that amino acid substitutions or insertions within the scope of the present invention can be made using naturally occurring or non-naturally occurring amino acids. Whether or not natural or synthetic amino acids are used, it is preferred that only L- amino acids are present.

Preferred features for the second and subsequent aspects of the invention are as for the first aspect mutatis mutandis.

The invention will now be further described by way of reference to the following Examples and Figures which are provided for the purposes of illustration only and are not to be construed as being limiting on the invention. Reference is made to a number of Figures in which:

FIGURE 1 shows a schematic diagram of chimeric genes comprising a patatin promoter (Rocha-Sosa et al (1989) supra) or ST-LS1 (Eckes et al (1985) supra), a 503bp antisense PCR fragment (PKIN503) derived from the potato SNFl -related gene PKIN1 (Man et al (1997) supra), and the octopine synthase gene termination sequence (Gielen et al (1984) supra).

FIGURE 2 shows the 503bp sequence called PKLN503 which was corresponds to nucleotide 608 to nucleotide 1111 of RKINl (Alderson et al (1991) supra).

FIGURE 3 shows a diagram of the design of a pBIN19-based vector for the introduction and expression of an antisense SnRK sequence in potato. Abbreviations: Kan = kanomycin-resistance gene; LB = left border; NOS = nopaline synthase; NPT = neomycin phosphotransferase; OCS octopic synthase.

FIGURE 4 is a photograph of some PAT 1J0 tubers (upper) and two groups of controls (lower) after 2 years at 4°C.

FIGURE 5 is a photograph of control potato plants (left hand side and right hand sides) and transgenic potato plant, PAT (centre).

FIGURE 6(a) to 6 (e). Figure 6(a) shows pollen from an untransformed barley plant. Figure 6(b) shows pollen from a transgenic barley plant expressing an antisense SnRKl sequence. Half of the pollen are small and are transparent because they contain no starch granules. Figure 6(c) shows pollen from a transgenic barley plant expressing an antisense SnRKl sequence stained for the presence of starch. Starch is indicated by a deep blue-black colour. The small, abnormal pollen grains contain little or no starch. Figure 6(d) shows GUS activity (shown by a blue colour) in pollen from a transgenic barley plant expressing an antisense SnRKl sequence. The abnormal pollen phenotype correlates exactly with presence of the transgenes. Figure 6(e) shows GUS activity (shown by a blue colour) in pollen from a plant transformed with the bar and UidA marker genes but not the SnRKl antisense sequence. Presence of the transgenes is indicated by the blue colour but is not associated with abnormal phenotype. References to colour in photographs is taken from the original prints. In the present application, the blue colour can be seen as darker grey or black tones in the Figures.

Example 1 : Production of transgenic potato plants expressing antisense SnRKl Transgenic potato plants expressing an antisense SnRKl sequence in the tubers were prepared according to Halford et al (Biochem. Soc. Trans. 22 953-957 (1994)) and Purcell et al (The Plant Journal 14 195-202 (1998)). Two chimeric genes were constructed and used to transform potato cv. Desiree and a schematic diagram of the chimeric gene constructs is shown in Figure 1. The constructs were introduced into the potato genome by Agrobacteήum-mediated transformation of leaf discs. The 503bp potato SNF7-related PCR fragment was inserted in an antisense orientation downstream of a promoter in a pBIΝ19-based vector (see Figure 3).

One construct consisted of 1.5kb of the promoter of a patatin gene (Rocha-Sosa et al (1989) supra), an antisense 503bp PCR product derived from the potato SNEi- related gene PKIN1 (Man et al (1997) supra), and the octopine synthase gene polyadenylation signal (Gielen et al (1984) supra). The other construct was identical except that the patatin promoter was replaced with 1.6kb of the promoter of ST-LS1, the gene which encodes the 10,000 molecular mass (lO.OkDa) protein of photosystem II. The patatin promoter is expressed only in the tubers, while the ST- LS1 promoter gives highest levels of expression in the leaves and is active to a lesser extent in stems (Εckes et al (1985), (1986) supra).

The 503bp sequence can be prepared by using PCR amplification of RKINl from potato (Solanum tuberosum) c.v. Desiree using oligonucleotide primers with sequences that are highly conserved in the plant SΝF1 -related gene family. The RKINl gene is the rye SnRKl that was the first plant SΝRK1 to be characterised (Alderson et al (1991) supra). The 503bp sequence called PKLΝ503 runs from nucleotide 608 to nucleotide 1111 of RKINl and is shown in Figure 2. The PCR product includes an EcoRl restriction site at the 5'-end, and a BamRI restriction site at the 3'-end. It shows 69.9% sequence identity with RKINl.

The template for the PCR reaction was cDNA prepared from total RNA extracted from potato leaf tissue. The oligonucleotides used to prime the reaction were:

5 ' -GGGGGAATTCGATGGTC ATTTTTTG AAG and, 5 ^* -GGGGGGATCCGGAAACGATTGTC .

The PCR product was 503bp in length and contained restriction sites as described above.

Transgenic lines transformed with the construct containing the patatin promoter were given the prefix PAT, those with the construct containing the ST-LS 1 promoter were given the prefix LS. Southern blot analysis to confirm the success of the transformations was as described in Purcell et al (1998) supra.

Example 2: Analysis of late sprouting in tubers of line PAT 1J0 Tubers from plants transgenic line PAT 1J0 were harvested, cold-treated at 4°C for 2 weeks and stored at 10-12°C. They were then planted in compost in pots in a greenhouse alongside control tubers. It was observed that sprouts from the PAT 1J0 tubers appears above the soil significantly later (approx. 2 weeks) than the controls. This lead to a preliminary study of the behaviour of the tubers in storage and it was found that tubers of line PAT 1J0 also sprouted later than controls when stored at 4°C or 12°C. Some batches of tubers failed to sprout at all at 4°C.

Figure 4 shows some PAT 1J0 tubers and controls after 2 years at 4°C, i.e. that engineering a reduction in SnRKl activity in potato tubers can be used to control sprouting during storage. The tubers of these plants show delayed sprouting in the soil and in storage, showing that genetic manipulation of SnRKl activity is a mechanism by which sprouting can be controlled in plants.

Figure 5 shows dramatically how sprouting in potato plants is reduced in transgenic plants compared to a control plant. The plant transformed with the PAT construct (centre) shows virtually no growth under optimal conditions. The plant transformed with the ST-LS construct (right hand side) shows slightly reduced leaf growth compared to the control plant (left hand side). Table 1 SnRKl family

Key to references:

(1) Le Guen et al.Gene 120 249-254 (1992)

(2) Huttly, A. K. and Philips A. L., Plant Molec. Biol. 27 1043-1052 (1995)

(3) Halford et al, The Plant Journal 2 791-797 (1992)

(4) Hannappel et al, Plant Molec. Biol. 27 1235-1240 (1995)

(5) Muranaka et al, Mol. Cell. Biol. 14 2958-2965 (1994)

(6) Man et al, Plant Molec. Biol. 34 31-43 (1997)

(7) Alderson et al, Proc. Nat'l Acad. Sci. USA 88 8602-8605 (1991)

(8) Monger etal.Plant Growth Regulation 22 181-188 (1997)

(9) Lakatos et al, Plant Physiol. 113 1004 (1997)

Table 2 SnRK2 family

Key to references:

(10) Bmi et al Plant Physiol. 106 1225-1226 (1994)

(11) Anderberg et alProc. Nat'l Acad. Sci. USA 89 10183-10187 (1992)

(12) Park et al Plant Mol. Biol. 22 615-624 (1993)

(13) Park et al Plant Mol. Biol. 27 829-833 (1995)

Table 3 SnRK3 family

Key to references:

(14) Sano, H. and Youssefian, S. Proc. Nat'l Acad. Sci. USA 91 2582-2586

Example 3: Inhibition of pollen tube formation

Inhibition of pollen tube formation was investigated in a study of transgenic barley plants expressing an antisense SnRKl sequence. The sequence was placed under the control of a wheat seed-specific promoter (high molecular weight glutenin subunit gene Glu-lDx5 (Halford et al Plant Science 62 207-216 (1989)). However, clear evidence of expression of the transgene was detected in anthers and approximately 50% of the pollen in 6 independent lines were abnormal.

Construction of expression vectors

An expression vector, PYZ1, was made that contained DNA fragments amplified from cDNA clone BKIN2 and genomic clone BKIN12 (Halford et al., 1992; Hannappel et al , 1995), respectively, by Polymerase Chain Reaction (PCR). The BKIN2 sequence was amplified using primers BA3,

5 ' -ACTGggatccGTGATATC AGGTAAACTG and BA4,

5 ' -ACTGtctagaGGTTTGGTATATTCTCAT

The BKIN12 sequence was amplified using primers BA1,

5 ^* -ACTGctgcagATTTTAGCTGGTGTTGAA and BA2,

5 ' -ACTGggatccCTCTGGTGC AGC ATAGTT-3 '

The BKIN2 and BKIN12 sequences were cloned in tandem in the antisense orientation between the wheat HMW glutenin subunit gene (Glu-lDxS) promoter (Halford et al., 1989) and the cauliflower mosaic virus 35S terminator in plasmid pLRPT.

Production of Transgenic Barley Plants

Transgenic barley plants were produced by particle bombardment of immature embryos followed by callus induction and plant regeneration. Plasmid PYZ1 was co-transformed with pAHC25, which carries the selectable gene bar encoding the enzyme phosphinotricin acetyltransferase (PAT) and the screenable gene uidA encoding β-glucuronidase (GUS), both under control of the maize ubiquitin (Ubi-l) promoter. Plants resistant to the selective agent Bialaphos were screened for the presence of bar and/or uidA, and then for the presence of the antisense SnRKl sequence, by PCR. Histochemical GUS assays of leaf tissues was also used for screening. Twenty-three transgenic lines were produced, of which 20 contained the antisense SnRKl sequence.

Morphological Changes of Pollen Development in Transgenic Lines

Among the 20 co-transformed lines containing the antisense SnRKl sequence, 14 lines were morphologically normal. However, cytological observation of pollen development showed that 6 of these independent transgenic lines had two remarkably distinct forms of pollen. Half of the pollen grains were smaller than normal and accumulated little or no starch (Figure 6(a) and 6(b)). The difference between normal and abnormal pollen in terms of starch accumulation was confirmed by iodine staining (Figure 6(c)). The normal pollen stained deep blue-black, as expected, whereas the abnormal pollen stained brown, indicating the presence of little or no starch.

Confirmation of expression of the antisense SnRKl sequence in anthers The Glu-lDx5 promoter is a wheat seed-specific promoter and an effect on pollen development had not been expected from this transgene. However, confirmation that the antisense SnRKl sequence was being expressed in anthers was obtained by RT- PCR amplification of the antisense SnRKl sequence from anther mRNA purified from the transgenic plants.

Pollen abnormality was due to presence of the antisense SnRKl transgene

Half of the pollen from the transgenic plants would be expected to carry the transgenes, while half would not, and this fitted with the observation that half of the pollen were normal, half abnormal. It was confirmed that the abnormal development was associated with presence of the transgenes by staining for activity of the co- transformed screenable marker gene, GUS (Figure 6(d)). GUS expression (indicated by a blue colour) was detectable in all of the abnormal pollen grains but not in the starch-filled, normal pollen grains. No abnormality was found in pollen produced by transgenic plants containing the bar and uidA marker genes but not the antisense SnRKl sequence. Half of the pollen from these plants stained blue when assayed for GUS activity but were otherwise normal (Figure 6(e)).

Pollen viability and lack of transmission of the transgenes to the Tl generation

The viability of the normal and abnormal pollen was examined using fluorescein diacetate (FDA) staining. Normal pollen grains looked brighter than the abnormal ones, indicating that the former were more viable than the latter. Pollen inviability was confirmed by looking for the presence of the transgenes in progeny of the transgenic plants. The transgenic lines set only approximately 50 % of the seed of normal plants. Tl seed were germinated and grown into plants. These Tl plants were shown by PCR to carry none of the three transgenes present in the TO plants, and produced normal pollen. This confirmed that the abnormal pollen of the TO plants were inviable, and that the embryo sac was also affected, since there was no transmission of the transgenes through either the male or female gametes.

Summary

Six independent transgenic lines were found to be abnormal in terms of pollen development and transgene transmission. The abnormal pollen, which made up 50% of the pollen population in these lines, were smaller and contained no starch grains. Antisense SnRKl transcripts were detected in the anthers of the transgenic lines using RT-PCR. GUS activity arising from one of the genes co-transformed with the antisense sequence was detected only in abnormal pollen, indicating that the abnormality was associated with transgene presence. All of the SnRKl-antisense lines gave approximately 50 % seed set, and no transgene transmission was detected in the next generation, indicating that pollen grains containing the transgenes were sterile. The data presented here indicate that SnRKl is essential for normal pollen development in barley and that antisense down-regulation of barley SnRKl in developing pollen results in pollen sterility.

METHODS

Plant Material

Barley plants (Hordeum vulgar e L. c.v. Golden Promise) were grown in an environmentally controlled plant growth chamber at 18 °C/16 °C (day /night) with a photoperiod of 16 hours.

Construction of expression vectors

Expression vector PYZ1 was made for plant transformation using standard molecular cloning procedures. It contained DNA fragments amplified from cDNA clones BKIN2 and genomic clone BKIN12 (Halford et al The Plant Journal 2(5) 791-797 (1992); Hannappel et al Plant Molecular Biology 271235-1240 (1995)), respectively, by Polymerase Chain Reaction (PCR).

B2 was amplified using primers BA3,

5 ' -ACTGggatccGTGATATCAGGTAAACTG and BA4,

5 ' -ACTGtctagaGGTTTGGTATATTCTC AT

B12J was amplified using primers BA1

5 ' -ACTGctgcag ATTTT AGCTGGTGTTGAA and BA2

5 ' -ACTGggatccCTCTGGTGCAGCATAGTT-3 ' Appropriate restriction sites were added at the 5' end of the primers to facilitate subcloning work (lettered in lower case). B2 was purified as a Xbal/BamBI restriction fragment (123 bp) cloned into expression vector pLRPT (containing the wheat Glu-lDx5 promoter and cauliflower mosaic virus 35S terminator) behind the wheat HMW promoter in an antisense orientation. B12J was purified as a BamHllPstl fragment (193 bp). It was then cloned directly alongside B2 in pLRPT in an antisense orientation. The resulting plasmid pYZl(-) contained fragments B2 and B12J in tandem in an antisense orientation.

Transformation

Barley spikes containing immature embryos of ca. 2.0mm in diameter were collected from the growth chamber. Immature seeds were separated and the lemma were peeled off using fine forceps. The isolated seeds were rinsed with 70% ethanol, surface-sterilized for 10 minutes in 10 % (v/v) bleach, and rinsed three to four times with sterile distilled water. Immature embryos were dissected aseptically with axis removed and the transformation procedure carried out using the method described by Wan and Lemaux (Wan, Y.C. and Lemaux, P.G. , Plant Physiology 104 37-48 (1994)) using a DuPont PDS-1000/He biolistic device at l lOOpsi. Gold particles were coated with a mixture of plasmids pAHC25 and pYZl at 1:2 molar ratio (pAHC25 contained the selectable gene bar encoding the enzyme phosphinotricin acetyltransferase (PAT) and the screenable gene uidA encoding β-glucuronidase (GUS), both under control of the maize ubiquitin (Ubi-l) promoter and first intron (Toki et al Plant Physiology 100 1503-1507 (1992)) followed by the 3' untranslated region and polyadenylation signal of the nopaline synthase gene (nos) from Agrobacterium tumefaciens (Bevan et al Nucleic acids Research 12 8711-8721 (1984)). All the culture steps were conducted at 25 ± 1 °C in darkness for embryogenesis or under light of a 16 hours photoperiod for plant regeneration.

PCR screening of transgenic lines

Total genomic DNA was isolated from leaf tissues of primary transformants and their progenies using a modified CTAB method of Stacey and Isaac (1994) and described in Barro et al. (Barro et al Nature Biotechnology 15 1295-1299 (1998)). The presence of transgenes was determined by PCR using the following primer pairs:

For bar, primers bar 1 and bar 2 were used, i.e. barl

5 ' -GTCTGC ACCATCGTC AACC and bar2

5 ' -GA AGTCC AGCTGCC AGA A AC

For uidA, primers gus5 and gus6 were used, i.e. gus 5

5 ' -AGTGTACGTATCACCGTTTGTGTGAAC and gusό,

5 ' -ATCGCCGCTTTGGACATACCATCCGTA

The amplified DNA fragments were approximately 420 bp and 1020 bp, respectively. For the antisense transgene B2B12J, primers 1D3,

5 ' -GTTGGC AAACTGCGC from the promoter region and BA1 or BA3 (see above) were used. A DNA fragment of about 525 bp was amplified when 1D3 / BA1 was used and a DNA fragment of about 336 bp was amplified when 1D3 / BA3 was used. For antisense transgene B12.2, the primers 1D3 and BA5 (see above) were used and a DNA fragment of about 337 bp was amplified. PCR reaction conditions were as described in Barro et al (1998).

Cytological observation of pollen grains

Anthers were collected at various developmental stages from transgenic plants grown in the glass house. Pollen grains were released on a glass slide by gently squashing anthers with fine forceps in a drop of staining solution (see below). Anther debris was removed carefully, and a cover slip was applied over the staining solution containing pollen grains before examination under microscope (Olympus, BH-2). Acetocarmine staining: The developmental stage of pollen grains was checked by staining in a drop of 1 % acetocarmine solution (lg acetocarmine dissolved in 100 ml 45% acetic acid). Iodine staining: To check starch accumulation, pollen grains from mature anthers were stained in a drop of I₂-IK solution (lg I₂ and 2g IK dissolved in 300 mL distilled water). Pollen grains accumulating starch turned a dark-blue colour, while pollen grains without starch were brown.

FDA staining: The viability of pollen grains from mature anthers was determined by staining in FDA (fluorescence diacetate) solution. Pollen grains were released in a drop of FDA working solution (FDA was first prepared in acetone at a concentration of 2 mg/iriL as a stock solution. The stock was diluted with 0.4 M mannitol before use. The viable pollen grains emitted yellowish green fluorescence under a UV light.

Histochemical assay of β-glucuronidase (GUS) activity in pollen

Histochemical staining for GUS expression was performed using 5-bromo-4-chloro- 3-indoxyl-β-D-glucuronic acid (X-gluc) as described by Jefferson et al. (Jefferson et al EMBO Journal 6 3901-3907 (1987)).

RT-PCR

RT-PCR was used to determine the expression of the antisense gene in anthers of transgenic plants. Total RNA was extracted from 120 to 180 mature or near mature anthers (corresponding 2 to 3 spikes), where the pollen grains inside were filled with starch, using a RNeasy plant mini kit (Qiagen Ltd. UK). The RNA was then quantified on a spectrophotometer at OD₂₆₀. Approximatly 50μg total RNA of high quality was generally obtained from each sample. The integrity of RNA was checked by running on a denaturing formaldehyde-agarose gel. Before RT-PCR, total RNA aliquots were treated with RQ RNase-free DNase I (Promega, UK). First-strand cDNA of the antisense transcripts was synthesised using gene-specific primer BAl (see section "construction of expression vectors") and C. therm. Polymerase (Roche Molecular Biochemicals) . The reaction was as described in the user's instruction manual provided by the manufacturer. The PCR reaction was carried out in lOOμl volume containing 1 x reaction buffer (75mM Tris-HCl, 20mM (NH₄)₂SO₄, 0.1 % Tween 20), 0.2μM dNTPs, 1.5mM MgCl₂, 0.3μM primers, O.όunits DNA polymerase (MBI Fermentas), and 5μl cDNA. Primers were BAO (forward), 5'-AAACTAGAGATCAATTCACTGATAG TCC-3' from the leader sequence of the transcript and BAl (see section 'construction of expression vectors'). The PCR profile was as follows: 94°C 3 minutes, 55°C 1 minute, 72°C 2 minutes, 1 cycle; 94°C 1 minute, 55°C 45 seconds, 72°C 2 minutes and 3 seconds delay, 35 cycles; 72°C extension for 10 minutes. The product length was approximately 358 bp.

Claims

1. The use of an antisense DNA sequence encoding a SNFl-related protein kinase (SnRK) for the inhibition of sprouting in plant tissues.

2. A use as claimed in claim 1, in which the SNFl-related protein kinase is SnRKl.

3. A use as claimed in claim 1 or claim 2, in which the SNFl-related protein kinase regulates the transcription of genes encoding enzymes of carbohydrate metabolism.

4. A use as claimed in claim 3, in which the enzyme of carbohydrate metabolism comprises Phosphofructokmase, pyruvate kinase, acid invertase, starch synthase, adenine diphosphoglucose pyrophosphorylase, sucrose synthase, 6-phospho- fructokinase (pyrophosphate) or sucrose phosphate synthetase.

5. A use as claimed in claim 4, in which the enzyme of carbohydrate metabolism is sucrose synthase.

6. A use as claimed in any preceding claim in which starch accumulation in the plant tissues is restricted.

7. A use as claimed in claim 6, in which the plant tissues do not contain starch.

8. A use as claimed in any preceding claim, in which the SNFl-related protein kinase is the SnRKl sequence shown in Figure 2.

9. A use as claimed in any preceding claim, in which the plant is of the family Leguminosea, Graminaceae, Saccharaceae, Solonaceae, Fabaceae, or Brassicaceae.

10. A use as claimed in any preceding claim, in which the plant is of the genera Lolium, Zea, Triticum, Sorghum, Triticale, Saccharum, Bromus, Oryzae, Avena, Hordeum, Secale, Setaria, or Solanum.

11. A use as claimed in any preceding claim in which the plant is Solanum tuberosum.

12. A use as claimed in any preceding claim in which the plant tissue comprises protoplasts, cells, calli, tubers, leaves, stems, seeds, buds, tissues, organs, seeds, embryos, egg cells, or zygotes.

13. A use as claimed in any one of claims 1 to 12, in which the inhibition of sprouting occurs prior to the harvest of the plant tissue.

14. A use as claimed in any one of claims 1 to 12, in which the inhibition of sprouting occurs post-harvest of the plant tissue.

15. A use as claimed in claim 14, in which the inhibition of sprouting occurs during storage of the plant tissue.

16. A use as claimed in any preceding claim, in which the inhibition of sprouting comprises inhibition of pollen development.

17. A use as claimed in claim 16, in which inhibition of pollen development comprises pollen sterility.

18. A use as claimed in any preceding claim, in which the inhibition of sprouting comprises the inhibition of transmission of the antisense DNA sequence through the gametes of the plant..

19. A use as claimed in any preceding claim, in which the progeny of the plant tissue are sterile.

20. The use as claimed in claim 1, in which the inhibition of sprouting comprises the inhibition of pollen tube formation.

21. A method for inhibiting sprouting in plant tissues, the method comprising the steps of (a) transforming a plant with an antisense DNA sequence encoding a SNFl- related protein kinase (SnRK); and (b) expressing said DNA sequence in a plant tissue.

22. The use of a DNA sequence encoding a SNFl-related protein kinase (SnRK) for the inhibition of sprouting in plant tissues

23. A method for inhibiting sprouting in plant tissues, the method comprising the steps of (a) transforming a plant with a DNA sequence encoding a SNFl-related protein kinase (SnRK); and (b) expressing said DNA sequence in a plant tissue.

24. A method for inhibiting sprouting in plant tissues through down regulation of gene expression, the method comprising transforming a plant with a DNA sequence encoding a SNFl-related protein kinase (SnRK); and (b) expressing said DNA sequence in a plant tissue.

25. The use of a DNA sequence encoding biologically inactive SNFl-related protein kinase (SnRK) for the inhibition of sprouting in plant tissues.