AU2003271420B2 - Use of bifunctional alpha-amylase subtilisin inhibitor promoter to direct expression in the maternal tissue of a plant seed - Google Patents

Description

WO 2004/035790 PCTiAU2003/001381 USE OF BIFUNCTIONAL ALPHA-AMYLASE SUBTILISIN INHIBITOR PROMOTER TO DIRECT EXPRESSION IN THE MATERNAL TISSUE OF A PLANT SEED Field of the Invention The present invention relates generally to a method of expressing nucleic acid or protein in the maternal tissue of a plant seed, especially the pericarp, and more particularly to a method wherein nucleic acid is expressed at the RNA or protein level operably under the control of a regulatory sequence o1 derived from a bifunctional alpha-amylase subtilisin inhibitor gene SEQ ID NOs: 1 or As exemplified herein, a wide range of structurally and functionally diverse genes are expressed in the pericarp of plants under the control of the regulatory sequence of the invention, in particular a gene selected from the group consisting of: a gene any one of SEQ ID NOs: 15, 17, 19, 21, 23, 25) encoding a wheat thaumatin-like protein any one of SEQ ID NOs: 16, 18, 20, 22, 24, 26) that confers protection against the fungal pathogen Fusarium graminearum (head scab) in wheat; (ii) a gene SEQ ID NO: 27) encoding a modified ribosomal protein L3 of wheat wRPL3:Cys 258; SEQ ID NO: 28) that is resistant to the action of a trichothecene produced by F. graminearum; (iii) a gene SEQ ID NO: 29) encoding a polypeptide having trichothecene O-acetyl transferase activity SEQ ID NO: 30) to thereby convert trichothecene produced by F. graminearum into a non-toxic product; (iv) a gene SEQ ID NO: 31) encoding the coat protein of barley stripe mosaic virus (SEQ ID NO: 32); a gene (SEQ ID NO: 31) encoding siRNA against the movement protein of BSMV (SEQ ID NO: 33); (vi) a gene (SEQ ID NO: 35) encoding an antigenic polypeptide of Transmissible Gastroenteritis Virus (SEQ ID NO: 36) for delivery to animals as a medicinal foodstuff or oral vaccine; and (vii) a gene (SEQ ID NO: 37) encoding a sulphurrich Brazil Nut Protein (BNP; SEQ ID NO: 38).

WO 2004/035790 PCT/AU2003/001381 2 Background to the invention 1. General This specification contains nucleotide and amino acid sequence information prepared using Patentln Version 3.1, presented herein after the claims. Each nucleotide sequence is identified in the sequence listing by the numeric indicator <210> followed by the sequence identifier <210>1, <210>2, <210>3, etc). The length and type of sequence (DNA, protein (PRT), etc), and source organism for each nucleotide sequence, are indicated by information provided in the numeric indicator fields <211>, <212> and <213>, respectively. Nucleotide sequences referred to in the specification are defined by the term "SEQ ID followed by the sequence identifier (eg. SEQ ID NO: 1 refers to the sequence in the sequence listing designated as <400>1).

The designation of nucleotide residues referred to herein are those recommended by the IUPAC-IUB Biochemical Nomenclature Commission, wherein A represents Adenine, C represents Cytosine, G represents Guanine, T represents thymine, Y represents a pyrimidine residue, R represents a purine residue, M represents Adenine or Cytosine, K represents Guanine or Thymine, S represents Guanine or Cytosine, W represents Adenine or Thymine, H represents a nucleotide other than Guanine, B represents a nucleotide other than Adenine, V represents a nucleotide other than Thymine, D represents a nucleotide other than Cytosine and N represents any nucleotide residue.

As used herein the term "derived from" shall be taken to indicate that a specified integer may be obtained from a particular source albeit not necessarily directly from that source.

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated step or element or integer or group WO 2004/035790 PCT/AU20031001381 3 of steps or elements or integers but not the exclusion of any other step or element or integer or group of elements or integers.

Unless specifically stated otherwise, each feature described herein with regard to a specific embodiment of the invention, shall be taken to apply mutatis mutandis to each and every other embodiment of the invention.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.

The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for the purposes of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the invention, as described herein.

The present invention is performed without undue experimentation using, unless otherwise indicated, conventional techniques of molecular biology, microbiology, virology, recombinant DNA technology, peptide synthesis in solution, solid phase peptide synthesis, and immunology. Such procedures are described, for example, in the following texts that are incorporated by reference: 1. Sambrook, Fritsch Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Second Edition (1989), whole of Vols I, II, and III; 2. DNA Cloning: A Practical Approach, Vols. I and II N. Glover, ed., 1985), IRL Press, Oxford, whole of text; WO 2004/035790 PCT/AU2003/001381 4 3. Oligonucleotide Synthesis: A Practical Approach J. Gait, ed., 1984) IRL Press, Oxford, whole of text, and particularly the papers therein by Gait, ppl-22; Atkinson et al., pp35-81; Sproat et al., pp 83-115; and Wu et al., pp 135-151; 4. Nucleic Acid Hybridization: A Practical Approach D. Hames S. J.

Higgins, eds., 1985) IRL Press, Oxford, whole of text; Immobilized Cells and Enzymes: A Practical Approach (1986) IRL Press, Oxford, whole of text; 6. Perbal, A Practical Guide to Molecular Cloning (1984); 7. Methods In Enzymology Colowick and N. Kaplan, eds., Academic Press, Inc.), whole of series; 8. J.F. Ramalho Ortigao, "The Chemistry of Peptide Synthesis" In: Knowledge database of Access to Virtual Laboratory website (Interactiva, Germany); 9. Sakakibara, Teichman, Lien, E. Land Fenichel, R.L. (1976).

Biochem. Biophys. Res. Commun. 73 336-342 Merrifield, R.B. (1963). J. Am. Chem. Soc. 85, 2149-2154.

11. Barany, G. and Merrifield, R.B. (1979) in The Peptides (Gross, E. and Meienhofer, J. eds.), vol. 2, pp. 1-284, Academic Press, New York.

12. W0nsch, ed. (1974) Synthese von Peptiden in Houben-Weyls Metoden der Organischen Chemie (MOler, vol. 15, 4th edn., Parts 1 and 2, Thieme, Stuttgart.

13. Bodanszky, M. (1984) Principles of Peptide Synthesis, Springer-Verlag, Heidelberg.

14. Bodanszky, M. Bodanszky, A. (1984) The Practice of Peptide Synthesis, Springer-Verlag, Heidelberg.

Bodanszky, M. (1985) Int. J. Peptide Protein Res. 25, 449-474.

16. Handbook of Experimental Immunology, Vols. I-IV M. Weir and C. C.

Blackwell, eds., 1986, Blackwell Scientific Publications).

WO 2004/035790 PCT/AU2003/001381 2. Description of the related art A major problem in the genetic improvement of agriculturally-important crops, in particular those crops producing metabolites (proteins and small peptides including antigens, essential amino acids, starches, oils, lipids, lignin, osmoprotectants such as betaines, phytosterols, calcium, sulphur, etc), is the manipulation of gene expression to produce plants which exhibit novel characteristics. In this respect, transgenic plants have been used to produce a wide range of structurally and functionally diverse proteins and nucleic acids.

Some examples to date are the production of interferon in tobacco (Goodman et al., 1987), encephalins in tobacco, Brassica napus and Ababidopsis thaliana (Vandekerchove et al., 1989), human serum albumin in tobacco and potato (Sijmons et al., 1990), antibodies in tobacco (Hiatt et al., 1990) and hepatitis B antigen (Mason et al., 1992).

However, the expression of novel characteristics is often required to be effected in specific cell types, tissues or organs of the plant, or under specific environmental or developmental conditions.

Advances in biotechnological research have produced an explosion of information in relation to the number of genetic sequences identified which, if appropriately expressed, are useful to produce improved crop plants, for example plants in which reproductive development is controlled, plants having altered shape or size characteristics, plants capable of rapid regeneration following harvest, or plants having improved resistance to pathogens, amongst others.

The application of biotechnology to the production of plants expressing novel traits is limited by the availability of genetic sequences which are capable of conferring appropriate expression patterns upon structural genes. Clearly, in the absence of appropriate regulatory sequences to confer expression in a particular cell type at a particular stage of development and/or in response to WO 2004/035790 PCT/AU2003/001381 6 specific environmental and hormonal stimuli, the potential of genetic sequences which encode novel proteins to express those proteins in planta cannot be realised.

Pericarp structure and function The maternal tissue is highly significant to the normal development of seeds. Pericarp strength and thickness are important quality traits for many agricultural crops, such as maize, barley, sorghum, wheat and other grain crops. For example, in maize seeds, the pericarp provides a barrier to water movement into and out of the kernel, and damaged pericarp greatly reduces germination in all endosperm types.

The pericarp is the outer layer of the ovary wall and, as a consequence is entirely maternally derived plant tissue. It comprises an inner area (endocarp), centre portion (mesocarp) and outer skin-like portion (exocarp).

The form of the pericarp varies in different types of plants. For example, in fleshy fruits, such as, for example, berries and tomato, the pericarp is soft and fleshy at maturity and may be edible. Pepos, such as, for example, watermelons, have a pericarp that form a hard rind surrounding the edible fruit, whilst Hesperidiums, such as, for example, citrus (oranges, lemons) have more leathery rinds. Drupe or stone fruits, such as, for example, peaches and plums, have a pericarp comprising a thin exocarp, a thick mesocarp, and a hard endocarp. In pome fruits, such as, for example, apples and pears, the pericarp is enclosed by fleshy parts that develop from parts of the flower other than the ovary. In contrast to fleshy fruits, dry fruits, such as, for example, nuts (macadamia, pecans, walnuts, acorns, etc) have a hard pericarp at maturity. In grain crops, such as, for example, wheat, barley and maize, the seed is completely fused to the pericarp.

Wheat bran is the outer layer of the wheat caryopsis and is itself composed of pericarp-testa and aleurone. The bran is rich in proteins of high WO 2004/035790 PCTiAU2003/001381 7 nutritional value and B-vitamins. During milling, the bran is generally removed and utilized for animal feed.

The lignin content of pericarp may be contributing factor to its protective characteristics. Accordingly, enhancing the lignin content of pericarp, such as by ectopically expressing one or more genes encoding lignin biosynthesis enzymes in the pericarp, may provide a means of protecting seeds against physical damage.

The pericarp also offers a significant post-harvest barrier to seed damage. For example, severe shrinking of the maize endosperm as it dries, such as in low sugar-containing lines (eg. sh2/sh2 plants), creates a number of structural problems for the seed, including the formation of air pockets between endosperm and pericarp and subsequent cracking of the pericarp, thereby increasing the susceptibility of the kernel to physical damage during handling.

Accordingly, it is important to maintain the structural integrity of the pericarp during seed development.

Notwithstanding the importance of pericarp strength, there are circumstances for which a reduced pericarp thickness is also beneficial. This is because a thick pericarp can adversely affect post-harvest processing of seed, such as, for example, making de-hulling and decortication processes more costly or difficult, or reducing yield of polished grain. The relatively low protein content of the pericarp relative to the endosperm and embryo of the seed, and poor digestibility of thick pericarps, can also make thick pericarp less desirable.

The role of the pericarp in phytoprotection against pathogens Maternal effects are important in resistance to several plant pathogens, such as, for example, the fungal pathogens causing kernel and ear rot in grain crops, Fusarium, Diplodia, and Gibberella) suggesting that the site of WO 2004/035790 PCT/AU2003/001381 8 resistance may be the pericarp. Accordingly, the pericarp offers a considerable barrier against pathogens, particularly fungal pathogens in many plants.

For example, latent infections of avocado by Colletotrichum gloeosporioides require secretion of pectic lyase by the fungus, which is pH dependent. Accordingly, genetic manipulation of the pericarp pH, such as, for example, by expressing acidic protein in the pericarp, can be used to produce a cultivar having enhanced resistance. Alternatively, pericarp-specific expression of nucleic acid capable of targeting the pectic lyase of the fungus may also produce a cultivar having enhanced resistance against C. gloeosporioides.

Exemplary pathogens of cereal crops, such as, for example, wheat and barley, that are seed borne, are discussed below.

1. Karnal bunt Karnal bunt is a severe disease of wheat in which the pericarp forms the infection site. Karnal bunt is caused by Tilletia indica, the symptoms of which are evident only when the grain fully develops. T. indica is pathogenic to Triticum aestivum, T. durum, T. boeticum, T. ovatum, T. variabilis and T.

shareo nensis, Triticale and Aegilops spp. The pathogen does not invade the embryo and the mycelial growth is limited to the pericarp, converting the infected ovary into a sorus where a mass of dark brown colored teliospores are produced. The mycelium proliferates in the pericarp by disintegrating the parenchymatous cells of the mesocarp and preventing the fusion of the ectocarp and endocarp with the seed coat, and rupturing the connection between the pericarp tissue surrounding the vascular bundle in the bottom of adaxil groove in the pericarp and the nuclear projection along the length of the developing seed. The consequence is atrophy of the seed through disruption of normal flow of nutrients from the pericarp, leading to starvation of the endosperm and the embryo.

WO 2004/035790 PCT/AU2003/001381 9 2. Barley Stripe Mosaic Virus Barley Stripe Mosaic Virus (BSMV) is a seed-borne hordeivirus that predominantly infects barley, and is also found in wheat and several grass species. The virus is seed-borne, translocated through the plant vasculature, and transferred from plant to plant when crop leaves rub against one another.

Experimentally, the virus can be transmitted by pollen, but since barley is selfpollinated this method of spread is generally of no consequence. Infected seeds produce infected plants. Seed from virus-infected plants is generally infected to a 60 per cent level. Disease builds up when infected seed is planted year after year. Symptoms may vary with the virulence of the BSMV strain and time of infection. Infections appear as chlorotic mottling with spots or stripes of a yellowish color. Infected plants may be stunted and may mature later than healthy plants.

Yield losses from BSMV are proportional to the level of infection in the seed lot. Losses are caused by reduced grain production, fewer heads per plant, semi-sterility and incomplete head emergence from the sheath. Heavily infected crops have had yield reductions of up to 25 per cent. The percentage of infected seedlings indicates the level of grain infection. Accordingly, there is a clear need to reduce the incidence of BSMV infection of seed lots.

The BSMV genome is composed of positive-sense ssRNA divided into three components designated a, P, and 7. The a- and y- RNAs are strictly required for replication, while RNA 3 is required for cell-to-cell movement. RNAP encodes a 5'-proximal coat protein (CP) separated from the triple gene block (TGB) by a short intergenic region. The TGB1 protein, formerly designated 3b, is expressed from the 2.45-kb sgRNApl to produce a 58-kDa TGB1 protein that binds to ssRNA and double-stranded RNA, to exhibit ATPase activity, and to bind nucleotides in vitro. Mutations of conserved amino acids within the TGB1 protein abrogate cell-to-cell movement of the virus. Infectivity results clearly demonstrate that the TGB1, TGB2, and TGB3 proteins are each WO 2004/035790 PCT/AU2003/001381 required for cell-to-cell movement in both monocotyledonous and dicotyledonous hosts.

3. Kernel and ear rot Fusarium kernel rot, also known as head scab or Fusarium head blight (FHB) is a devastating disease of corn, wheat and barley that is primarily caused by the fungus Gibberella zeae (anamorph=Fusarium graminearum).

This disease can reach epidemic levels and causes extensive damage to wheat and barley in humid and semi-humid wheat growing areas of the world, in particular the United States, Canada, China, Australia, India, Russia, France, Germany and the United Kingdom. The infection of seed by F. graminearum reduces seed germination, seedling vigor and plant emergence (Bechtel et al., 1985) Cereal Chem. 62:191-197. Infection of wheat kernels by F. graminearum reduces grain yield and affects grain quality (Clear et al., 1990) Can. J. Plant Sci. 70:1057-1069. F. graminearum also has a strong adverse effect on pasta color when Fusarium-damaged kernels make up as little as 2% of a seed lot (Dexter et al., 1997 Cereal Chem. 74:519-525).

Reductions in grain yield by F. graminearum are at least partially attributable to the pathogen producing mycotoxins, such as the trichothecenes, which are retained in the grain at high levels following infection. The major trichothecene produced by F. graminearum is deoxynivalenol (DON), also known as vomitoxin). Trichothecenes are potent protein synthesis inhibitors and are quite toxic to humans and livestock (Snijders, 1990 Neth J. Plant Pathol. 96: 187-198; Proctor et al., 1995 MPMI 8: 593-601; Casale et al., 1988, Phytopathology 78:1673-1677).

The pericarp as a source of secondary metabolites The maternal tissue also produces several secondary metabolites that are useful end-products, or provide a defense mechanism against pathogens or WO 2004/035790 PCT/AU2003/001381 11 predators, or contribute to the physiological function and development of the seed, including seed maturation, dormancy and germination.

For example, the seed coat, nucellar (hyaline) layer and aleurone layer have a high protein content and are useful as ingredients in breakfast cereals, binders, breads and snack foods, premium feeds and rusk, thereby resulting in value-added products for the mill.

A variety of activites have been ascribed to the hydrotannins (tannic acid and the ellagitannins) which are formed from gallic acid and its derivatives, and the proanthocyanidins or condensed tannins, produced by the pericarp of monocotyledonous plants. For example, the proanthocyanidins provide phytoalexin properties.

Similarly, grain flavonoids inhibit the growth of various pathogenic microbes and may stimulate the growth of symbiotic bacteria.

Anthocyanin pigments such as, for example, pelargonidin, cyanidin and delphinidin derivatives, can have potent anti-oxidant activity. Isoflavones in soybean seeds have been shown to have antifungal and anti-oxidant activities.

Resistance to grain mold in sorghum is also attributed to enhanced levels of specific phenolic compounds, such as, for example, apigeninidin, flavan-4-ols and proanthocyanidins.

Isoflavones are also of particular interest as phytoestrogens that protect against heart disease or cancer.

On the other hand, certain secondary metabolites produced by the pericarp, such as, for example, proanthocyanidins or condensed tannins produced in sorghum and barley pericarp tissues, contribute to the WO 2004/035790 PCT/AU2003/001381 12 unpalatability of seed. Such favonoids also cause bloat in animals fed on whole seed rather than processed or de-hulled grain. The proanthocyanidins of barley have undesirable consequences for beer production, and the isolation of proanthocyanidin-deficient mutants has improved malting qualities of barley.

The pericarp as a factory for producing non-endogenous proteins It will also be apparent to the skilled artisan that there is an enormous potential for expressing a wide range of structurally and functionally diverse proteins and nucleic acids in the pericarp of a plant, particularly in view of the utility of the pericarp in animal feed. By virtue of the high pericarp content of bran, the potential also exists for improving the nutritional value of the bran for humans by expressing proteins in the pericarp.

1. Oral vaccines It is widely recognized that mucosal immunity is generally best induced by direct immunization of the mucosal tissue. Preferred oral vaccines against mucosal diseases, vaccines should stimulate the mucosal system and generate an SIgA immune response. Once mucosal immunity is established in an animal it can be advantageously transferred to the offspring through colostrum and/or milk lactogenic immunity) and is an efficient way to protect animals during early life. SIgA is the major immunoglobulin in milk and is most efficiently induced by mucosal immunization.

One way of achieving lactogenic immunity is by administering a vaccine orally to a lactating mammal wherein the vaccine comprises an antigen targeted to the mucosal tissue lining the gastrointestinal tract.

Studies support the potential of inducing SIgA antibody formation and immune protection in "distant" extra-intestinal mucosal sites after oral vaccination. Activated lymphocytes from the gut can disseminate immunity to other mucosal and glandular tissues. Therefore, oral vaccines can protect WO 2004/035790 PCT/AU2003/001381 13 against infections at sites remote from the antigenic stimulation, for example in the respiratory and urogenital tracts.

The principal challenge of delivering an oral vaccine is to be able to present adequate amounts of the antigen to the intestinal mucosa where it can stimulate the gut mucosal system to generate SIgA and induce lasting immunity.

Thus, there is a need for a method of delivering oral vaccines to animals and presenting large doses of the antigens to mucosal surfaces without having to extract and purify the protein. There is a need to deliver an animal vaccine by directly feeding transgenic plants, plant organs or seeds containing the vaccine antigen to domestic animals. There is a need to provide an immunogenic composition comprising a vaccine antigen in a transgenic plant or seed. The vaccine antigen can be used as oral vaccine in the transgenic plant or seed or extracted and purified for other uses.

2. Phytosterols Plants produce more than 250 different phytosterols and as many as different phytosterols have been identified in corn. However, insects, fungi and nematodes, as well as many other sterol-less parasitic organisms, do not synthesize all of their necessary sterols de novo. Rather, they satisfy their nutritional requirements for sterols by feeding on plants. This fact has been utilized in the development of commercial agrochemicals such as triazoles, pyrimidines and azasterols, which act by interfering with production of sterols within parasitic organisms.

Recent advances in molecular biology have made it possible to introduce advantageous traits into plants via genetic engineering. Some forms of insect resistance have been introduced into plants by genetic approaches. For example, transgenic plants expressing foreign genes encoding endotoxins of WO 2004/035790 PCT/AU20031001381 14 Bacillus thuringiensis (Bt) can confer on the plants the ability to kill pests which feed on them. Unfortunately, approaches such as this are effective only against the particular insects susceptible to the endotoxin. There remains in the agricultural industries a continual need for alternative pest control strategies, particularly those that could be broadly effective against numerous pests/pathogens.

3. Proteins that enhance the nutritional value of bran Chimeric genes encoding sulfur-rich proteins, such as, for example, the sunflower (Helianthus annuus) seed albumin (SSA), Brazil Nut Protein (BNP), glycinin, and Kunitz trypsin inhibitor, have been expressed in plant seeds for the purpose of increasing the sulfur amino acid content and/or total protein content, to thereby influence the nutritive value of the plant seed. There remains a need for enhancing the nutritional quality of animal feed by expressing high sulphur proteins in the bran.

It will be apparent from the preceding description that the genetic manipulation of maternal tissue in plants offers wide-ranging benefits to agriculture in terms of phytoprotection through enhanced physical protection of seed during seed development and in a post-harvest context, enhanced resistance against pathogen infection, pharmaceuticals for human or veterinary use, and nutritional benefits. The availability of promoter sequences that regulate expression of desirable traits specifically in maternal tissue is clearly a rate-limiting step in providing these benefits.

Summary of the Invention In work leading up to the present invention the inventors sought to identify useful regulatory sequences which were capable of conferring expression on structural gene sequences to which they are operably connected in the maternal tissues of a plant, in particular the seed pericarp. Expression of WO 2004/035790 PCT/AU2003/001381 defense genes against seed-borne pathogens in the maternal tissue, specifically the pericarp, offer an advantage over expression in other tissues in so far as such expression targets the primary infection site of the pathogen in the plant. The pericarp expression of genes that are required for the production of secondary products pharmaceuticals, nutriceuticals, immunogenic proteins, etc) for use in farming, veterinary or medical applications, or to improve nutrition of the bran, is also advantageous because it conserves the resources of the plant compared to expression throughout the plant; and (ii) the pericarp is generally extracted from the mature endosperm during milling processes thereby permitting administration of the secondary products to humans and animals in feed lots without additional processing steps being required.

Surprisingly, the inventors found that, contrary to conventional wisdom, the bifunctional a-amylase subtilisin inhibitor (ASI) gene promoter sequence from a monocotyledonous plant species is capable of conferring expression on a structural gene sequence in the maternal tissue.

Accordingly, one aspect of the present invention provides a method of expressing nucleic acid or protein in the maternal tissue of a plant seed comprising expressing isolated nucleic acid operably under the control of a bifunctional a-amylase subtilisin inhibitor (ASI) gene promoter that is operable in the maternal tissue.

By "bifunctional a-amylase subtilisin inhibitor (ASI) gene promoter" is meant any promoter derived from a bifunctional a-amylase subtilisin inhibitor (ASI) gene, including any fragments and synthetic molecules, that are functional in the pericarp of a plant. For the purposes of nomenclature, the nucleotide sequence of the ASI gene promoter from barley is set forth in SEQ ID NO: 1. The nucleotide sequence of the rice ASI gene promoter is set forth in SEQ ID NO: 2. The invention clearly extends to the use of any functionally WO 2004/035790 PCT/AU20031001381 16 homologous promoter sequences to the exemplified barley or rice ASI gene promoters, the only requirement being that said homologous promoter sequence is operable in seed maternal tissue.

By "maternal tissue of a plant seed" is meant the tissue which forms the outer protective layer of the seed including the pericarp, vascular tissue, nucellar projection cells and endosperm transfer cells. In a preferred embodiment of the present invention the maternal tissue comprises or consists of the pericarp.

Preferably, the method further comprises determining that the pericarp contains an amount or an effective amount of nucleic acid or protein encoded by the expressed nucleic acid. by "effective amount" is meant an amount of nucliec acid or protein sufficient to achieve a stated prupose eg. as determined using a functional assay.

In one embodiment, the method of the present invention further comprises obtaining the pericarp containing a nucleic acid or protein encoded by the expressed nucleic acid.

Preferably, the ASI gene promoter is from a cereal plant. In one embodiment, the cereal plant is selected from the group consisting of rice, wheat, barley, sorghum, maize, millet, rye and oats. Preferably, the ASI gene promoter is from a rice plant or a barley plant.

In another embodiment, the ASI gene promoter comprises a nucleotide sequence selected from the group consisting of: the sequence set forth in SEQ ID NO: 1; the sequence set forth in SEQ ID NO: 2; a sequence of a fragment of or that is operable in the maternal tissue of a plant seed; and WO 2004/035790 PCT/AU2003/001381 17 a sequence that in its native context regulates the expression of a protein-encoding region of an ASI gene of a plant wherein said sequence is operable in the maternal tissue of a seed of the plant.

In a further embodiment, the ASI gene promoter comprises the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2.

In a preferred embodiment of the invention, the expressed nucleic acid operably under the control of the ASI promoter comprises a structural gene that encodes a polypeptide. In one embodiment, the structural gene comprises or consists of a reporter gene. In a preferred embodiment of the present invention, the structural gene encodes a protein that confers or enhances protection against a plant pathogen.

In one embodiment, the plant pathogen is a seed-borne fungus.

In a particularly preferred embodiment, the seed-borne fungus is Fusarium graminearum (head scab) and wherein nucleic acid operably under the control of the ASI promoter consists of a structural gene comprising a nucleotide sequence selected from the group consisting of: a nucleotide sequence selected from the group consisting of SEQ ID NOs: 15, 17, 19, 21, 23, and 25; and a nucleotide sequence that encodes an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 18, 20, 22, 24, and 26.

In another particularly preferred embodiment, the seed-borne fungus is Fusarium graminearum (head scab) and nucleic acid operably under the control of the ASI promoter consists of a structural gene encoding a modified ribosomal protein L3 comprising a nucleotide sequence selected from the group consisting of: the nucleotide sequence set forth in SEQ ID NO: 27; and WO 2004/035790 PCT/AU2003/001381 18 a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 28.

In a still further particularly preferred embodiment. the seed-borne fungus is Fusarium graminearum (head scab) and the nucleic acid operably under the control of the ASI promoter consists of a structural gene encoding a polypeptide having trichothecene acetyl transferase enzyme activity, said structural gene comprising a nucleotide sequence selected from the group consisting of: the nucleotide sequence set forth in SEQ ID NO: 29; and a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: In an alternative embodiment, the plant pathogen is a seed-borne virus.

In a particularly preferred embodiment, the seed-borne virus is barley stripe mosaic virus (BSMV) and wherein nucleic acid operably under the control of the ASI promoter consists of a structural gene encoding a coat protein of BSMV, said structural gene comprising a nucleotide sequence selected from the group consisting of: a nucleotide sequence comprising residues from about position 90 to about position 683 of SEQ ID NO: 31; and a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 32.

In another embodiment of the present invention, a structural gene encodes an immunogenic protein. Preferably, the immunogenic protein is a protein from a pathogen of a human or animal.

In a preferred embodiment, the pathogen is Transmissible Gastro Enteritis Virus (TGEV). In accordance with this embodiment, nucleic acid WO 2004/035790 PCT/AU2003/001381 19 expressed operably under the control of the ASI promoter consists of a structural gene comprising a nucleotide sequence selected from the group consisting of: a nucleotide sequence set forth in SEQ ID NO: 35; and a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 36.

In a still further embodiment of the invention, a structural gene expressed under control of the ASI promoter encodes a nutritional protein to enhance the nutritional quality of the seed. In accordance with this embodiment, nucleic acid operably under the control of the ASI promoter consists of a structural gene encoding a sulphur rich protein. Preferably, the structural gene encodes Brazil Nut Protein. More preferably, the structural gene comprising a nucleotide sequence selected from the group consisting of: a nucleotide sequence set forth in SEQ ID NO: 37; and a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 38.

In yet another embodiment of the present invention, a nucleic acid operably under the control of the ASI promoter consists of a structural gene encoding a nutritional protein selected from a calcium-binding protein and an iron-binding protein.

In still other preferred embodiments of the invention, a nucleic acid operably under the control of the ASI promoter consists of a structural gene encoding an enzyme that is used in a range of industrial and laboratory contexts.

Preferred enymes for use in a laboratory context include, for example, an enzyme used in performing a molecular biological technique a restriction WO 2004/035790 PCT/AU2003/001381 enzyme, ligases, polymerase, reverse transcriptase), or other biochemical enzyme reagent.

Preferred enymes for use in an industrial context include, for example, those used in a wide range of food production processes. One example is beta-glucanases and xylanases, used for degrading the cell wall in the production of beer. Similarly, thermostable enzymes such as alpha-amylases are used to assist in conversion of starch to sugars for subsequent application in beer brewing. Alpha-amylases are also used to achieve optimum performance in the baking of bread: starch is broken down to disaccharides to just the extent needed to produce the required substrate for yeast, to ferment the dough and cause the bread to rise.

In other preferred embodiments of the invention, a nucleic acid operably under the control of the ASI promoter consists of a structural gene encoding a biosynthetic enzyme that is required for the production of an osmoprotectant, a fatty acid, a phytosterol, an anthocyanin, lignin, an anti-nutritional protein, an enzyme capable of altering a substrate in the phenylpropanoid pathway, a choline metabolizing enzyme capable of acting upon choline to modify the use of choline by other enzymes in the phenylpropanoid pathway, an enzyme involved in the malting process, an enzyme capable of acting upon a sugar alcohol, or an enzyme capable of acting upon myo-inositol.

In another embodiment, the expressed nucleic acid operably under the control of the ASI promoter comprises nucleic acid encoding inhibitory RNA, an antisense molecule, ribozyme, abzyme, co-suppression molecule, genesilencing molecule or gene-targeting molecule. Preferably, the nucleic acid operably under the control of the ASI promoter targets expression in the plant of a gene of a plant pathogen that is required for infection the plant by the pathogen or transmission of the pathogen in the plant.

WO 2004/035790 PCT/AU20031001381 21 In a particularly preferred embodiment of the invention, the nucleic acid operably under the control of the ASI promoter reduces expression of a movement protein of BSMV. In one particularly preferred embodiment, the nucleic acid that reduces expression of a movement protein of BSMVS comprises a nucleotide sequence selected from the group consisting of: a sequence that is complementary to at least about 20 contiguous nucleotides from about position 804 to about position 2387 of SEQ ID NO: 31; a sequence that is complementary to at least 20 contiguous nucleotides of a sequence that encodes the amino acid sequence set forth in SEQ ID NO: 33.

In each of its various embodiments described herein, the method of the present invention can preferably further comprise introducing to a cell, tissue or organ of a plant the isolated nucleic acid operably under the control of a bifunctional a-amylase subtilisin inhibitor (ASI) gene promoter that is operable in the maternal tissue. This produces a transformed cell comprising the introduced nucleic acid, either stably integrated into the genome or present as extrachromosomal mucleic acid.

Preferably, the method of the present invention further comprises regenerating a whole plant from the transformed cell, tissue or organ (expecially if the nucleic acid is stably integrated) and growing the whole plant for a time and under conditions sufficient for seed to be produced that express the isolated nucleic acid operably under the control of the ASI gene promoter.

Preferably, the transformed plant is a monocotyledonous plant expecially a cereal plant. More preferably, the monocotyledonous plant is selected from the group consisting of wheat, oats, maize, barley, rice, sorghum, millet and rye. In a particularly referred embodiment, the plant is barley or wheat.

WO 2004/035790 PCT/AU2003/001381 22 A further aspect of the present invention provides a method of enhancing or conferring resistance of a plant against Fusarium graminearum (head scab) comprising expressing isolated nucleic acid in the maternal tissue of the plant that encodes a defence protein operably under the control of a bifunctional aamylase subtilisin inhibitor (ASI) gene promoter that is operable in the maternal tissue of the plant. Preferably, the nucleic acid operably under control of the ASI promoter comprises a nucleotide sequence that encodes a thaumatin-like protein.

Preferably, the nucleic acid operably under control of the ASI promoter comprises a nucleotide sequence selected from the group consisting of: a nucleotide sequence selected from the group consisting of SEQ ID NOs: 15, 17, 19, 21, 23, and 25; and a nucleotide sequence that encodes an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 18, 20, 22, 24, and 26.

In another embodiment, a nucleic acid operably under the control of the ASI promoter comprises a nucleotide sequence that encodes a modified ribosomal protein L3 comprising a nucleotide sequence selected from the group consisting of: the nucleotide sequence set forth in SEQ ID NO: 27; and a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 28.

In a further embodiment, a nucleic acid operably under the control of the ASI promoter comprises a nucleotide sequence that a polypeptide having trichothecene acetyl transferase enzyme activity, said sequence selected from the group consisting of: the nucleotide sequence set forth in SEQ ID NO: 29; and a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: WO 2004/035790 PCT/AU20031001381 23 In one embodiment the maternal tissue comprises a seed tissue selected from the group consisting of pericarp, vascular tissue, nucellar projection cells and endosperm transfer cells. Preferably, the maternal tissue comprises pericarp.

Preferably, the method of enhancing or conferring resistance of a plant against Fusarium graminearum (head scab) further comprises determining that the pericarp contains an effective amount of nucleic acid or protein encoded by the expressed nucleic acid eg., as determined in a bioassay for Fusariumn head blight developed and/or DON growth assay and/or protoplast assay.

Preferably, the method described herein for enhancing or conferring resistance against head scals further comprises introducing to a cell, tissue or organ of a plant the isolated nucleic acid encoding the defense protein operably under the control of the ASI gene promoter.

Preferably, the method further comprises regenerating a whole plant from the cell, tissue or organ and growing the whole plant for a time and under conditions sufficient for seed to be produced that express the isolated nucleic acid operably under the control of the ASI gene promoter.

In a particularly preferred embodiment, the plant on which protection is conferred or enhanced is a wheat plant.

The present invention also provides an isolated transformed wheat seed that expresses isolated nucleic acid encoding a plant defense protein placed operably under the control of the ASI gene promoter wherein said seed exhibits enhanced resistance against Fusarium graminearum (head scab) by virtue of the presence of said nucleic acid compared to an otherwise isogenic line that does not express said nucleic acid placed operably under the control of the ASI WO 2004/035790 PCT/AU2003/001381 24 gene promoter. Preferably, the seed is substantially free of Fusarium graminearum (head scab) or a mycotoxin thereof. Preferably, the seed is free of a tricothecene. More preferably, the seed is free of deoxynivalenol (DON).

Another aspect of the present invention provides a method of enhancing or conferring resistance of a plant against barley stripe mosaic virus (BSMV) comprising expressing isolated nucleic acid in the maternal tissue of the plant that encodes a BSMV coat protein operably under the control of a bifunctional a-amylase subtilisin inhibitor (ASI) gene promoter that is operable in the maternal tissue of the plant. Preferably, the nucleic acid encoding the coat protein of BSMV comprises a nucleotide sequence selected from the group consisting of: a nucleotide sequence comprising residues from about position 90 to about position 683 of SEQ ID NO: 31; and a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 32.

Preferably, the maternal tissue comprises a seed tissue selected from the group consisting of pericarp, vascular tissue, nucellar projection cells and endosperm transfer cells. More preferably, the maternal tissue comprises pericarp.

Preferably, the method further comprises determining that the pericarp contains an effective amount of nucleic acid or protein encoded by the expressed nucleic acid, eg., as deteremined by suitable bioassay.

In a preferred embodiment of this method for conferring or enhancing BSMV resistance, the method further comprises introducing to a cell, tissue or organ of a plant the isolated nucleic acid encoding the BSMV coat protein operably under the control of the ASI gene promoter.

WO 2004/035790 PCT/AU20031001381 Preferably, the method further comprises regenerating a whole plant from the cell, tissue or organ and growing the whole plant for at ime and under conditions sufficient for seed to be produced that express the isolated nucleic acid operably under the control of the ASI gene promoter.

In a particularly preferred embodiment, the plant on which BSMV resistance is conferred or enhanced is a barley plant.

The present invention also provides an isolated transformed barley seed that expresses isolated nucleic acid encoding a barley stripe mosaic virus (BSMV) coat protein placed operably under the control of the ASI gene promoter wherein said seed exhibits enhanced resistance against BSMV by virtue of the presence of said nucleic acid compared to an otherwise isogenic line that does not express said nucleic acid placed operably under the control of the ASI gene promoter. Preferably, the isolated transformed barley seed is substantially free, or free, of BSMV.

A further aspect of the present invention provides a method of enhancing or conferring resistance of a plant against barley stripe mosaic virus (BSMV) comprising expressing isolated nucleic acid in the maternal tissue of the plant that encodes inhibitory RNA that prevents, inhibits or reduces expression of a BSMV movement protein operably under the control of a bifunctional aamylase subtilisin inhibitor (ASI) gene promoter that is operable in the maternal tissue of the plant. Preferably, the nucleic acid encoding inhibitory RNA that prevents, inhibits or reduces expression of a BSMV movement protein comprises a nucleotide sequence selected from the group consisting of: a sequence that is complementary to at least about 20 contiguous nucleotides from about position 804 to about position 2387 of SEQ ID NO: 31; and WO 2004/035790 PCT/AU20031001381 26 a sequence that is complementary to at least 20 contiguous nucleotides of a sequence that encodes the amino acid sequence set forth in SEQ ID NO: 33.

In one embodiment, the maternal tissue comprises a seed tissue selected from the group consisting of pericarp, vascular tissue, nucellar projection cells and endosperm transfer cells. Preferably, the maternal tissue comprises pericarp.

Preferably, the method of the present invention further comprises determining that the pericarp contains an effective amount of the inhibitory

RNA.

In a preferred embodiment, this method of enhancing/conferring BSMV resistance further comprises introducing to a cell, tissue or organ of a plant the isolated nucleic acid encoding the inhibitory RNA operably under the control of the ASI gene promoter. Preferably, the method further comprises regenerating a whole plant from the cell, tissue or organ and growing the whole plant for at ime and under conditions sufficient for seed to be produced that express the inhibitory RNA operably under the control of the ASI gene promoter.

In a particularly preferred embodiment, the plant on which resistance is conferred or enhanced is barley.

The present invention also provides an isolated transformed barley seed that expresses isolated nucleic acid encoding inhibitory RNA that prevents, inhibits or reduces expression of a BSMV movement protein placed operably under the control of the ASI gene promoter wherein said seed exhibits enhanced resistance against BSMV by virtue of the presence of said nucleic acid compared to an otherwise isogenic line that does not express said nucleic WO 2004/035790 PCTiAU2003/001381 27 acid placed operably under the control of the ASI gene promoter. Preferably, the isolated transformed barley seed is substantially free, or free of BSMV.

A still further aspect of the present invention provides a method of producing an immunogenic composition comprising expressing isolated nucleic acid in the maternal tissue of the plant that encodes an immunogenic polypeptide operably under the control of a bifunctional a-amylase subtilisin inhibitor (ASI) gene promoter that is operable in the maternal tissue of the plant.

In one embodiment, the immunogenic polypeptide is from an animal pathogen, such as, for example, a virus and more particularly, from an entric virus. In a particularly preferred embodiment, the immunogenic protein is from

TGEV.

Preferably, the nucleic acid that encodes an immunogenic polypeptide comprises a sequence selected from the group consisting of: the sequence set forth in SEQ ID NO: 35; and a sequence that encodes the amino acid sequence set forth in SEQ ID NO: 36.

In one embodiment, the maternal tissue comprises a seed tissue selected from the group consisting of pericarp, vascular tissue, nucellar projection cells and endosperm transfer cells. Preferably, the maternal tissue comprises pericarp.

In one embodiment, the method further comprises determining that the pericarp contains an effective amount of the encoded immunogenic polypeptide, eg., by immunoassay and/or appearance of neutralizing antibodies against the pathogen and/or IgA antibody titer and/or by pathogen challenge.

WO 2004/035790 PCT/AU20031001381 28 Preferably, the method of producing an immunogenic composition further comprises introducing to a cell, tissue or organ of a plant the isolated nucleic acid encoding the immunogenic polypeptide operably under the control of the ASI gene promoter. Preferably, the method further comprises regenerating a whole plant from the cell, tissue or organ and growing the whole plant for at ime and under conditions sufficient for seed to be produced that express the immunogenic polypeptide operably under the control of the ASI gene promoter.

Preferably, the plant in which the composition is produced is a wheat plant.

In one embodiment, the method of the present invention further comprises obtaining the maternal tissue of the plant eg., the brain or pericarp.

Preferably, the maternal tissue is fed to a human or animal in need thereof in an amount sufficient to induce an immune response against the immunogenic protein (eg IgA production).

The present invention also provides an immunogenic composition comprising the maternal tissue of a plant seed produced by a process that comprises a method of producing an immunogenic composition comprising expressing isolated nucleic acid in the maternal tissue of the plant that encodes an immunogenic polypeptide operably under the control of a bifunctional aamylase subtilisin inhibitor (ASI) gene promoter that is operable in the maternal tissue of the plant.

A further aspect of the present invention provides a method of enhancing the nutritional quality of bran or animal fodder comprising expressing isolated nucleic acid in the maternal tissue of the plant that encodes a nutritional protein operably under the control of a bifunctional a-amylase subtilisin inhibitor (ASI) gene promoter that is operable in the maternal tissue of the plant. Preferably, the nutritional protein is a sulphur-rich protein, calcium-binding protein, iron- WO 2004/035790 PCT/AU2003/001381 29 binding protein, a fatty acid biosynthetic enzyme, anthocryanin biosynthetic enzyme, or matting enzyme.

In one embodiment, the nucleic acid encodes a sulphur-rich protein comprising a sequence selected from the group consisting of: the sequence set forth in SEQ ID NO: 37; and a sequence that encodes the amino acid sequence set forth in SEQ ID NO: 38.

In one embodiment, the maternal tissue comprises a seed tissue selected from the group consisting of pericarp, vascular tissue, nucellar projection cells and endosperm transfer cells. Preferably, the maternal tissue comprises pericarp.

In one embodiment, the method of the present invention further comprises determining that the pericarp contains an effective amount of the encoded nutritional protein, eg., by ELISA or other umminoassay, by direct measurement of sulfur content or iron content or calcium content of the pericarp or brain as appropriate, or by enzyme assay or by determining the amount of a particular metabolite in the pericarp or brain.

Preferably, the method further comprises introducing to a cell, tissue or organ of a plant the isolated nucleic acid encoding the nutritional protein operably under the control of the ASI gene promoter. Preferably, the method further comprises regenerating a whole plant from the cell, tissue or organ and growing the whole plant for at ime and under conditions sufficient for seed to be produced that express the nutritional protein operably under the control of the ASI gene promoter.

WO 2004/035790 PCT/AU2003/001381 In another embodiment of the invention, the method further comprises obtaining the maternal tissue of the plant. Preferably, the method further comprises feeding the maternal tissue to a human or animal in need thereof.

The present invention also provides isolated bran or maternal tissue of a seed produced by a process that comprises performing a method of enhancing the nutritional quality of bran or animal fodder comprising comprising expressing isolated nucleic acid in the maternal tissue of the plant that encodes a nutritional protein operably under the control of a bifunctional a-amylase subtilisin inhibitor (ASI) gene promoter that is operable in the maternal tissue of the plant.

A further aspect of the present invention is directed to the use of a genetic construct comprising an isolated ASI gene promoter sequence to confer expression of a gene in the maternal tissue of a plant seed or a cell or tissue derived from the maternal tissue.

A further aspect of the present invention provides a transfected or transformed plant which expresses a nucleic acid or protein operably under the control of an ASI promoter sequence in the maternal tissue, and/or maternal tissue derived from said plant. Preferably, the nucleic acid encodes a recombinant polypeptide or comprises or consists of a ribozyme, antisense, gene-targetting molecule, gene-silencing molecule or co-suppression molecule.

Brief Description of the Drawings Figure 1 is a schematic representation showing the nucleotide sequence of the 1033 bp 5'-upstream region of the asi gene from barley (SEQ ID NOs: 1 or Various putative regulatory elements present in this sequence are shown.

Symbols indicate: TATA-box; AAAA, CAAT-box; E-motif of the -300 element; abscisic acid responsive element (ABRE)-like; truncated forms of the E-motif; RY-repeat element (Sph-element); WO 2004/035790 PCT/AU2003/001381 31 sugar responsive element (SRE). Truncated form of GARE, CA-rich and TArich regions are underlined by a fine line, small stretches of repeats are double underlined Figure 2 is a schematic representation showing the nucleotide sequence of the 655 bp 3'-downstream region of the asi gene from barley (SEQ ID NO: This sequence contains the 3'-untranslated sequence (77 bp, 3'-UTR, underlined) of the asi gene, and 577 bp sequence downstream from the 3'-

UTR.

Figure 3a is a graphical representation showing the activity of the 1033asi promoter from barley and deletions thereof in aleurone cells. Measurement of green fluorescent protein (GFP) transiently expressed in aleurone cells under control of the promoter or deletion fragments thereof was carried out by "Image analysis". Particle bombardment was used to transfect plasmid DNA into cells of mature barley aleurone layers. Promoter constructs (x-axis) comprised the Em promoter the 1033-asi promoter (SEQ ID NOs: 1 or 2; 1033 bp asi); or various truncated versions of SEQ ID NOs: 1 or 2 (668bp asi; 496bp asi; 272bp asi; 220bp asi; and 172bp asi). GFP concentration is indicative of expression and is indicated on the ordinate. Data represent values from 3 to 5 independent experiments. Data indicate low level transient expression of the reporter gene in aleurone cells under control of the ASI promoter.

Figure 3b is a graphical representation showing the activity of several promoters in barley aleurone cells. Measurement of green fluorescent protein (GFP) transiently expressed in aleurone cells under control of the promoters indicated on the x-axis by "Image analysis". Particle bombardment was used to transfect plasmid DNA constructs pUbi.gfp.nos, pEm.gfp-s65t.nos, and pAlpha-amy1.gfp.nos) into cells of mature barley aleurone layers in the absence of phytohormone, or alternatively, in the WO 2004/035790 PCT/AU2003/001381 32 presence of gibberellin or abscisic acid Promoters are indicated on the x-axis. GFP concentration (IpM) is indicative of expression and is indicated on the ordinate. Data represent values from 3 to 5 independent experiments. Data indicate that the Em promoter directs low levels of transient gfp expression but is induced 13-times in the presence of abscisic acid (ABA).

The a-amy1 promoter is induced 11- to 18-times in the presence of gibberellic acid The ubiquitin and the CaMV35S promoters direct high level of transient gfp expression.

Figure 4a is a copy of a photographic representation showing background expression of gfp in mature leaf tissue of untransformed barley c.v.

Golden Promise. Observations were carried out under blue light and using a fluorescence compound microscope (ex: 489 nm and em: 510 and images were captured at identical exposure times.

Figure 4b is a copy of a photographic representation showing expression of gfp under control of the full-length asi promoter in the 1033asi.gfp.nos transgene in mature leaf tissue of transgenic barley c.v. Golden Promise.

Observations were carried out under blue light and using a fluorescence compound microscope (ex: 489 nm and em: 510 and images were captured at identical exposure times.

Figure 4c is a copy of a photographic representation showing expression of gfp under control of the hordein gene promoter in the B-hordein.gfp.nos transgene in mature leaf tissue of transgenic barley c.v. Golden Promise.

Observations were carried out under blue light and using a fluorescence compound microscope (ex: 489 nm and em: 510 and images were captured at identical exposure times.

Figure 4d is a copy of a photographic representation showing expression of gfp under control of the ubiquitin gene promoter in the ubi.gfp.nos transgene WO 2004/035790 PCT/AU2003/001381 33 in mature leaf tissue of transgenic barley c.v. Golden Promise. Observations were carried out under blue light and using a fluorescence compound microscope (ex: 489 nm and em: 510 and images were captured at identical exposure times. Green fluorescent protein was detected in leaf tissue of transgenic barley transformed with the ubi.gfp.nos transgene.

Figure 5a is a copy of a photographic representation showing expression of gfp under control of the ASI promoter of barley in the pericarp of transgenic barley plants. Immature grain of barley that had been transformed with the 1033asi.gfp.nos gene was observed for gfp expression. Data indicate that the 1033-asi promoter (SEQ ID NOs: 1 or 2) directs gfp expression in the pericarp.

Observations were carried out under blue light using a fluorescence compound microscope (ex: 489 nm and em: 510 GL, glume; P, pericarp; T, testa Figure 5b is a copy of a photographic representation showing expression of gfp under control the ASI promoter of barley in the pericarp of transgenic barley plants. Immature grain of barley that had been transformed with the 1033asi.gfp.nos gene was observed for gfp expression. Results indicate that the 1033-asi promoter (SEQ ID NOs: 1 or 2) directs gfp expression in the pericarp at 28 days post anthesis (DPA). As shown little or no gfp expression was observed in the embryo axis scutellum or testa Observations were carried out under blue light using a fluorescence compound microscope (ex: 489 nm and em: 510 nm.).

Figure 5c is a copy of a photographic representation showing expression of gfp under control of the ASI promoter of barley in the crease or groove of a transgenic barley seed. Immature grain of barley that had been transformed with the 1033asi.gfp.nos gene was observed for gfp expression. Data indicate that the 1033-asi promoter (SEQ ID NOs: 1 or 2) directs gfp expression in various tissues of the crease of the seed. Observations were carried out under WO 2004/035790 PCT/AU2003/001381 34 blue light using a fluorescence compound microscope (ex: 489 nm and em: 510 CR, crease; P, pericarp; T, testa.

Figure 5d is a copy of a photographic representation of a longitudinal section of the crease of a barley grain showing expression of gfp under control of the ASI promoter of barley. An immature grain of barley that had been transformed with the 1033asi.gfp.nos gene was observed for gfp expression.

Data indicate that the 1033-asi promoter (SEQ ID NOs: 1 or 2) directs gfp expression in nuclear projection cells (NPC) and pericarp Observations were carried out under blue light using a fluorescence compound microscope (ex: 489 nm and em: 510 T, testa; V, vacuole or endosperm cavity.

Figure 5e is a copy of a photographic representation of a transverse section of a barley grain showing expression of gfp under control of the ASI promoter of barley in the crease of the grain. Observation of an immature grain of barley that had been transformed with the 1033asi.gfp.nos gene indicated that the 1033-asi promoter (SEQ ID NOs: 1 or 2) directs gfp expression in pericarp and vascular tissue Observations were carried out under blue light using a fluorescence compound microscope (ex: 489 nm and em: 510 ET, embryo transfer cells; NP, nucellar projection cells; SE, starchy endosperm; V, vacuole or endosperm cavity.

Figure 5f is a copy of a photographic representation of a transverse section through the crease of a barley grain showing expression of gfp under control of the ASI promoter of barley. An immature grain of barley that had been transformed with the 1033asi.gfp.nos gene was observed for gfp expression. Data indicate that the 1033-asi promoter (SEQ ID NOs: 1 or 2) directs gfp expression in pericarp embryo transfer cells nucellar projection cells (NP) and vascular tissue Observations were carried out under blue light using a fluorescence compound microscope (ex: 489 nm and em: 510 A, aleurone; T, testa; and V, vacuole or endosperm cavity.

WO 2004/035790 PCT/AU2003/001381 Figure 5g is a copy of a photographic representation showing expression of gfp under control of the ASI promoter of barley in a transgenic barley grain.

An immature grain of barley that had been transformed with the 1033asi.gfp.nos gene was observed for gfp expression. Data indicate that there is no gfp expression in testa aleurone or starchy endosperm (SE) of transgenic barley grain. Observations were carried out under blue light using a fluorescence compound microscope (ex: 489 nm and em: 510 nm.).

Figure 5h is a copy of a photographic representation of a transverse section of immature grain from barley that was not transformed with a transgenic construct a negative control grain). No gfp expression was observed in the negative control grain. Observations were carried out under blue light using a fluorescence compound microscope (ex: 489 nm and em: 510 ET; endosperm transfer cells; NP; Nucellar projection cells, P; pericarp; SE; starchy endosperm; V; vacuole or endosperm cavity; and VT; vascular tissue.

Figure 5i is a copy of a photographic representation showing a transverse section of an immature barley grain transformed with gfp under control of the D-hordein promoter. An immature grain of barley that had been transformed with the D-hordein.gfp.nos gene was observed for gfp expression.

Data indicate that the D-hordein promoter directs gfp expression in starchy endosperm (SE) and aleurone Observations were carried out under blue light using a fluorescence compound microscope (ex: 489 nm and em: 510 nm.).

Figure 6a is a copy of a photographic representation showing expression of gfp under control of the 1033-asi promoter (SEQ ID NOs: 1 or 2) in an immature seed of transgenic barley at 7 days post anthesis (dpa). Barley c.v.

Golden promise was transformed by Agrobacterium tumefaciens containing the WO 2004/035790 PCT/AU2003/001381 36 1033asi.gfp.nos gene. At this stage of development, only very low levels of expression are able to be observed.

Figure 6b is a copy of a photographic representation showing expression of gfp under control of the 1033-asi promoter (SEQ ID NOs: 1 or 2) in an immature seed of transgenic barley at 10 days post anthesis (dpa). Barley c.v.

Golden promise was transformed by Agrobacterium tumefaciens containing the 1033asi.gfp.nos gene. Data indicate that the 1033-asi promoter drives expression of gfp in nucellar projection cells (NP) at this stage of development.

Observations were carried out under blue light using a fluorescence compound microscope (ex: 489 nm and em: 510 ET; endosperm transfer cells; P; pericarp; T; testa; and VT; vascular tissue.

Figure 6c is a copy of a photographic representation showing expression of gfp under control of the 1033-asi promoter (SEQ ID NOs: 1 or 2) in an immature seed of transgenic barley at 12 days post anthesis (dpa). Barley c.v.

Golden promise was transformed by Agrobacterium tumefaciens containing the 1033asi.gfp.nos gene. By 12 dpa, expression of gfp appears to be declining in NP cells, and is beginning to appear in VT and ET cells. Very faint expression is also discernible in the pericarp.

Figure 6d is a copy of a photographic representation showing expression of gfp under control of the 1033-asi promoter (SEQ ID NOs: 1 or 2) in an immature seed of transgenic barley at 18 days post anthesis (dpa). Barley c.v.

Golden promise was transformed by Agrobacterium tumefaciens containing the 1033asi.gfp.nos gene. Data indicate that the 1033-asi promoter drives expression of gfp in endosperm transfer cells pericarp and vascular tissue (VT) at this stage of development. Observations were carried out under blue light using a fluorescence compound microscope (ex: 489 nm and em: 510 NP, nucellar projection cells; SE, starchy endosperm; and T, testa.

WO 2004/035790 PCT/AU2003/001381 37 Figure 6e is a copy of a photographic representation showing expression of gfp under control of the 1033-asi promoter (SEQ ID NOs: 1 or 2) in an immature seed of transgenic barley at 22 days post anthesis (dpa). Barley c.v.

Golden promise was transformed by Agrobacterium tumefaciens containing the 1033asi.gfp.nos gene. Data indicate that the 1033-asi promoter drives expression of gfp in endosperm transfer cells nucellar projection cells (NP) and pericarp at this stage of development. Observations were carried out under blue light using a fluorescence compound microscope (ex: 489 nm and em: 510 SE, starchy endosperm; and GL, glume.

Figure 6f is a copy of a photographic representation showing expression of gfp under control of the 1033-asi promoter (SEQ ID NOs: 1 or 2) in an immature seed of transgenic barley at 28 days post anthesis (dpa). Barley c.v.

Golden promise was transformed by Agrobacterium tumefaciens containing the 1033asi.gfp.nos gene. Data indicate that the 1033-asi promoter drives expression of gfp in pericarp and vascular tissue (VT) at this stage of development. Observations were carried out under blue light using a fluorescence compound microscope (ex: 489 nm and em: 510 ET, endosperm transfer cells; V, vacuole or endosperm cavity; and SE, starchy endosperm.

Figure 7a is a copy of a photographic representation showing expression of gfp under control of the 1033-asi promoter of barley in a transgenic barley embryo. A germinating seed of a barley plant that had been transformed with the 1033.asi.gfp.nos was sectioned at 24 hours after germination and observed for gfp expression. Data indicate that the 1033-asi promoter drives expression of gfp in the entire germinating embryo at this developmental stage.

Observations were carried out under blue light using a fluorescence compound microscope (ex: 489 nm and em: 510 SE, starchy endosperm.

WO 2004/035790 PCT/AU2003/001381 38 Figure 7b is a copy of a photographic representation showing expression of gfp under control of the 1033-asi promoter of barley in a transgenic barley embryo. A germinating seed of a barley plant that had been transformed with the 1033.asi.gfp.nos was sectioned at 72 hours after germination and observed for gfp expression. Data indicate that the 1033-asi promoter drives expression of gfp in the entire germinating embryo at this stage of development.

Observations were carried out under blue light using a fluorescence compound microscope (ex: 489 nm and em: 510 SE, starchy endosperm; and GL, glume.

Figure 7c is a copy of a photographic representation showing expression of gfp under control of the 1033-asi promoter of barley in a seed containing a transgenic barley embryo. A germinating seed of a barley plant that had been transformed with the 1033.asi.gfp.nos gene was sectioned at 72 hours after germination and observed for gfp expression. Data indicate that the 1033-asi promoter drives expression of gfp in the region of the aleurone (sub-aleurone) adjoining the embryo. Observations were carried out under blue light using a fluorescence compound microscope (ex: 489 nm and em: 510 SE, starchy endosperm.

Figure 7d is a copy of a photographic representation showing expression of under control of the 1033-asi promoter of barley in a longitudinal section of a de-embryonated seed. The de-embryonated seed carrying the 1033.asi.gfp.nos gene was imbibed with a buffer containing gibberellic acid 24 hours after incubation in the buffer containing GA the gfp expression was observed in the de-embryonated seed. Observations were carried out under blue light using a fluorescence compound microscope (ex: 489 nm and em: 510 nm.).

Figure 7e is a copy of a photographic representation showing expression of gfp under control of the 1033-asi promoter of barley in a longitudinal section WO 2004/035790 PCT/AU2003/001381 39 of a de-embryonated seed. The de-embryonated seed carrying the 1033.asi.gfp.nos gene was imbibed with a buffer containing gibberellic acid Expression of gfp at 48 hours following incubation of the deembryonated seed with GA was observed to be upregulated compared to the level of expression at 24 hours (Figure 7d). Observations were carried out under blue light using a fluorescence compound microscope (ex: 489 nm and em: 510 nm.).

Figure 7f is a copy of a photographic representation showing expression of gfp under control of the 1033-asi promoter of barley in a longitudinal section of a de-embryonated seed. The de-embryonated seed carrying the 1033.asi.gfp.nos gene was imbibed with a buffer containing gibberellic acid Expression of gfp at 72 hours following incubation of the deembryonated seed with GA was observed to be upregulated compared to the level of expression at 24 hours (Figure 7d) and 48 hours (Figure 7e).

Observations were carried out under blue light using a fluorescence compound microscope (ex: 489 nm and em: 510 nm.).

Figure 7g is a copy of a photographic representation showing expression of gfp under control of the 1033-asi promoter of barley in a transverse section of a de-embryonated seed. The de-embryonated seed carrying the 1033.asi.gfp.nos gene was imbibed with a buffer containing gibberellic acid 72 hours after incubation in buffer containing GA gfp expression was observed in aleurone tissue Observations were carried out under blue light using a fluorescence compound microscope (ex: 489 nm and em: 510 nm.).

SE, starchy endosperm.

Figure 7h is a copy of a photographic representation showing expression of gfp under control of the 1033-asi promoter of barley in a de-embryonated seed. The de-embryonated seed carrying the 1033.asi.gfp.nos gene was imbibed with a buffer containing abscisic acid (ABA) 72 hours after incubation WO 2004/035790 PCT/AU2003/001381 in buffer containing ABA gfp expression was observed in aleurone tissue Observations were carried out under blue light using a fluorescence compound microscope (ex: 489 nm and em: 510 SE, starchy endosperm.

Figure 7i is a copy of a photographic representation showing expression of gfp under control of the 1033-asi promoter of barley in a transverse section of a de-embryonated seed. The de-embryonated seed carrying the 1033.asi.gfp.nos gene was imbibed with a buffer containing abscisic acid (ABA) 72 hours after incubation in buffer containing ABA gfp expression was observed in aleurone tissue Observations were carried out under blue light using a fluorescence compound microscope (ex: 489 nm and em: 510 nm.).

SE, starchy endosperm.

Color representations of the photgraphic representations supplied herein are available from the inventors on request.

Detailed Description of the Preferred Embodiments One aspect of the present invention provides a method of expressing nucleic acid or protein in the maternal tissue of a plant seed comprising expressing nucleic acid operably under the control of a bifunctional a-amylase subtilisin inhibitor (ASI) gene promoter that is operable in the maternal tissue.

Reference herein to a "promoter" is to be taken in its broadest context and includes the transcriptional regulatory sequences of a classical genomic gene, including the TATA box which is required for accurate transcription initiation, with or without a CCAAT box sequence and additional regulatory elements upstream activating sequences, enhancers and silencers) which alter gene expression in response to developmental and/or hormonal and/or environmental stimuli, or in a tissue-specific or cell-type-specific manner. A promoter is usually, but not necessarily, positioned upstream, or of a structural gene, the expression of which it regulates. Furthermore, the WO 2004/035790 PCTAU2003/001381 41 regulatory elements comprising a promoter are usually positioned within 2 kb of the start site of transcription of the gene.

The bifunctional a-amylase subtilisin inhibitor (ASI) gene promoter that is operable in the maternal tissue is preferably derived from a bifunctional aamylase subtilisin inhibitor (ASI) gene of a monocotyledonous plant, more preferably a cereal such as, for example, wheat, barley, rice, sorghum, maize, millet, rye or oats. In a particularly preferred embodiment, the promoter is derived from barley or rice. Additional sources are not excluded.

Particularly preferred maternal tissue-operable promoters comprise the nucleotide sequence set forth in SEQ ID NO: 1 or 2 or a fragment thereof comprising at least about 500 contiguous nucleotides or at least about 1000 contiguous nucleotides or about 1500 contiguous nucleotides derived from any one of said sequences.

The present invention clearly encompasses the use of derivatives of the nucleotide sequence set forth in SEQ ID NOs: 1 or 2 to regulate expression in the maternal tissue.

As used herein "derivatives" of the promoter sequence shall be taken to refer to any isolated nucleic acid molecule which comprises at least 10 and preferably at least 20 contiguous nucleotides, and more preferably at least contiguous nucleotides, derived from an ASI promoter, in particular the rice or barley ASI promoter exemplified herein. In the present context, the term "derivative" is also used to describe a synthetic or fusion molecule or a derivative of the nucleotide sequence set forth in SEQ ID NOs: 1 or 2 or a complementary nucleotide sequence thereto, which possesses the same expression function as the exemplified promoter sequences.

WO 2004/035790 PCTIAU2003001381 42 In one embodiment, the promoter is derived from SEQ ID NOs: 1 or 2 or a part thereof or a complementary nucleotide sequence thereto and possesses the same function as said promoter sequence or part or complement, however comprises one or more additional regulatory elements, derived from either the exemplified promoter sequence or a heterologous promoter sequence, to further enhance expression of a genetic sequence to which it is operably connected and/or to alter the timing of expression of a genetic sequence to which it is operably connected. For example, chimeric promoter sequences that comprise the nucleotide sequence set forth in SEQ ID NOs: 1 or 2 may be modified by the inclusion of nucleotide sequences derived from a different maternal tissue-operable promoter to further enhance expression of a genetic sequence to which the promoter is operably connected in the maternal tissue.

The performance of such embodiments is readily achievable by those skilled in the art. For example, other maternal tissue-operable promoters include those promoters derived from the pea gibberellin 20-oxidase gene, barley germin gene, tomato ACC synthase gene, tomato endo-l ,4-beta-glucanase gene, tomato polygalacturonase gene, maize Myb gene, and tomato fruit beta-subunit protein gene, amongst others.

Those skilled in the art will be aware that it is also possible to modify the level of structural gene expression and/or the timing of structural gene expression and/or the regulation of structural gene expression, by mutation of a regulatory genetic sequence cis-regulatory region or 5'-non-coding region, etc) within the promoter sequence to which the structural gene is operably connected. In particular, to achieve such an objective, the promoter sequence of the present invention may be subjected to mutagenesis to produce single or multiple nucleotide substitutions, deletions and/or additions.

Alternatively, or in addition, the arrangement of specific regulatory sequences within the promoter sequence may be altered, including the deletion WO 2004/035790 PCT/AU2003/001381 43 therefrom of certain regulatory sequences and/or the addition thereto of regulatory sequences derived from the same or a different promoter sequence.

Preferred derivatives of the ASI promoter comprise one or more functional cis-acting elements present in an ASI promoter and required for maternal tissue expression operably connected to one or more heterologous nucleotide sequences. In a aprticularly preferred embodiment, the derivative comprises a regulatory motif depicted in Figure 1 in combination with a functional'TATA-box motif. Additional integers are not excluded.

Derivatives of the promoter can be produced by synthetic means or alternatively, derived from naturally-occurring sources.

For example, the promoter sequence may be derivativatized without complete loss of function such that it at least comprises one or more of the following sequences: a 5' non-coding region; and/or (ii) one or more cis-regulatory regions, such as one or more functional binding sites for a transcriptional regulatory proteins or translational regulatory proteins, one or more upstream activator sequences, enhancer elements or silencer elements; and/or (iii) a TATA box motif; and/or (iv) a CCAAT box motif; and/or an upstream open reading frame (uORF);and/or (vi) a transcriptional start site; and/or (vii) a translational start site; and/or (viii) a nucleotide sequence which encodes a leader sequence.

As used herein, the term non-coding region" shall be taken in its broadest context to include all nucleotide sequences which are derived from the upstream region of a maternal tissue-expressed gene, preferably derived WO 2004/035790 PCT/AU20031001381 44 from the ASI gene exemplified herein, other than those sequences which encode amino acid residues comprising the polypeptide product of said gene.

As used herein, the term "uORF" refers to a nucleotide sequence localised upstream of a functional translation start site in a gene and generally within the 5'-transcribed region leader sequence), which encodes an amino acid sequence. Whilst not being bound by any theory or mode of action, a uORF functions to prevent over-expression of a structural gene sequence to which it is operably connected or altemrnatively, to reduce or prevent such expression.

As used herein, the term "cis-acting sequence" or "cis-regulatory region" or similar term shall be taken to mean any sequence of nucleotides which is derived from a promoter sequence wherein the timing, level or regulation of expression conferred by said promoter in a particular cell, tissue or organ is conferred at least in part by said sequence of nucleotides. Those skilled in the art will be aware that a cis-regulatory region may be capable of activating, silencing, enhancing, repressing or otherwise altering the level of expression and/or cell-type-specificity and/or developmental specificity of any structural gene sequence to which it is operably connected. In general, a single cisregulatory region may be responsible for conferring one mode of regulation on a structural gene sequence to which it is operably connected, however the occurrence of several cis-regulatory regions in operable connection with a single structural gene sequence may confer multiple regulatory modes on said structural gene, which are not necessarily the mere summation of the individual regulatory modes there may be interaction between individual cisregulatory regions). Furthermore, such cis-acting regions generally, but not necessarily, comprise a linear array of groups of nucleotides which each comprise at least four and preferably at least six contiguous nucleotide residues.

WO 2004/035790 PCT/AU2003/001381 Preferred cis-regulatory regions according to the invention comprise a linear array of one or more silencer, enhancer, or upstream activating sequences, not necessarily juxtaposed, however in sufficiently close association to be at least capable of conferring, either in concert or independently of each other, one or more regulated modes of expression on a structural gene sequence to which they are operably connected, such as, for example, a sequence selected from the list comprising the GARE, ABRE, Sph element, CA-rich element, SRE, pyrimidine box, MYB-transcription factor binding site, endosperm box and .TT-box.

Preferred homologues of the exemplified ASI promoter include those promoters that share structural features with the barley or rice ASI promoters set forth in SEQ ID NOs: 1 or 2 and that are useful in conferring the maternal tissue expression on nucleic acid or protein. To isolate such homologues, genomic DNA is hybridized to nucleic acid derived from the structural proteinencoding region of an ASI gene and the promoter region of the hybridizing nucleic acid is isolated. Alternatively, polymerase chain reaction is employed to isolate the structural protein-encoding region of an ASI gene, which is subsequently used to isolate the 5'-upstream region of the corresponding genomic gene. Such procedures are well-known to the skilled artisan.

For the purposes of defining the level of stringency, those skilled in the art will be aware that several different hybridisation conditions may be employed. As used herein, a low stringency hybridisation may comprise the standard reaction buffer used in a polymerase chain reaction (PCR) to anneal an oligonucleotide primer to template DNA at temperatures in the range 250C to 37 0 C or higher, or alternatively, a standard DNA/DNA hybridisation and/or wash carried out in a buffer comprising 6xSSC buffer, 0.1% SDS at ambient temperature, or equivalent annealing/hybridisation conditions. In the present context, references to "hybridisation" conditions clearly refer to both a standard "Southern" or "northern" type of hybridisation, and to the conditions WO 2004/035790 PCT/AU2003/001381 46 required for annealing of a primer to template nucleic acid in a polymerase chain reaction.

A medium stringency may comprise the standard reaction buffer used in a polymerase chain reaction (PCR) to anneal an oligonucleotide primer to template DNA at temperatures in the range 370C to 42 0 C or higher, or alternatively, a standard DNA/DNA hybridisation and/or wash carried out in 2xSSC buffer, 0.1% SDS at a temperature in the range 45°C to 650C or equivalent annealing/hybridisation conditions.

A high stringency may comprise the standard reaction buffer used in a polymerase chain reaction (PCR) to anneal an oligonucleotide primer to template DNA at temperatures higher than 420C, or alternatively, a standard DNA/DNA hybridisation and/or wash carried out in O.1xSSC buffer, 0.1% (w/v) SDS at a temperature of at least 65 0 C, or equivalent annealing/hybridisation conditions.

As will be known to those skilled in the art, the stringency is increased by reducing the concentration of SSC buffer, and/or increasing the concentration of SDS in a standard hybridisation, and/or increasing the temperature of the annealing/hybridisation of PCR or a standard hybridisation, and/or increasing the temperature of the wash in a standard hybridisation. Conditions for hybridisations and washes are well understood by one normally skilled in the art. For the purposes of clarification of parameters affecting hybridisation between nucleic acid molecules, reference is found in pages 2.10.8 to 2.10.16.

of Ausubel et al. (1987). In: Current Protocols in Mol. Biol. Wiley Interscience, which is herein incorporated by reference.

As will be known to those skilled in the art, the specificity of PCR may also be increased by reducing the number of cycles, or the time per cycle, or by the use of specific PCR formats, such as, for example, a nested PCR, a format WO 2004/035790 PCT/AU20031001381 47 that is well-known to those skilled in the art. For the purposes of clarification of the parameters affecting the specificity of PCR, reference is made herein to McPherson et al. (1991) PCR, A Practical Approach, IRL Press, Oxford, which is incorporated by way of reference.

By placing the nucleic acid to be expressed "operably" under the control of the ASI gene promoter or in "operable" connection with the promoter is meant that the promoter is positioned in an appropriate proximityand orientation to the nucleic acid such expression of the nucleic acid is controlled by the promoter. Promoters are generally positioned 5' (upstream) to the structural genes that they control. 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, 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 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, the genes from which it is derived. Again, as is known in the art and demonstrated herein with multiple copies of regulatory elements, some variation in this distance can occur.

By "expressing nucleic acid or protein" is meant that nucleic acid operably connected to the promoter is transcribed and optionally translated thereby yielding nucleic acid consisting of RNA or protein, as appropriate.

In one embodiment, the nucleic acid to which the promoter is operably connected comprises or consists of a structural gene. In the present context, the term "structural gene" shall be taken to refer to nucleic acid comprising a nucleotide sequence that encodes a peptide, oligopeptide, polypeptide, protein WO 2004/035790 PCT/AU2003/001381 48 or enzyme or a portion thereof. Accordingly, structural genes may include the protein-encoding regions of genomic genes, cDNA molecules and other DNA molecules, including fusion molecules.

In one embodiment, the structural gene comprises or consists of a reporter gene, such as but not limited to the green fluorescent protein (gfp) gene, the P-glucuronidase gene, the chloramphenicol acetyl transferase gene, or the firefly luciferase gene, amongst others.

Without detracting from the general applicability of the promoter sequence of the invention, one embodiment provides for expression of a structural gene that encodes a protein that confers or enhances protection against a plant pathogen, such as, for example, a seed-borne fungus, seedborne virus, seed-borne bacterium, or insect that feeds on the seed. Such proteins are known to those skilled in the art and include, for example, a range of structurally and functionally diverse plant defense proteins or pathogenesisrelated proteins chitinase, in particular acid chitinase or endochitinase; Pglucanase in particular p-1,3-glucanase; ribosome-inactivating protein (RIP); ykafirin; Hevea brasiliensis hevein; potato win1 or win2 proteins, or related protein from wheat such as, for example, wheatwin or WPR4 or, related protein from barley such as, for example, barwin); thionin, in particular y-thionin; thaumatin or thaumatin-like protein such as zeamatin; a proteinase inhibitor such as, for example, trypsin or chymotrypsin; or sormatin), virus coat proteins, and proteins that convert one or more pathogen toxins to non-toxic products.

Nucleic acid encoding such proteins are publicly available and/or described in the scientific literature. The structures of such genes and their encoded proteins are fully described in the database of the National Center for Biotechnology Information of the US National Library of Medicine, 8600 Rockville Pike, Bethesda, MD 20894, USA.

WO 2004/035790 PCT/AU2003/001381 49 As exemplified herein, a wide range of structurally and functionally diverse proteins are expressed in the pericarp in operable connection with the promoter of the invention to confer or enhance resistance of a wheat plant to Fusarium graminearum (head scab). In accordance with this embodiment, the protein conferring or enhancing protection against F. graminearum is selected from the group consisting of: a wheat thaumatin-like protein that confers protection against the fungal pathogen Fusarium graminearum (head scab) in wheat SEQ ID NOs: 16, 18, 20, 22, 24, or 26); (ii) a modified ribosomal protein L3 of wheat wRPL3:Cys 258; SEQ ID NO: 28) that is resistant to the action of a trichothecene produced by F. graminearum; and (iii) a polypeptide having trichothecene O-acetyl transferase activity and capable of converting trichothecene produced by F. graminearum into a non-toxic product SEQ ID NO: Also exemplified herein, the coat protein of BSMV (SEQ ID NO: 32) is expressed in barley pericarp in operable connection with the promoter of the invention to confer or enhance resistance of barley to BSMV.

Also without detracting from the general applicability of the promoter sequence of the invention, an alternative embodiment provides for expression of a structural gene that encodes a pharmaceutically, immunologically or nutritionally useful protein, or an enzyme that is required for production of a pharmaceutically, immunologically or nutritionally useful secondary product, or a protein capable of modifying the utilization of a substrate in a secondary metabolic pathway. Such proteins are known to those skilled in the art and include, for example, a range of structurally and functionally diverse antigenic proteins an antigenic protein derived from a pathogen that infects a human or animal to be fed on the bran product of the grain), a sulphur-rich protein Brazil Nut Protein, sunflower seed albumin, 2S protein, Aspl synthetic protein), a calcium-binding protein calmodulin, calreticulin, or calsequestrin), an iron-binding proteins hemoglobin), and a biosynthetic WO 2004/035790 PCT/AU2003/001381 enzyme that is required for the production of an osmoprotectant such as betaine choline oxidase, betaine aldehyde dehydrogenase), a fatty acid delta-12 desaturase), a phytosterol S-adenosyl-L-methionine-A 2 4 sterol methyl transferases (SMT, or SMTn), a C-4 demethylase, a cycloeucalenol to obtusifoliol-isomerase, a 14a-methyl demethylase, a A 8 to A isomerase, a A 7 -sterol-C-5-desaturase, or a 24,25-reductase), an anthocyanin or other pigment (proanthocyaninidin reductase), lignin cinnamoyl alcohol dehydrogenase, caffeic acid O-methyl-transferase, or phenylalanine ammonia lyase), an anti-nutritional protein, an enzyme capable of altering a substrate in the phenylpropanoid pathway choline oxidase, betaine aldehyde dehydrogenase, ferulic acid decarboxylase), a choline metabolizing enzyme capable of acting upon choline to modify the use of choline by other enzymes in the phenylpropanoid pathway choline oxidase, betaine aldehyde dehydrogenase, ferulic acid decarboxylase), an enzyme involved in the malting process high pl a-amylase, low pi a-amylase, Ell-(1-3,1-4)-p-glucanase, Cathepsin p-like proteases, a-glucosidase, xylanase or arabinofuranosidase), an enzyme capable of acting upon a sugar alcohol, or an enzyme capable of acting upon myo-inositol, etc. Nucleic acid encoding such proteins are publicly available and/or described in the scientific literature. The structures of such genes and their encoded proteins are fully described in the database of the National Center for Biotechnology Information of the US National Library of Medicine, 8600 Rockville Pike, Bethesda, MD 20894, USA.

As exemplified herein, an antigenic polypeptide of Transmissible Gastroenteritis Virus (TGEV) SEQ ID NO: 36) is expressed in the pericarp in operable connection with the promoter of the invention to confer or enhance resistance of pigs to TGEV. In accordance with this embodiment, the bran or pericarp is administered to animals as a medicinal foodstuff or oral vaccine.

WO 2004/035790 PCT/AU2003/001381 51 Also exemplified herein, the Brazil Nut Protein (SEQ ID NO: 38) is expressed in the pericarp in operable connection with the promoter of the invention to enhance the sulphur content of fodder comprising wheat bran.

It is to be understood that the invention also extends to the expression of nucleic acid that does not comprise or consist of a protein-encoding structural gene. The nucleic acid to which the promoter of the invention is operably connected can clearly comprise a nucleotide sequence that does not encode a peptide, oligopeptide, polypeptide, protein or enzyme or a portion thereof.

Accordingly, in an alternative embodiment of the invention, the promoter is operably connected to nucleic acid that is expressed to produce inhibitory RNA, an antisense molecule, ribozyme, abzyme, co-suppression molecule, genesilencing molecule or gene-targeting molecule, which targets the expression of a gene the expression of which is to be reduced or down-regulated in the maternal tissue.

For example, it may be desirable to target expression of a fungal gene required for infection of the plant, or alternatively, to reduce expression of a pericarp-expressed gene in the plant (eg. reducing expression of a ligninbiosynthesis gene or proanthocyanidin biosynthesis gene to enhance palatability or digestibility of the pericarp, thereby reducing the need for downstream processing to remove the pericarp). According to this embodiment, expression of such nucleic acid under the control of the ASI promoter will partially or completely reduce, delay or inhibit the expression of the endogenous gene against which the nucleic acid is targeted in the pericarp.

Wherein the endogenous gene being targeted is normally expressed in more than one cell type, the expression of said gene under control of the promoter sequence may further result in said gene being expressed in cells other than merely the maternal tissue cells of the plant.

WO 2004/035790 PCT/AU2003/001381 52 In a particularly preferred embodiment, nucleic acid that is capable of down-regulating or inhibiting expression of a gene that is required for the life cycle or infectivity or transmission of a seed-borne plant pathogen is expressed operably under control of the promoter.

For example, S-adenosyl-L-methionine-A 24 -sterol methyl transferases (SMTI or SMTII) is required for the life cycle of many insects and fungal pathogens to be completed, and expression of inhibitory RNA against this enzyme in the pericarp can prevent the pathogen from maturing into an adult, thereby preventing pathogen spread.

As exemplified herein, a gene encoding siRNA against the movement protein of BSMV (SEQ ID NO: 33) is expressed in the pericarp of barley to inhibit virus movement from the pericarp through the vasculature of the plant.

Preferably, the plant in which the nucleic acid or protein is expressed operably under control of the bifunctional a-amylase subtilisin inhibitor (ASI) gene promoter is a monocotyledonous plant of agronomic importance, for example a grain crop such as wheat, oats, maize, barley, rice, sorghum, millet or rye, amongst others. In a particularly preferred embodiment, the promoter sequence is at least capable of conferring expression in the maternal tissue of rice, wheat or barley. As exemplified herein, the promoter is operable in barley and wheat pericarp in connection with a wide range of structurally and functionally diverse nucleic acids.

In an alternative embodiment, the present invention provides a method of expressing nucleic acid or protein in the maternal tissue of a plant seed comprising expressing isolated nucleic acid operably under the control of a bifunctional a-amylase subtilisin inhibitor (ASI) gene promoter that is operable in the maternal tissue and determining that the pericarp contains an effective amount of nucleic acid or protein encoded by the expressed nucleic acid.

WO 2004/035790 PCTIAU2003001381 53 Means for determining that the pericarp contains an amount or an effective amount of nucleic acid or protein encoded by the expressed nucleic acid will vary depending upon the function of the expressed nucleic acid and the context in which the nucleic acid is employed i.e. to encode a protein or an inhibitory nucleic acid). Such means will be readily apparent to the skilled artisan and include, for example, immunoassays such as ELISA or Western blot analysis, to detect an expressed protein or PCR, Northern hybridization or dot blot analysis to detect expressed RNA. In the case of calcium-binding proteins or iron-binding proteins, expression of an effective amount of the protein may also be confirmed by directly assaying the level of the ion against an internationally-acceptable pharmacological standard.

For example, the expression of nucleic acid in the maternal tissue can be readily determined by any means known to the skilled artisan, such as, for example, using polymerase chain reaction (PCR) following reverse transcription of an mRNA template molecule, essentially as described by McPherson et aL. (1991) PCR, A Practical Approach, IRL Press, Oxford.

Alternatively, expression may be determined by northern hybridisation analysis or dot-blot hybridisation analysis or in situ hybridisation analysis or similar technique, wherein mRNA is transferred to a membrane support and hybridised to a nucleic acid probe comprising a nucleotide sequence complementary to the nucleotide sequence of the expressible mRNA transcript, labelled with a suitable reporter molecule such as a radioactively-labelled dNTP (eg [a- 32 P]dCTP or [a- 35 S]dCTP) or biotinylated dNTP, amongst others.

Expression is then determined by detecting the appearance of a signal produced by the reporter molecule bound to the hybridised probe.

Alternatively, the rate of transcription of the expressible nucleic acid may be determined by nuclear run-on and/or nuclear run-off experiments, wherein WO 2004/035790 PCT/AU2003/001381 54 nuclei are isolated from a particular cell or tissue and the rate of incorporation of rNTPs into specific mRNA molecules is determined.

Alternatively, expression may be determined by RNase protection assay, wherein a labelled RNA probe or riboprobe having a sequence complementary to the transcribable mRNA is annealed to said mRNA for a time and under conditions sufficient for a double-stranded mRNA molecule to form, after which time the sample is subjected to digestion by RNase to remove single-stranded RNA molecules and in particular, to remove excess unhybridised riboprobe.

Those skilled in the art will also be aware of various immunological and enzymatic methods for detecting the level of expression of a particular gene at the protein level, for example using rocket immunoelectrophoresis, ELISA, radioimmunoassay and western blot immunoelectrophoresis techniques, amongst others.

Such approaches supra are described by Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed, NY. and Ausubel et al.

(1987). In: Current Protocols in Mol. Biol. Wiley Interscience.

Functional assays may also be employed, such as, for example, determining the ability of pericarp expressing an immunogenic protein or peptide to confer oral immunity on a human or animal subject. In accordance with this embodiment, the ability of transgenic seed expressing the immunogenic protein to confer oral immunity is determined in a process comprising providing the seed or bran of a transgenic plant to an animal subject and then determining the immune response of the subject to the immunogenic protein, wherein the production of antibodies and/or the eliciting of a CTL response against the immunogenic protein indicates that an effective amount of the protein is produced. Suitable control seed for such functional tests include WO 2004/035790 PCT/AU20031001381 seed from a substantially isogenic plant that does not express the immunogenic protein operably under the control of the promoter.

In a particularly preferred embodiment, the ability of transgenic seed expressing a protein or nucleic acid that is intended to enhance resistance against a seed-borne pathogen is determined in a process comprising infecting the seed of a transgenic plant with the pathogen and determining the infectivity or transmission of the pathogen, wherein a reduced infectivity or transmission of the pathogen indicates that an effective amount of the nucleic acid or protein is produced. Suitable control seed for such functional tests include seed from a substantially isogenic plant that does not express the nucleic acid or protein operably under the control of the promoter.

Extracted or isolated pericarp tissue or bran is preferably used for such analyses. In the case of expression in mature seed, extracted bran, such as obtained during milling of the grain, is the preferred analyte. It is particularly preferred that the pericarp or bran be substantially free of endosperm.

In an alternative embodiment, the present invention provides a method of expressing nucleic acid or protein in the maternal tissue of a plant seed comprising expressing isolated nucleic acid operably under the control of a bifunctional a-amylase subtilisin inhibitor (ASI) gene promoter that is operable in the maternal tissue and obtaining the pericarp containing a nucleic acid or protein encoded by the expressed nucleic acid.

As will be apparent from the preceding description, pericarp can be obtained from mature seed in the form of bran, such as obtained during milling of the grain. In the case of immature seed prior to the cessation of grain filling), the pericarp is readily removed by any mechanical means known to the skilled artisan, and can be readily removed from contaminating aleurone and WO 2004/035790 PCTIAU2003001381 56 endosperm tissues. It is particularly preferred that the pericarp that is obtained is substantially free of endosperm.

In a preferred embodiment, the present invention provides a method of expressing nucleic acid or protein in the maternal tissue of a plant seed comprising expressing isolated nucleic acid operably under the control of a bifunctional a-amylase subtilisin inhibitor (ASI) gene promoter that is operable in the maternal tissue, obtaining the pericarp containing a nucleic acid or protein encoded by the expressed nucleic acid and determining that the pericarp contains an effective amount of nucleic acid or protein encoded by the expressed nucleic acid.

In a preferred embodiment, the inventive method further comprises introducing to a plant cell, tissue or organ an isolated nucleic acid comprising a bifunctional a-amylase subtilisin inhibitor (ASI) gene promoter that is operable in the maternal tissue of a plant.

In one embodiment, the present invention provides a method of expressing nucleic acid or protein in the maternal tissue of a plant seed comprising introducing to a plant cell a nucleic acid operably linked to a bifunctional aamylase subtilisin inhibitor (ASI) gene promoter that is operable in the maternal tissue and expressing the introduced nucleic acid operably under the control of the promoter.

In an alternative embodiment, the present invention provides a method of expressing nucleic acid or protein in the maternal tissue of a plant seed comprising introducing to a plant cell a nucleic acid operably linked to a bifunctional a-amylase subtilisin inhibitor (ASI) gene promoter that is operable in the seed, regenerating a plant therefrom and expressing the introduced nucleic acid in the maternal tissue of the plant seed.

WO 2004/035790 PCT/AU2003/001381 57 A chimeric gene comprising the isolated promoter operably linked to nucleic acid to be expressed in the maternal tissue, or a genetic construct comprising same, may be introduced into a plant cell by various techniques known to those skilled in the art. The technique used may vary depending on the known successful techniques for that particular organism.

Means for introducing recombinant DNA into bacterial cells or plant cells include, but are not limited to, transformation using CaCI 2 and variations thereof, in particular the method described by Hanahan, D. (1983) J. Molec.

Biol. 166: 557-560, direct DNA uptake into protoplasts (Krens et al, 1982 Nature 296: 72-74; Paszkowski et al, 1984), PEG-mediated uptake to protoplasts (Armstrong, et al, 1990. Plant Cell Reports 9:335-339), electroporation (Fromm et al., 1985, Proc. Natl. Acad. Sci. (USA) 82: 5824- 5828), microinjection of DNA (Crossway et al., 1986 Mol. Gen. Genet. 202, 179-185), microparticle bombardment of tissue explants or cells (Christou, P., et al, 1988. Plant Physiol 87, 671-674; Sanford et al., 1987 Particulate Sci.

Tech. 5: 27-37; Finer J.J. and McMullen, 1990 Plant Cell Reports 8:586- 589; Finer et al., 1992, Plant Cell Rep. 11: 323-328; Sanford et al., 1993 Methods Enzymol. 217: 483-509; Karunaratne et al., 1996 Aust. J. Plant Physiol. 23: 429-435; and Abedinia et al., (1997) J. Plant Physiol. 24: 133-141, vacuum-infiltration of tissue with nucleic acid, or T-DNA-mediated transfer from Agrobacterium to the plant tissue (An etal.1985, EMBO J. 4:277-284); Herrera- Estrella et al., 1983a Nature 303: 209-213; Herrera-Estrella et al., 1983b EMBO J. 2: 987-995; Herrera-Estrella et al., 1985 In: Plant Genetic Engineering, Cambridge University Press, NY, pp 63-93.

For the transformation of monocotyledonous plants, microparticle bombardment of cells or tissues is preferred, particularly in cases where plant cells are not amenable to transformation mediated by A. tumefaciens. In such procedures, microparticle is propelled into a cell to produce a transformed cell.

Any suitable ballistic cell transformation methodology and apparatus can be WO 2004/035790 PCT/AU2003/001381 58 used in performing the present invention. Exemplary apparatus and procedures are disclosed by Stomp et al. Patent No. 5,122,466) and Sanford and Wolf Patent No. 4,945,050). When using ballistic transformation procedures, the genetic construct may incorporate a plasmid capable of replicating in the cell to be transformed. Examples of microparticles suitable for use in such systems include 0.5 to 5 micron gold spheres. The DNA construct may be deposited on the microparticle by any suitable technique, such as by precipitation.

Once introduced into the plant tissue, the expression of nucleic acid or protein under control of the promoter sequence may be assayed in a transient expression system, or it may be determined after selection for stable integration within the plant genome.

Whole plants are regenerated from the transformed 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 genetic 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, scutella, cotyledons, hypocotyls, megagametophytes, callus tissue including embryogenic callus, existing meristematic tissue apical meristems, axillary buds, and root meristems), and induced meristem tissue cotyledon seed and hypocotyl seed).

The term "organogenesis", as used herein, means a process by which shoots and roots are developed sequentially from meristematic centres.

WO 2004/035790 PCTIAU2003001381 59 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.

The regenerated 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) transformant, and the T2 plants further propagated through classical breeding techniques.

The regenerated transformed plants contemplated herein may take a variety of forms. For example, they may be chimeras of transformed cells and non-transformed cells; clonal transformants all cells transformed to contain the expression cassette); grafts of transformed and untransformed tissues in plants, a transformed root stock grafted to an untransformed scion A second aspect of the present invention is directed to the use of a genetic construct comprising an ASI promoter or a derivative or homologue thereof operably connected to an expressible nucleic acid for expression in the maternal tissue of a plant seed. As with the other embodiments described herein, this invention is not to be limited by the nature of the expressible nucleic acid, the only requirement being that it is nucleic acid capable of being transcribed or translated.

Exemplary expressible nucleic acids in this context are described supra and in the examples that follow.

The genetic construct used to confer maternal tissue expression may further comprise a transcription termination sequence placed operably in connection with the expressible nucleic acid. As will be known to those skilled WO 2004/035790 PCT/AU2003/001381 in the art, a "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. Preferably, the transcription termination sequence is placed downstream of the promoter sequence, and spaced therefrom by the expressible nucleic acid.

Terminators active in cells derived from viruses, yeasts, moulds, 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 genetic constructs of the present invention include the nopaline synthase (NOS) gene terminator of Agrobacterium tumefaciens, the tumor morphology large (tml) gene terminator of Agrobacterium tumefaciens, the terminator of the Cauliflower mosaic virus (CaMV) 35S gene, the ADP-glucose pyrophosphorylase gene terminator (t3'bt2) derived from Oryza sativa, the zein gene terminator from Zea mays, the HMW glutenin gene terminator derived from Triticum aestivum, the Rubisco small subunit (SSU) gene terminator sequences, subclover stunt virus (SCSV) gene sequence terminators, any rhoindependent E. coli terminator, amongst others. Alternatively or in addition, the ASI 3'utr 3' untranslated region of the ASI gene) may be used, particularly to provide for optimum stability of the mRNA encoded by the expressible nucleic acid placed under control of the promoter. For example, the modulation of expression by phytohormones under control of the ASI promoter may be enhanced by including the ASI 3' utr downstream of the translation stop codon of the expressible nucleic acid, with or without additional transcription termination sequences placed downstream thereof. Those skilled in the art will be aware of additional terminator sequences which may be suitable for use in performing the invention. Such sequences may readily be used without any undue experimentation.

WO 2004/035790 PCT/AU2003/001381 61 The genetic constructs may further include an origin of replication sequence which is required for replication in a specific cell type, for example a bacterial cell, when said genetic construct is required to be maintained as an episomal genetic element (eg. plasmid or cosmid molecule) in said cell.

Preferred origins of replication include, but are not limited to, the fl-ori and colE1 origins of replication.

In a further alternative embodiment, the genetic construct of the invention further comprises one or more selectable marker gene or reporter gene sequences, placed operably in connection with a suitable promoter sequence which is operable in a plant cell and optionally further comprising a transcription termination sequence placed downstream of said selectable marker gene or reporter gene sequences.

As used herein, the term "selectable marker gene" includes any gene which confers a phenotype on a cell in which it is expressed to facilitate the identification and/or selection of transfected or transformed cells. Suitable selectable marker genes contemplated herein include the ampicillin resistance tetracycline resistance gene (Tcr), bacterial kanamycin resistance gene (Kanr), phosphinothricin resistance gene, neomycin phosphotransferase gene (nptll), hygromycin resistance gene (hph), p-glucuronidase (GUS) gene, chloramphenicol acetyltransferase (CAT) gene and luciferase gene, amongst others.

Those skilled in the art will be aware that the choice of promoter for expressing a selectable marker gene or reporter gene sequence may vary depending upon the level of expression required and/or the species from which the host cell is derived and/or the tissue-specificity or development-specificity of expression which is required. Examples of promoters suitable for expressing a selectable marker gene include promoters derived from the genes of viruses, WO 2004/035790 PCT/AU2003/001381 62 yeasts, moulds, bacteria, insects, birds, mammals and plants which are capable of functioning in the maternal tissue or whole plants. The promoter may regulate the expression of the selectable marker gene constitutively, or differentially with respect to the tissue in which expression occurs or, with respect to the developmental stage at which expression occurs, or in response to external stimuli such as physiological stresses, pathogens, or metal ions, amongst others.

Examples of suitable promoters in this context include the CaMV promoter, NOS promoter, octopine synthase (OCS) promoter, Arabidopsis thaliana SSU gene promoter, napin seed-specific promoter, and the like. In addition to the specific promoters identified herein, cellular promoters for socalled housekeeping genes are useful. Those skilled in the art will be aware of additional promoter sequences which may be suitable for use in expressing the selectable marker gene. Such sequences may readily be used without any undue experimentation.

The genetic construct may further comprise one or more integration sequences. As used herein, the term "integration sequence" shall be taken to refer to a nucleotide sequence which facilitates the integration into plant genomic DNA of a promoter sequence with optional other integers referred to herein. Particularly preferred integration sequences according to this embodiment include the left border (LB) and right border (RB) sequences of T- DNA derived from the Ti plasmid of Agrobacterium tumefaciens or a functional equivalent thereof.

A further aspect of the present invention provides transfected or transformed maternal tissue of a seed or a cell thereof, or a whole plant having transformed maternal tissue in its seed, wherein said maternal tissue or cell or whole plant expresses nucleic acid or protein operably under the control of a heterologous ASI promoter sequence described herein.

WO 2004/035790 PCT/AU2003/001381 63 The present invention clearly extends to the progeny plants and plant parts, in particular maternal tissue derived from the transformed plants, the only requirement being that said progeny plants and plant parts also comprise the introduced ASI promoter sequence.

The present invention is further described by reference to the accompanying non-limiting Examples.

EXAMPLE 1 Isolation and characterization of the promoter of the barley asi gene.

1.1 Methods Seeds of barley (Hordeum vulgare c.v's Grimmett and Himalaya were obtained from "The Australian Winter Cereal Collection", Tamworth, Australia, and c.v. Golden promise from Plant Industry, CSIRO, Canberra, Australia.

The barley seeds were sown in potting mix, in a glasshouse with day and night temperatures of 22°C and 19 0 C respectively, and whole shoots of 10 to 15 day old seedlings were taken for extraction (essentially as described by Carroll, BJ. 1995. Genetics 139:407-420).

The 5'-upstream sequence of the asi gene was isolated by using the "Universal Genome Walker" kit (Clontech laboratories, CA, USA). Five DNA libraries henceforth referred to as Dra I, Eco RV, Stu I, Scal, and Pvu II libraries respectively were prepared and DNA from each of the five libraries was used as template in a nested PCR to amplify 5'-upstream region of the asi gene.

Combinations of primers AP1 and BGP (SEQ ID NO: 4) was used in the primary PCR while AP2 and BGPN (SEQ ID NO: 5) was used in the secondary PCR. Using the Scal library as template-DNA, a ca. 1.2 kb fragment WO 2004/035790 PCTIAU2003/001381 64 (henceforth refereed to as the Scal fragment) was amplified by PCR. The Scal fragment was cloned into pGEMT-easy, a TA-type cloning vector (pGEMTeasy, Promega Corporation, MI, USA), which resulted in the construct pA17.

Similarly, primer combinations of BG (SEQ ID NO: 6) and AP1 (primary PCR), and BGN (SEQ ID NO: 7) and AP2 (secondary PCR) were used to isolate a 655 bp 3' downstream sequence (850 bp PCR fragment from the Stu I library) from the stop codon of the asi gene. Again, this PCR product was cloned into the pGEMT-easy vector, resulting in the construct pAC1.

The primary PCR and secondary PCR, to isolate the 5' and 3' upstream sequences of the asi gene, was carried out according to the manufactures recommendations (Genome Walker, Clontech, USA). PCR to amplify the 1033 bp 5'-upstream region of the asi gene and five of its 5'-deletions, using pA1 7 as template DNA, was carried out using the DNA-polymerase Pfu-turbo (Clontech, USA) according to the manufacturers recommendations.

Oligonucleotides BF1 (SEQ ID NO: BF2 (SEQ ID NO: BF3 (SEQ ID NO: 10), BF4 (SEQ ID NO: 11), BF5 (SEQ ID NO: 12) and BF6 (SEQ ID NO: 13) each in combination with the primer BR (SEQ ID NO: 14) were used to amplify a 1033 bp 5'-upstream regions of the asi gene (-959 to +74) (SEQ ID NO: 1) and five progressive 5'-deletions corresponding to 666 bp (-592 to +74), 496 bp (-422 to 272 bp (-198 to 220 bp (-146 to +74) and 173 bp 99 to respectively, collectively called the asi-promoter constructs.

BGP: 5'-GCGGTTGGCCGAGAGGACGTAGTAGTTC-3' (SEQ ID NO: 4); BGPN: 5'-CGCGAGAGGGCGGTGCTGGCCAGAATAAGG-3' (SEQ ID NO: BG: CCGAGGTGCACGAGTACAAGCTGATGTCG (SEQ ID NO: 6); BGN: CGAGCCATACCATGTCGTCGTGTTCAAG (SEQ ID NO: 7); BF1: 5'-ATCGGAAGCTTACTGGGCTCGAAACTAAATAAGAACATG-3' (SEQ ID NO: 8); BF2: 5'-ATCGGAAGCTTCATACCACCATCGAAGATGCCCTAAG-3 (SEQ ID NO: 9); BF3: 5'-ATCGGAAGCTTGGAGGATGTGGAACGCAGATAGTGAC-3 (SEQ ID NO: BF4: 5'-ATCGGAAGCTTGGAGTGGTAGGATATAACATTGCTCTG-3' (SEQ ID NO: 11); 5'-ATCGGAAGCTTCGGTGTACGCACTTACTGGATGCCAC-3' (SEQ ID NO: 12); BF6: 5'-ATCGGAAGCTTCACATCACGCAATCCACCAGAAG-3' (SEQ ID NO: 13); and BR: 5'-ACCTATTCATGATGAAACCTCTGCTGGAGGTCC-3' (SEQ ID NO: 14).

WO 2004/035790 PCT/AU2003/001381 Oligonucleotides were obtained from Pacific Oligos, Australia.

The sequence of the cloned PCR fragments was determined using the dideoxy method (essentially as described by Sanger et al., 1977. Proc. Natl Acad. Sci. USA 74: 5463-5467) using the Big Dye Terminator kit (Perkin Elmer, USA). A commercial provider (AGRF, Brisbane, Australia) was used to obtain sequence data. Analysis of the sequence data was performed using the facilities provided by the Australian National Genomic Information Service

(ANGIS).

1.2 Results PCR reaction using each five libraries as template DNA yielded DNA fragments of one or more sizes. In the nested PCR reaction the Scal and the Stul libraries yielded ca 1.2 kb and 0.85 bp fragments representing 1033 bp upstream (SEQ ID NO: 1) and 655 bp 3'-downstream (SEQ ID NO: 3) sequences respectively, of the asi gene.

The sequence of the 1033-asi promoter (SEQ ID NO: 1) is shown in Figure 1. Based on previous reports (Leah, R. and Mundy, J. 1989. Plant Molecular Biology 12:673-682) the transcription start site position) was assigned to the adenine nucleotide (74th base upstream from the translation initiation codon of the asi gene). Sequence homology to published DNAelements was used to locate a number of putative elements along the promoter region of the asi gene (Figurel). A putative TATA box and CAAT box were located at positions -34 to -27 and -86 to -83 respectively. The sequence TGTAAAGG (-112 to -105) matched the consensus sequence of the E-motif TG(T/A/C)AAA(G/A)(GIT) (Huang et al., 1992) Proc. Nat/ Acad. Sci. USA 89: 7526-7530, while truncated forms of the E-motif sequence were located at positions -220 to -213 (TGATAAAT) and -272 to -265 (TGTAAATT). A sequence with high homology to the ABA-responsive element WO 2004/035790 PCT/AU2003/001381 66 (GTACGTGGCGC) (Marcotte et al., 1989, Plant Cell 1: 969-976; Mundy et al., 1990. Proc. Natl Acad. Sci. USA 87: 1406-1410; Skriver et al., 1991. Proc. Nati Acad. Sci. USA 88: 7266-7270) was located at -542 to -534 (TACTGTGGC).

Sequences similar to the GA-responsive element (GARE) -TAACAAA- (Skriver et al., 1991 Proc. NatlAcad. Sci. USA 88: 7266-7270) were located at -377 to 371 (TCACAAA), -569 to -563 (TAAGAAC) and -751 to -745 (TAACTAA). A pyrimidine box (Huang et al., 1992. Proc. NatI Acad. Sci USA 89:7526-7530) was located at -333 to -327 (TCTTTTT) and also in reverse orientation at -67 to -72 (TCTTTTT), while truncated forms were located at positions -261 to -255 (TCATTTT) and -650 to -644 (TCTTTTA). The RY-repeat element also called the Sph-element (Huang et al., 1992. Proc. Natl Acad. Sci. USA 89:7526-7530) was found at position -531 to -525 (CATGCATG) and truncated forms were found at positions -161 to -153 (CACCGCATG), -601 to -594 (CAATGATG) and -635 to -627 (CATGTCATC). A CT-rich region (-665 to-645) and a TA-rich region (-346 to -255) were also been identified.

The sequence of DNA, 3'-downstream of the asi gene is shown in Figure 2 (SEQ ID NO: It contains the 3'-untranslated sequence (77 bp, 3'-UTR) and contains 577 bp sequence downstream from the 3'-UTR.

EXAMPLE 2 Production and analysis of cells transiently expressing gfp controlled by the barley asi gene promoter 2.1 Methods 2.1.1 Preparation of transgenic constructs The plasmid pAGN (WO 00/18926; SEQ ID NO: 39), a pGEM3Zf+ based vector (Promega Corporation, MI, USA) that contains the gfp and nos terminator sequence, was used as the cloning vector to generate the asi promoter constructs for particle bombardment. The asi-promoter constructs were transcriptional fusions of the 1033-asi promoter (SEQ ID NO: 1) and five WO 2004/035790 PCT/AU2003/001381 67 of its progressive 5'-deletions (as described in Example and the green fluorescent protein (gfp) (Patterson et al., 1997 Biophysical Journal 73: 2782- 2790) reporter gene. The construct pA17 (Sca I fragment) was used as template DNA in a PCR with primers BF1 (SEQ ID NO: 8) and BR (SEQ ID NO: 14) to amplify the 1033-asi promoter fragment (SEQ ID NO: which was directionally cloned into the Hind III and Nco I sites of pAGN resulting in the construct pA57 (SEQ ID NO: 40). The construct pA57 (SEQ ID NO: corresponding to position -959 to +74 of the 1033-asi promoter was then used to amplify progressive 5-deletions of the 1033-asi promoter. Deletions corresponding to positions -959, -592, -422, -198, -146, and -99 and position +74 respectively, were directionally cloned (as performed for construct pA57) to generate constructs pA58, pA61, pA64, pA67 and pA70 respectively. Ligation reactions were carried out using T4 DNA ligase (Promega Corporation, USA) and constructs were verified by sequence analysis. The plasmid pAGN was also used as the cloning vector to generate the pubi.gfp.nos, the pa-amyl.gfp.nos and the pEm.gfp.nos gene constructs. The constructs pubigfp.nos, pCaMV35S.gfp.nos, the paamyl.gfp.nos and the pEm.gfp.nos contain the ubiquitin promoter (Christensen, A.H. and Quail, P.H. 1996. Transgenic Research 5:213-218), the cauliflower mosaic virus 35S RNA promoter (Guilley et al., 1982. Cell 30:763-770), the aamy1 promoter from barley (Gubler, F. and Jacobsen, J.V. 1992. Plant Cell 4: 1435-1441) and the Em promoter (extending from -554 to 92) (Marcotte et al., 1989. Plant Cell 1: 969-976) respectively, linked to the gfp gene and nos terminator sequence. Plasmid pDP687 (Ludwig et al., 1990) was used as a control for bombardment experiments and to assess viability of cells. The pDP687 vector contains the cauliflower mosaic virus 35S RNA promoter (Guilley et al., 1982. Cell 30:763-770) which controls the constitutive expression of two genes, each encoding transcription factors which regulate synthesis of the red anthocyanin pigment.

WO 2004/035790 PCT/AU2003/001381 68 2.1.2 Tissue preparation Aleurone layers from mature seeds of barley were prepared essentially according to established procedures (Chrispeels et al and Varner 1976. Plant Physiology 42: 398-406; Mundy et al., 1986. Plant Physiology 81: 630-636).

Seeds of barley, c.v. Himalaya, were cut in half and the ends containing the embryo were discarded. The other half of the seeds were sterilised sodium hypochlorite for 5 min) and imbibed for three days on sterile sand moistened with sterile tap water. Three days after imbibition, aleurone layers were prepared aseptically as follows prior to bombardment. Incisions were made with a scalpel blade on either side of the crease of the half-grain and the part of the seed with the crease was discarded. The layers of pericarp and testa were peeled away, while the transparent nucellus layer was removed by scraping gently with the back of a scalpel blade, thus exposing the upper side of the aleurone layer. The endosperm tissue in contact with the aleurone layer was gently removed by first soaking for 15 minutes in phosphate buffer (10.0 mM Na 3

PO

4 150 mM NaCI, pH 7.4) (Mundy et al., 1986. Plant Physiology 81: 630- 636) followed by gently scraping the endosperm with the back of a scalpel blade. The aleurone layer from each seed was further cut into 4 pieces prior to particle bombardment. Immature seeds (21-25 DPA) of barley c.v. Golden promise, were prepared as follows prior to bombardment of the pericarp tissue.

Immature seeds were surface sterilised with 1% sodium hypochlorite for 2 min followed by rinsing five times in sterile distilled water. Intact secondary leaves of 12-day-old seedlings (two seeds sown in soil contained in a 50 ml Falcon tube) of barley cv Golden promise, were taken for bombardment.

2.1.3 Particle bombardment of barley cells Nine immature barley seeds or aleurone layers were arranged in the centre of a plastic culture plate (8.5 cm) prior to bombardment. To bombard leaf tissue, the 50 ml Falcon tube containing the 10-day-old seedling growing in soil, was placed in the chamber of the particle inflow gun. Four leaves from two plants were arranged side-by-side between two plastic sheets and maintained WO 2004/035790 PCT/AU2003/001381 69 in this position by clamping the two sheets of plastic with paper clips. The two plastic sheets (exposed X-ray film of 9 cm X 5 cm) had an internal rectangular piece (3.5 cm X 2.0 cm) cut out thereby exposing a fixed area of the leaf tissue for bombardment. Particle bombardment (Klein et al., 1987. Nature 327: 70-73) using a particle inflow gun (Finer, J.J. and McMullen, M.D. 1990. Plant Cell Reports 8:586-589) was used as the method of gene delivery. Construct DNA (4 ig), of either pubi.gfp.nos or each of the asi-promoter constructs were precipitated, and wherever necessary was co-precipitated with pDP687 (2 [g onto 2.5 gg of tungsten (Tg) particles (1.2 pm). The Tg-construct DNA precipitate was finally suspended in 30 pl of 90% ethanol yielding 83.3 ng of Tg coated with 200 ng of construct DNA for 1 Ip of suspension (133.3 ng 66.7 ng of test and control construct DNA respectively). The Tg-construct-DNA suspension (3 il) was placed on the grid of a 13 mm-Sweeny holder (Gelman Sciences, MI, USA), and bombarded under vacuum (-91 KPa) using pressure of 1500 KPa for aleurone layers or immature seeds, and 1000 KPa for leaf tissue. A nylon mesh (500 pm) was kept at a distance of 5.5 cm above the explant tissue. The explant tissues were kept on a platform in the gun chamber such that the distance between the explant tissue and the Sweeny holder was 12.8 cm for mature barley seeds or aleurone layers and 14.9 cm for leaf tissue.

2.1.4 Analysis of transient gfp expression Measurement of GFP protein transiently expressed in aleurone cells was carried out by image analysis according to Furtado A. and Henry R. (Analytical Biochemistry 310, 84-92, 2002).

Image capture was carried using a microscope mounted CCD colour camera (Cool-SNAP-Pro, Media Cybernetics, USA) controlled by it's dedicated software (Image-Pro Plus, version 5.1, Media Cybernetics, USA). All images were captured in colour images at a resolution of 1392 x 1040.

WO 2004/035790 PCT/AU2003/001381 2.2 Results and discussion The 1033 asi promoter or its deletions directed low-level transient expression of the gfp gene in barley aleurone layers (Figure 3a). The hormones ABA or GA had no effect on the induction or repression of the asi promoter (data not shown). The Em and the a-amy1 promoters, used as controls in the experiment, were induced by ABA and GA respectively (Figure 3b).

Constitutive expression of gfp by the ubiquitin promoter was higher than the promoter (Figure 3b).

Using a transient expression system we demonstrated that the1033-asi promoter and its deletions directed low level of gfp expression in mature aleurone tissue, and ABA or GA had no effect on the reporter gene expression (Figure 3 The Em and the a-amy1 promoters, used as control, were substantially induced to expected levels by ABA (Jacobsen, J.V. and Beach, L.

1985. Nature 316:275-277) and GA (Gubler et al., 1995. Plant Cell 7: 1879- 1891; Jacobsen, J.V. and Beach, L. 1985. Nature 316:275-277)] respectively (Figure 3b). Using the construct pubi.gfp.655asi3'downstream, cells of mature aleurone transiently expressing GFP protein were detected indicating that the 655 bp 3'-downstream region contained signals for termination of transcription.

Furthermore, the transient expression assay demonstrated that the 1033-asi promoter and its deletions do not direct gfp expression in leaf tissue of barley (Figure However, the asi promoter directed low level of transient gfp expression in the mature imbibed aleurone tissue, and observably stronger transient gfp expression in pericarp tissue of immature barley seeds (data not shown).

WO 2004/035790 PCT/AU2003/001381 71 EXAMPLE 3 Production and analysis of transgenic barley stably expressing gfp controlled by the barley asi gene promoter 3.1 Methods 3.1.1 Production of transgenic constructs for the expression of gfp controlled by the barley asi promoter The construct pEvec202N nos (SEQ ID NO: 41) was used to prepare all the constructs used for transformation of barley. Transcriptional fusions between the 1033-asi promoter, the D-hordein (Cho etal., 1999. Theor. Applied Genetics 98: 1253-1262) and the ubiquitin (Christensen and Quail 1996) promoter sequences, and the gfp sequence were generated by PCR (Mike, J.Y. 1989. Nucleic Acids Research 17: 4895) using Pfx polymerase (Life Technologies, USA). The promoter-gfp fragments were directionally cloned into the Hind 111 and Apa I sites of the pEvec202Nnos construct. The pEvec202Nnos construct was prepared by directional cloning of the Not I-Apa I-Hind Ill-Kpn 1-nos terminator-Xba I fragment into the Not I and Xba I sites of the pMNLig101N construct, which is a modification of the pWBVec8 construct (Wang et al., 1998. Acta Horticulturae 461: 401-407) containing the flipped Eco RI-CaMV35S-hph-nos-Eco RI fragment.

3.1.2 Transformation of barley Transgenic barley was generated by Agrobacterium-mediated transformation of embryogenic callus essentially according to a modified form of a published method (Tingay et al., 1997. Plant Journal 11: 1369-1376). We adapted our own modification at the step of Agrobacterium inoculation.

Following removal of the embryo axis, the immature embryos (barley c.v.

Golden promise) were placed (cut side up) in the centre of a circular filter paper (Wattman No.1) kept on solid BCI-DM (5 mg/L dicamba) solid medium.

Agrobacterium tumefaciens AGLO (Lazo et al., 1991. Bio/Technology 9:963- 967), transformed with various plasmids, was grown overnight in MGL medium WO 2004/035790 PCT/AU2003/001381 72 (Garfinkel, M. 1980. J. Bacteriology 144: 732-743) and used for inoculation. An Eppendorf pipette was used to place drops of the Agrobacterium culture on the cut side of the immature embryos. After incubation of the plates in the dark at 24" C, the embryos were transferred into plates containing BCI-DM medium containing 50 mg/L Hygromycin and 150 mg/L Timentin. After six weeks of dark incubation, with transfers in fresh medium every two weeks, the callus produced was transferred to FHG medium containing 20-mg/L hygromycin.

Regenerated shoots were transferred into BCI medium for development of roots before transferring in soil.

3.1.3 PCR and Southern blot screening of transgenic barley.

PCR screening of transgenic plants was carried out using purified genomic DNA. All hygromycin resistant plants were screened for the gfp-nos sequence using the primers SGFPF1 and NOSR1 to generate a 767 bp fragment. PCR reaction for 25 cycles commenced by denaturing at 94° C for s, followed by annealing at 50" C for 45 s, followed by extension at 72' C for 1 min.

Southern blot hybridisation was carried out essentially according to established procedures (Maniatis and Fritsch 1982. Molecular Cloning: A Laboratory Manual. Cold Spring Harbour Laboratory Press). Genomic DNA from non-transformed or transformed plants was digested with Hind III and checked for digestion before resolving on a 0.7% agarose gel, followed by transfer onto a nylon membrane (Nylon-hybond, Roche, Germany).

Hybridisation was carried out using Dig-labelled probe (720 bp) corresponding to the gfp gene and the Dig-detection system (Roche, Germany).

3.1.4 Fluorescence Microscopy for Detection of GFP Fluorescence.

Detection of GFP fluorescence was carried out using a compound microscope equipped with an attachment for fluorescence observations (model E600, Nikon, Japan). Observations and image-captures were carried out at 2X, WO 2004/035790 PCT/AU2003/001381 73 4X or 10X magnification using Plan-Flor objectives. White light was provided by a super high-pressure mercury lamp (100 and an appropriate filter set was used (450 490 nm band pass excitation filter, a dichroic mirror of 505 nm and a 520 nm emission filter) for observations under blue light.

3.1.5 Image Capture Image capture was carried using a microscope mounted CCD colour camera (Cool-SNAP-Pro, Media Cybernetics, USA) controlled by it's dedicated software (Image-Pro Plus, version 5.1, Media Cybernetics, USA). All images o1 were captured in colour images at a resolution of 1392 x 1040.

3.2 Results 3.2.1 Analysis of transgenic plants for transgene integration Six hygromycin-resistant barley plants, identified as transformed by PCR analysis, were investigated by Southern blot analysis to determine for transgene copy number and integration events. Four plants were confirmed to contain the gfp transgene with single or multiple copies (data not shown). In addition the Southern blot data indicated that these four plants were generated from two independently transformed callus lines (data not shown).

3.2.2 Analysis of transgenic plants for 1033-asi promoter-directed gfp expression Different tissues of barley, transformed with the 1033-asi.gfp.nos, Dhordein.gfp.nos and the ubi.gfp.nos transgenes, were observed for expression of GFP protein. Unlike the ubiquitin promoter, the 1033-asi and D-hordein promoters do not direct gfp expression in the leaf (Figure the root tissue and anthers (data not shown) of transgenic barley. In developing seeds the 1033asi promoter directs gfp expression in the pericarp tissue (Figure 5a and b), and abundant expression in tissues comprising the crease or groove of the seed (Figure 5c). Transverse and longitudinal sections of the seed through the crease indicate that gfp expression in different tissue including the pericarp WO 2004/035790 PCT/AU2003/001381 74 vascular tissue nucellar projection cells (NP) and endosperm transfer cells (ET) (Figure 5d, e and Expression of GFP in vascular tissue, nucellar projection cells and the nucellar epidermis was detected as early as 10 DPA and in the pericarp as early as 12 DPA, and expression continued even at 28 DPA (Figure Expression of GFP was not detected in the testa, aleurone and the endosperm (Figure 5) tissues of developing barley transformed with the 1033-asi.gfp.no gene. The D-hordein promoter directs gfp expression only in the developing endosperm and aleurone tissue of transgenic barley (Figure 3.2.3 Activity of the 1033-asi promoter in germinating seeds In germinating seeds of barley transformed with the 1033-asi promoter, gfp expression was observed in the entire germinating embryo (Figure 7a and b) and also in that region of the aleurone (sub-aleurone) adjoining the embryo (Figure 6c). Expression of gfp was observed in the coleoptile and coleorhiza and the root tip.

3.2.4 Activity of the 1033-asi promoter in de-embryonated germinating seeds In de-embryonated seeds of barley, the 1033-asi promoter-directed gfp expression was observed in aleurone tissue when imbibed with buffer containing GA (Figure 7d to but not with ABA or buffer or water. GA induced expression of gfp was observed after 24 h of incubation and increased progressively even till 72 h. In de-embryonated seeds of transgenic barley transformed with the Em.gfp.nos gene, gfp expression was observed in the aleurone tissue when imbibed with buffer containingABA (Figure 7g and h) but not with buffer or water.

3.3 Discussion In developing seeds of transgenic barley, the 1033-asi-promoter directed gfp gene expression in the pericarp nucellar projection cells (NP), endosperm transfer cells (ET) and the vascular tissue (VT) (Figure Expression of GFP was detected in VT, NP and the ET as early as 10 DPA and WO 2004/035790 PCT/AU2003/001381 in the pericarp as early as 12 DPA, and this expression continued even at 28 DPA (Figure However, gfp expression was not detected in the starchy endosperm and the aleurone cells (Figure 5 g and The D-hordein promoter directed high-level gfp expression in the aleurone and endosperm of developing barley grain (Figure Our results are in agreement with reports that the asi mRNA is expressed in developing endosperm but not the aleurone (Leah, R. and Mundy, J. 1989 Plant Molecular Biology 12: 673-682; Mundy et al., 1986. Plant Physiology 81: 630-636). Aleurone tissue preparation in these early studies to 30 DPA) excluded the pericarp from the assay by peeling, and further separation was achieved during the process of washing the aleurone (Mundy et al., 1986. Plant Physiology 81: 630-636). In addition, no mention was made in the prior art as to whether or not the crease was included or excluded with the aleurone tissue for detection of asi mRNA. However, our studies indicate that activity of the 1033-asi promoter was detected in specific tissues or cell types of developing barley grain. The lack of 1033-asi promoter activity in the endosperm and embryo in our experiments seem to be in contrast to the cDNA array experiment described by Sreenivasulu et al. (2002) Molecular Genetics and Genomics 266: 758-767, where asi mRNA was detected in the embryo sac, which consists of the embryo and the endosperm. However, Sreenivasulu et al. (2002) Molecular Genetics and Genomics 266: 758-767, have indicated that the embryo-sac tissue preparation had adhering vascular tissue and nucellar projection cells, both of which were difficult to separate from the embryo sac tissue. In our study, observably high gfp expression was detected in the crease of the grain, which is comprised of the vascular tissue, the nucellar projection cells and epidermal transfer cells (Figure 5f). Thus, the detection of the asi mRNA in the embryo sac (Molecular Genetics and Genomics 266: 758-767 vasulu et al., 2002), could have been essentially due to the contaminating vascular and nucellar projection cells.

WO 2004/035790 PCT/AU2003/001381 76 In mature aleurone tissue of de-embryonated transgenic barley seeds, our results indicate that the 1033- asi promoter is induced by GA but not by ABA (Figure 6 In the presence of GA, gfp expression was observed after 24 h in aleurone cells adjoining the cut end of the mature de-embryonated seed, and could be at the posttranscriptional level, as is the case in immature barley embryos (Liu, J.H. and Hill, 1995. Plant Molecular Biology 29: 1087-1091).

By virtue of inhibiting AMY2, research on asi gene has been focused on studying its expression in the seed, specifically in the aleurone and endosperm this expression progressively spread throughout the aleurone layer by 72 h of incubation (Figure 6 d-f).

Immature grain tissue was also incubated in ABA to study if the 1033-asi promoter was induced by ABA. Our studies (Figure 6h and i) indicate that ABA had no effect on the induction of gfp expression in the pericarp, endosperm and other tissues of the developing grain (28 DAF).

From this study we conclude that in the immature barley grain, the 1033asi promoter is not induced by ABA and hence asi gene expression is not induced by ABA at the transcriptional level. We also conclude that in the developing barley grain, activity of the 1033-asi promoter is specific to the pericarp, modified nucellar and endosperm cells and the vascular tissue, but not the aleurone and endosperm.

WO 2004/035790 PCT/AU2003/001381 77 EXAMPLE 4 Production of transgenic wheat stably expressing gfp controlled by the barley asi gene promoter 4.1 Preparation of plant material The 129 'Bobwhite' accessions were grown in a greenhouse under controlled conditions with day temperatures of 24-28 0 C and night temperatures of 15-180C. To ensure continuous production of immature embryos, seeds of each variety were planted every 2-3 days. Fifteen days after heading, the immature seeds were harvested, sterilized, and a minimum of 200 immature embryos/accession were isolated for bombardment.

4.2 DNA plasmid vector and preparation of the microcarrier Plasmid DNA pA57 (SEQ ID NO: 40; Example was engineered to contain the selectable bar gene that confers resistance to the herbicide Basta (Hoechst, Frankfurt, Germany) under the control of the maize ubiquitin promoter (Christensen et al., Transgenic Res., 5: 213-218, 1996). The resultant plasmid is designated pUbi-bar::A57. Five micrograms of plasmid DNA were precipitated onto gold particles following the protocol described by Pellegrineschi et al (2000). For each bombardment, 10 pL of microparticle DNA were placed onto the macrocarrier. Bombardments were conducted at a distance of 5 cm from the stopping plate using a PDS 1000/He microprojectile gun (Bio-Rad, Hercules, Calif.) with 900 psi (1 psi 6.895 kPa), according to the procedure described by Pellegrineschi et al (2000).

4.3 Culture conditions and recovery of transformed plants A minimum of 200 freshly isolated embryos (between 0.7 and 1.2 mm long) from each accession were isolated and the zygotic meristem then removed. 200 isolated scutella per petri dish were placed on osmotic basal salt medium (Murashige and Skoog 1962) without modifications other than the addition of 15% w/v maltose for 4 h prior to bombardment. As a control for each WO 2004/035790 PCT/AU2003/001381 78 experiment, 200 additional scutella from the same accession were transferred to the osmotic medium without bombardment. The day after bombardment, the embryos were placed on MS medium (Murashige and Skoog 1962) containing mg 2,4-dichlorophenoxyacetic acid 30 g sucrose/L, and 8 g Bacto-Agar for somatic embryo induction. Between 12 and 14 days after transfer to the induction medium the induced embryos were scored for the somatic embryo formation. The induced explants were then transferred in MS medium containing 5 mg PPT/L (DL-phosphinothricin; Sigma, St. Louis, MO) to a growth chamber with a photoperiod of 16 h light 8 h dark at 25 0 C for selection. The light was provided by white cool florescent tubes (Solar) and the light intensity was 200 MJ/m 2 s. After 30 days, healthy, fully differentiated plantlets were scored and transferred to the same medium for further selection during development. Surviving green-rooted plantlets were transferred to a soil mixture and placed in the growth room. Herbicide resistance of the putative transgenic wheat plants was determined by spraying the leaves of plants at the fifth or sixth leaf stage twice with Basta w/v) with 7 days between applications to miminize escapes. Plants were scored as susceptible or resistant according to the degree of leaf desiccation after 7 days.

4.4 Recovery and analysis of T1 generation seed Putatively transgenic plants 102 lines were grown to maturity under the same conditions and seeds were harvested.

GFP expression in seeds through development is monitored using flurosecence microscopy, analogously as was carried out for barley, to identify the pattern of expression from, in particular, 7 days post-flowering through to seed maturity. The pattern of expression confirms the pericarp specificity already found in barley.

WO 2004/035790 PCT/AU2003/001381 79 EXAMPLE Use of the barley ASI promoter to confer protection against head scab in wheat.

5.1 Production of wheat stably expressing a thaumatin-like gene under control of the barley ASI promoter Plasmid DNA pUbi-bar::A57 (Example 4.2) is engineered to replace the gfp encoding nucleic acid with a nucleic acid encoding a thaumatin-like protein (SEQ ID NO: 15, 17, 19, 21, 23 or 25) obtained essentially as described by Kuwabara et al., Physiol. Plantarum 115, 101-110, 2002; Mingeot, Theor. Appl.

Genet., 95, 822-827, 1998; Dudlet Plant Mol.Biol.17, 283-285, 1991.

Thaumatin-like proteins are stress response proteins that are particularly effective in the treatment of plant pathogens, as they are capable of inhibiting the infection of the plant by such a pathogen.

The resultant plasmids are designated pUbi-bar::AT 1 N (comprising the thaumatin-like gene represented by SEQ ID NO: 15), pUbi-bar::AT 2

N

(comprising the thaumatin-like gene represented by SEQ ID NO: 17), pUbibar::AT 3 N (comprising the thaumatin-like gene represented by SEQ ID NO: 19), pUbi-bar::AT 4 N (comprising the thaumatin-like gene represented by SEQ ID NO: 21), pUbi-bar::AT 5 N (comprising the thaumatin-like gene represented by SEQ ID NO: 23), pUbi-bar::AT 6 N (comprising the thaumatin-like gene represented by SEQ ID NO: Each of these plasmids is then used to transform wheat essentially as described in Examples 4.2 and 4.3.

5.2 Production of wheat stably expressing RPL3:Cys258 protein Plasmid DNA pUbi-bar::A57 (as described in Example 4.2) is engineered to replace the gfp encoding nucleic acid with a nucleic acid encoding a RPL3:Cys258 protein (SEQ ID NO: 27). The resultant plasmid is designated pUbi-bar::AR 1

N.

WO 2004/035790 PCT/AU2003/001381 The RPL3:Cys258 protein is a modified form the ribosomal protein L3, ie the target of the tricothecene deoxynivalenol produced by head scab producing fungi. However the RPL3:Cys258 protein has been mutated at amino acid residue 258 to introduce an amino acid base change (W258C) that reduced or inhibits the ability of DON to bind to the protein while not affecting the ribosomal activity of the protein. Accordingly, the maternal tissue, in particular the pericarp, of a plant expressing the RPL3:Cys258 protein is effectively resistant to DON.

The pUbi-bar::AR 1 N plasmid is then used to transform wheat essentially as described in Examples 4.2 and 4.3.

5.3 Production of wheat stably expressing tricothecene Plasmid DNA pUbi-bar::A57 (as described in Example 4.2) is engineered to replace the gfp encoding nucleic acid with a nucleic acid encoding a tricothecene 3-0-acetyltransferase (SEQ ID NO: 29). The resultant plasmid is designated pUbi-bar::ATacN.

Tricothecene 3-0-acetyltransferase is a protein that converts tricothecenes to a non-toxic product.

The pUbi-bar::ATaclN plasmid is then used to transform wheat essentially as described in Examples 4.2 and 4.3.

5.4 Assay for wheat-scab resistance Seedlings of transformed wheat previously described (Example 5.1-5.3) are grown in air-steam pasteurized (60 0 C. for 30 minutes) potting mix (Terra-lite Rediearth, W. R. Grace, Cambridge, Mass.) in a growth chamber at 250 C, 14 h light/day for approximately 8 weeks prior to use in bioassays. Conidial inoculum of Fusarium graminearum isolate Z3639 are produced on clarified V-8 juice WO 2004/035790 PCT/AU2003/001381 81 agar at 250 C, 12 h light/day for 7 days while biomass of each strain of microorganism is produced on TSA/5 by inoculating plates and incubating at 250C for 48 h. Conidia of F. graminearum 3639 are used to inoculate the middle floret of two wheat heads per microbial strain. Inoculated wheat plants are placed in a clear plastic enclosure on greenhouse benches for 72 h to promote high relative humidity. The enclosure is then removed and wheat heads are scored for visual symptoms of Fusarium head blight 16 days after inoculation. Those that show no sign of Fusarium head blight are considered to express a protein that confers protection against head scab.

Wheat heads from plants expressing thaumatin-like proteins in the pericarp exhibit reduced head blight compared to non-transformed lines that are isogenic except for the transgene.

5.5 Greenhouse Assays of resistance to head scab Transformed and wild-type seedlings are grown two to a pot in pasteurized potting mix in a growth chamber for 8 weeks as described above.

Conidia of F. graminearum isolates Z3639, DOAM, and Fg-9-96 are produced on CV-8 agar as described above. After 8 weeks, wheat plants are transferred to greenhouse benches for approximately 1 week. At the onset of wheat head flowering, generally by the end of 1 week on greenhouse benches, biocontrol bioassays are initiated. The middle floret of a wheat head is inoculated with F.

graminearum. Inoculated wheat plants are then placed in a plastic enclosure on greenhouse benches for 72 h to promote high relative humidity and free moisture necessary for optimal Fusarium head blight development. Sixteen days after inoculation, wheat heads are scored for disease severity on a 0 to 100% bleached wheat head scale [Stack et al., (1995) North Dakota State University Extension Service Bulletin PP-1095], and a 0 to 100% disease incidence scale. Kernel weights are determined after heads have matured.

Fully developed kernels in healthy heads have high 100 kernel weights, while shriveled kernels in heads infected by F. graminearum have lower 100 kernel WO 2004/035790 PCT/AU2003/001381 82 weights. F. graminearum is recovered from randomly selected heads showing symptoms of disease development.

Wheat heads from plants expressing thaumatin-like proteins in the pericarp exhibit reduced head blight compared to non-transformed lines that are isogenic except for the transgene.

5.6 DON growth assay Plants expressing transgene constructs that confer resistance to the tricothecene, deoxynivalenol (DON) (ie as described in Examples 5.2 and 5.3) are further assayed to determine their ability to grow in the presence of DON.

5.6.1 Protoplast Isolation and Culture Seed harvested from transgenic wheat are surface sterilized in Javex solution.

Protoplasts are isolated from pericarp cells following the lead mesophyl described by Sproule et al. (1991, Theor. Appl. Genet. 82:450-456). Briefly, an enzyme solution of 1% cellulase R-10 and macerozyme R-10 in 0.45M mannitol salt solution is filter sterilized and aliquoted to sterile petri dishes.

Pericarps are excised and floated abaxial side down over the enzyme solution.

Petri dishes are sealed, and incubated in a humid box in a dark growth chamber at 280 C for 17 hrs with gentle agitation. The liberated protoplasts are separated from tissue debris by filtration through a sterile 88pm mesh nylon funnel. The protoplast-enzyme solution is aliquoted into round-bottom sterile glass test tubes and centrifuged. Isolated protoplasts are separated from cellular debris by flotation on the surface of 0.6M sucrose solution with an overlay of 0.5 ml of SCM (0.45M sorbitol, 10 pg/ml CaCI 2

H

2 0, 5 pg/ml MES morpholinoethane sulfonic acid; pH Purified protoplasts are recovered from the SCM interface with a pipette. Protoplasts are suspended in liquid NT WO 2004/035790 PCT/AU2003/001381 83 medium (Nagata and Takebe, 1991, Planta 99: 12-20) containing 0.4M glucose as osmoticum.

A stock solution of DON, produced according to the method of Greenhalgh et al. (1986, J. Agric. Food Chem. 34: 98-102) is used to adjust the concentration of DON toxin in some protoplast cultures to either 0, 0.1, 1.0, or 10.0 ppm. All protoplast cultures are incubated at 280 C in darkness. After one week of culture, the osmotic concentration of the medium is adjusted by the addition of 0.5 ml of NT medium containing 0.3M glucose, and the protoplast cultures are moved to low light (10 iE m 2 sec 1 at 250 C.

Wild-type non-transgenic protoplasts incubated in the presence of DON show a reduced the ability to reform cell walls, reduced division frequency (mitotic index of the cells), and reduced plating efficiency (number of micro colonies formed) of protoplasts relative to those cultured in the absence of DON, or transgenic protoplasts expessing tricothecene and/or or a modified RPL3:Cys258 protein when cultured in the presence of

DON.

Protoplasts are also cultured over 2 ml agarose underlayers w/v) inside sterile petri dishes. The agarose underlayers contain either 0, 0.1, or 25 ppm DON. Protoplasts in these cultures are suspended in liquid NT medium and cultured as in Sproule et al. (1991, Theor. Appl. Genet. 82:450- 456). When protoplasts are cultured on medium supplemented with DON, noticeable differences are observed in micro-colony formation (cell colonies from isolated protoplasts), for transofrmed and non-transformed lines consistent with enhanced resistence in lines expressing trichothecene acetyltransferase and/or modified RPL3:Cys258.

WO 2004/035790 PCT/AU2003/001381 84 5.6.2 Cell Suspension Cultures Cell suspension cultures from primary transgenic or wild-type wheat plants are initiated from leaf callus cultures. Calli are ground in a sterile blender, and the homogenized tissue used to inoculate liquid MS medium containing 2 pg/ml 2,4-D in a sterile erlenmeyer flask. Cell suspensions are maintained on an orbital shaker under a 16 hr day length at 250 C with a weekly sub-culture of fresh medium.

5.6.3 DON growth assays Growth measurements of cell suspensions of wild-type and transgenic wheat are taken after the cultures have equilibrated in growth conditions for at least 12 weeks. The measure of weight gain is determined by plating 1 ml of finely filtered cell suspensions on sterile HA filters inside sterile petri dishes containing 2 ml of liquid MS medium with 2 pg/ml 2,4-D, supplemented with either 0, 10, 25 or 50 pg/ml DON. At 5 day intervals the fresh weight of each filter unit is determined under aseptic conditions and then the cells are recultured on the same medium with fresh DON added. Leaf explants of all genotypes are evaluated for the ability to regenerate shoots on shoot regeneration medium (MS medium containing 3% sucrose, 1 pg/ml benzylaminopurine, 0.1 [pg/ml naphthalene acetic acid, 0.8% agar) supplemented with 0, 5, or 10 .ig/ml DON.

EXAMPLE 6 Use of the barley ASI promoter to confer resistance against Barley mosaic stripe virus 6.1 Production of barley stably expressing the BSMV coat protein Plasmid DNA pEvec202N nos (as described in Example 2.1.1) is engineered to replace the gfp encoding nucleic acid with a nucleic acid WO 2004/035790 PCT/AU2003/001381 encoding a BSMV coat protein (SEQ ID NO: 31). The resultant plasmid is designated pEvec::BSMV nos.

pEvec::BSMV nos plasmid is then used to transform barley essentially as described in Example 3.1.2.

6.2 Production of barley stably expressing siRNA to inhibit expression of the BSMV movement protein Plasmid DNA pEvec202N nos (as described in Example 2.1.1) is engineered to replace the gfp encoding nucleic acid with a nucleic acid encoding a siRNA derived from a movement protein-encoding region of SEQ ID NO: 31. The resultant plasmid is designated pEvec::siBSMV nos. The expression of this construct causes the mRNA encoding the BSMV movement protein to be cleaved, thereby silencing this gene and inhibiting the ability of the virus to replicate.

The pEvec::siBSMV nos plasmid is then used to transform barley essentially as described in Example 3.1.2.

6.3 Assay to determine resistance to BSMV Seeds are isolated from transgenic barley plants produced in Example 6.2 and/or 6.3 and wild-type (untransformed) barley plants. Seeds are mechanically inoculated with a solution comprising BSMV. Seeds are then planted and wild-type and transgenic seedlings grown in a growth chamber.

Following sufficient growth to allow leaf formation and leaves observed for visual symptoms of BSMV infection, such as, for example, leaf yellowing, leaf malformation and leaf curling. Furthermore, tissue from the plants are assayed for expression of the BSMV coat protein using a sandwich ELISA essentially as described by Savenkov, E. and J. P. Valkonen Virology 283:285-293, 2001.

WO 2004/035790 PCT/AU2003/001381 86 Provided that wild-type plants develop symptoms of BSMV infection and show expression of BSMV coat protein, it is presumed that those transgenic plants that do not demonstrate such symptoms are resistant to this pathogen.

EXAMPLE 7 Use of the ASI promoter to produce an oral vaccine against transmissible gastroenteritis virus (TGEV).

7.1 Production of wheat stably expressing TGEV spike E2 protein Plasmid DNA pUbi-bar::A57 (as described in Example 4.2) is engineered to replace the gfp encoding nucleic acid with the nucleic acid encoding the TGEV spike E2 protein represented by SEQ ID NO: 35. The resultant plasmid is designated pUbi-bar::ATGEVN. The TGEV spike E2 protein is essential for the replication of TGEV. Accordingly, an immune response against such a protein will protect a subject from TGEV infection.

The pUbi-bar::ATGEVN plasmid is then used to transform wheat essentially as described in Examples 4.2 and 4.3.

7.2 Immunization of Pigs Against TGEV Virus Transmissible Gastroenteritis Virus (TGEV) causes an acute and fatal enteric disease in newborn piglets. In adult pigs, the infection with the virus is characterized by anorexia, dehydration, severe diarrhea followed by death.

Pigs at 5-7 days old are fed wheat which includes the TGEV spike E2 protein in order to immunize and protect the pigs from enteric disease and symptoms caused by TGEV.

Transgenic wheat plants carrying an expression cassette comprising a DNA sequence coding for TGEV (E2) spike protein are produced as described WO 2004/035790 PCT/AU2003/001381 87 in Example 7.1. The levels of expression of the TGEV (E2) spike protein in the pericarp is assessed using quantitative western blots with monoclonal antibodies to the TGEV (E2) spike protein. Following determining the level of expression of the TGEV (E2) spike protein in the pericarp, the amount of transgenic plant material to be administered to the animal to achieve doses in the range of 0.01 to 50 mg/kg is determined.

A standard dose response immunization schedule is employed to determine the optimal dosages for oral immunization to induce protection against TGE virus. Groups of pigs 5-7 days old are fed different doses such as 0.1, 1.0, 5.0, and 25.0 mg/kg of the TGEV (E2) spike protein daily for 5 days.

The development of protective immunity in the pigs is evaluated by examining the pigs for the development of neutralizing antibodies and/or IgA antibodies to TGEV (E2) spike protein. Immunized pigs are challenged with the TGE virus and the level of infection and symptoms such as diarrhea or death are monitored.

EXAMPLE 8 Use of the barley ASI promoter to express sulfur-rich brazil nut protein 8.1 Production of wheat stably expressing sulfur-rich brazil nut protein Plasmid DNA pUbi-bar::A57 (as described in Example 4.2) is engineered to replace the gfp encoding nucleic acid with the nucleic acid encoding the sulphur-rich brazil nut protein represented by SEQ ID NO: 37. The resultant plasmid is designated pUbi-bar::ASN. This protein is enriched in sulfur, and, as a consequence, wheat expressing this protein is useful for sulphur supplementation.

The pUbi-bar::ASN plasmid is then used to transform wheat essentially as described in Examples 4.2 and 4.3.

00 88 c The wheat produced is then useful as feed for livestock, and, in particular )j sheep to enhance the sulphur content and thereby quality of the wool.

00o 0Any description of prior art documents herein is not an admission that the documents form part of the common general knowledge of the relevant art in Australia.

.(N

Claims (14)

1. A method of expressing nucleic acid or protein in the maternal tissue of a plant 00 0seed comprising expressing isolated nucleic acid operably under the control of a bifunctional a-amylase subtilisin inhibitor (ASI) gene promoter that is operable in the maternal tissue. S2. The method of claim 1 wherein the maternal tissue comprises a seed tissue Sselected from the group consisting of pericarp, vascular tissue, nucellar projection cells t'q and endosperm transfer cells.

3. The method of claim 1 wherein the ASI gene promoter is from a cereal plant.

4. The method of claim 3 wherein the cereal plant is selected from the group consisting of rice, wheat, barley, sorghum, maize, millet, rye and oats. The method of claim 1 wherein the ASI gene promoter comprises a nucleotide sequence selected from the group consisting of: the sequence set forth in SEQ ID NO: 1; the sequence set forth in SEQ ID NO: 2; a sequence of a fragment of (a) or that is operable in the maternal tissue of a plant seed; a sequence that in its native context regulates the expression of a protein-encoding region of an ASI gene of a plant wherein said sequence is operable in the maternal tissue of a seed of the plant.

6. The method of claim 1 wherein the expressed nucleic acid operably under the control of the ASI promoter comprises a structural gene that encodes a polypeptide.

7. The method of claim 6 wherein the structural gene comprises or consists of a reporter gene.

8. The method of claim 6 wherein the structural gene is selected from the group consisting of: a structural gene which encodes a protein that confers or enhances protection against a plant pathogen; (ii) a structural gene encoding an immunogenic protein; (iii) a structural gene enjcoding a sulphur rich protein; (iv) a structural gene encoding a calcium-binding protein; 00 YU a structural gene encoding an iron-binding protein. e(

9. The method of claim 8 wherein the plant pathogen is a seed-borne fungus, or a 00 seed- borne virus The method of claim 8 wherein the immunogenic protein is a protein from a pathogen of a human or animal. (11. The method of claim 6 wherein nucleic acid operably under the control of the ASI promoter consists of a structural gene encoding a biosynthetic enzyme that is required C for the production of an osmoprotectant, a fatty acid, a phytosterol, an anthocyanin, lignin, an anti-nutritional protein, an enzyme capable of altering a substrate in the phenylpropanoid pathway, a choline metabolizing enzyme capable of acting upon choline to modify the use of choline by other enzymes in the phenylpropanoid pathway, an enzyme involved in the malting process, an enzyme capable of acting upon a sugar alcohol, or an enzyme capable of acting upon myo-inositol.

12. The method of claim 1 wherein the expressed nucleic acid operably under the control of the ASI promoter comprises nucleic acid encoding inhibitory RNA, an antisense molecule, ribozyme, abzyme, co-suppression molecule, gene-silencing molecule or gene-targeting molecule.

13. The method of claim 12 wherein the nucleic acid operably under the control of the ASI promoter targets expression in the plant of a gene of a plant pathogen that is required for infection the plant by the pathogen or transmission of the pathogen in the plant.

14. The method of claim 1 further comprising introducing to a cell, tissue or organ of a plant an isolated nucleic acid operably under the control of a bifunctional a-amylase subtilisin inhibitor (ASI) gene promoter that is operable in the maternal tissue. The method of claim 14 further comprising introducing to a cell, tissue or organ of a plant the isolated nucleic acid operably under the control of the ASI gene promoter.

16. The method of claim 15 further comprising regenerating a whole plant from the cell, tissue or organ and growing the whole plant for a time and under conditions 00 91 Ssufficient for seed to be produced that express the isolated nucleic acid operably under e( d the control of the ASI gene promoter. 00

17. The method of claim 14 wherein the plant is a monocotyledonous plant.

18. The method of claim 17 wherein the monocotyledonous plant is selected from the group consisting of wheat, oats, maize, barley, rice, sorghum, millet and rye.

19. The method of claim 18 wherein the plant is barley or wheat. The method of expressing nucleic acid or protein in the maternal tissue of a plant seed according to any one of claims 1 to 19, substantially as herein before described with reference to the Examples and/or Figures. DATED this FOURTH day SEPTEMBER of 2008 Molecular Plant Breeding Nominees Ltd, Grains Research and Development Corporation. Patent Attorneys for the Applicant: F.B. RICE CO.