BACKGROUND OF THE INVENTION

The present invention relates to a polygenic DNA construct consisting of three abiotic stress tolerance genes, separated by nucleic acids encoding FMDV 2A peptides under the controi of a stress inducible promoter. The stable insertion of the construct into plants confers drought tolerance to the plants. The invention also provides for vectors, host ceils, transgenic plants and transgenic seeds containing the construct.

The invention specifically relates to a polygenic DNA construct consisting of three genes from the plant Xerophyta viscosa controlled by a stress inducible promoter. The stable insertion of the construct into plants has shown that transgenic plants containing the construct are more drought tolerant compared to the wild type plants which do not contain the construct. Plant promoters play an important role in the process of plant gene expression and regulation. The use of constitutive promoters to drive gene expression in transgenic plants often results in stunted growth and reduction of yield. Accordingly, in order to prevent over-expression of genes of interest inducible promoters have significant advantages. Proteins expressed under the control of stress inducible promoters are only expressed when the plant is exposed to a stress.

Abiotic stresses include inter alia drought, salinity, cold and extreme temperatures. Drought has been the major cause of crop losses in agriculture. It is widely known that genes act together rather than in isolation in order to counteract the effects of dehydration due to water deficit in a plant. A need therefore exists for a number of genes to be switched on by a stress inducible promoter to counteract the effects of abiotic stress.

The stacking of multiple genes in plants has become an increasing area of study of modern plant research and biotechnology. Several methods have been used to stack genes into various plant genomes and then coordinate their expression. However, many of these strategies are unreliable because of the co-expression of the heterologous proteins.

The use of a self-processing viral 2A peptide bridge such as the Foot and Mouth Disease Virus 2A (FMDV 2A) polyprotein manipulates the ribosome to skip the synthesis of the glycyl-prolyl peptide bond at its C terminus ieading to the release of the nascent protein and allowing translation of the downstream sequence. The FMDV 2A oligopeptide is only 23 amino acids (aa) long. The FMDV 2A oligopeptide comprises the following amino acid sequence GSGQLLNFDLLKLAGDVESNPGP (SEQ ID NO: 12) and has a co-translational cleavage at its C terminus and a post trans!ational cleavage at its N terminus mediated by the virus encoded proteinase.

SUMMARY OF THE INVENTION

The present invention relates to a polygenic DNA construct consisting of three abiotic stress tolerance genes, separated by nucleic acids encoding FMDV 2A peptides under the control of a stress inducible promoter. The stable insertion of the construct into plants confers drought tolerance to the plants. The invention also provides for vectors, host cells, transgenic plants and transgenic seeds containing the construct.

According to a first aspect of the invention there is provided for a recombinant nucleic acid molecule comprising a polygenic nucleic acid construct operably linked to an abiotic stress inducible promoter. The polygenic nucleic acid construct comprises: a nucleotide sequence encoding a Xvsapi poiypeptide, a nucleotide sequence encoding a first 2A element peptide, a nucleotide sequence encoding a XvAld polypeptide, a nucleotide sequence encoding a second 2A element peptide, a nucleotide sequence encoding a XvPrx2 polypeptide, and a terminator.

In one embodiment of the invention the first and second 2A element peptides are Foot-and-Mouth disease virus 2A element peptides.

In another embodiment of the invention the abiotic stress may be selected from the group consisting of osmotic stress, dehydration stress, temperature stress, drought, salinity and desiccation.

In a second aspect of the invention the polygenic nucleic acid construct comprises a nucleotide sequence encoding a first polypeptide of interest, a nucleotide sequence encoding a first 2A element peptide, nucleotide sequence encoding a second polypeptide of interest, a nucleotide sequence encoding a second 2A element peptide, a nucleotide sequence encoding a third poiypeptide of interest, and a terminator. In this aspect of the invention the abiotic stress inducible promoter comprises a sequence of SEQ ID NO:2.

In one embodiment of the invention the polygenic nucleic acid construct comprises a sequence of SEQ ID NO.1.

In another embodiment of the invention the first, second and third polypeptides of interest are abiotic stress tolerance polypeptides. The abiotic stress tolerance peptides may be selected from the group consisting of XvSapl , XvPrx2 and XvAld.

in a third aspect of the invention there is provided for a vector comprising a recombinant nucleic acid molecule of the invention.

A forth aspect of the invention provides for a host cell transformed with the vector of the present invention. Preferably the host cell is a plant cell.

In one embodiment of the invention the host ceil is stably transformed with the recombinant nucleic acid molecule of the invention. Those of skill in the art will however appreciate that it will be possible to also transiently express the recombinant nucleic acid molecule of the invention.

In yet another aspect of the invention there is provided for a transgenic plant comprising the recombinant nucleic acid molecule of the invention or the host ceil as described herein. The transgenic plant may be selected from the group consisting of alfalfa, barley, canola, cassava, cotton, maize, oats, rye, sorghum, soybean, sunflower, sweet potato, tobacco and wheat.

The invention aiso provides for a transgenic seed comprising the recombinant nucleic acid molecule the invention.

A further aspect of the invention provides for a method of producing an abiotic stress tolerant transgenic plant, the method comprising obtaining a recombinant nucieic acid molecule as described herein and stably transforming a plant with the recombinant nucleic acid molecule.

BRIEF DESCRIPTION OF THE FIGURES

Non-limiting embodiments of the invention will now be described by way of example only and with reference to the following figures:

Figure 1 : Schematic illustration of the polygenic DNA construct in the plant transformation vector pTF101.1.

Figure 2: Photos of 4 replicates of the wild-type (upper set) and transgenic plants (lowers set; MG2), respectively. These were dehydrated for seven days and then tested for recovery by rehydrating the plants for five days.

Figure 3: The effect of dehydration on seed pod formation of WT and transgenic plants. The bars reflect a shift in pod formation over a 11 day period in mature plants.

Figure 4: The effect of dehydration on flower formation of WT and transgenic plants. The bars reflect a shift in pod formation over a 11 day period in mature plants.

SEQUENCE LISTING

The nucleic acid and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and the standard three letter abbreviations for amino acids. It will be understood by those of skill in the art that only one strand of each nucieic acid sequence is shown, but that the complementary strand is included by any reference to the displayed strand. In the accompanying sequence listing:

SEQ ID NO:1 - Complete nucleotide sequence of polygenic DNA construct (3792 bp)

SEQ ID NO;2 - Nucleotide sequence of truncated promoter PsapI D (1120 bp) SEQ ID NO:3 - Nucleotide sequence of XvSapl gene (798 bp)

SEQ ID NO:4 ~ Nucleotide sequence of XvPrx2 gene (489 bp)

SEQ ID NO:5 - Nucleotide sequence of XvAldmut2 gene (960 bp)

SEQ !D NO:6 - Nucleotide sequence of FMDV 2A region (69 bp)

SEQ !D NO:7 - Nucleotide sequence of nosT terminator (257 bp)

SEQ ID NO:8 - Nucleotide sequence of the pTF101.1 vector

SEQ ID NO:9 - Amino acid sequence of XvSapl polypeptide

SEQ ID NO:10 - Amino acid sequence of XvPrx2 polypeptide

SEQ ID NO:11 - Amino acid sequence of XvAldmut2 polypeptide

SEQ ID NO: 12 - Amino acid sequence of the FMDV 2A linker

SEQ ID NO: 13 - Nucleotide sequence of the Psap1-RB F5 oligonucleotide primer

SEQ ID NO: 14 - Nucleotide sequence of the Psap1-RB R5 oligonucleotide primer

SEQ !D NO:15 - Nucleotide sequence of the Bar i-F oligonucleotide primer SEQ ID NO:16 - Nucleotide sequence of the Bar i-R oligonucleotide primer SEQ ID NO: 17 - Nucleotide sequence of the M13F oligonucleotide primer SEQ ID NO:18 - Nucleotide sequence of the M13R oligonucleotide primer SEQ ID NO:19 - Nucleotide sequence of the XvSapl Bel F oligonucleotide primer

SEQ ID NO:20 - Nucleotide sequence of the XvPrx2 C/al R oligonucleotide primer

SEQ ID NO:21 - Nucleotide sequence of the XvSapl C/al R oligonucleotide primer

DETAILED DESCRIPTION OF THE INVENTION

The present invention will now be described more fully hereinafter with reference to the accompanying drawings, in which some, but not ail embodiments of the invention are shown.

The invention as described should not be limited to the specific embodiments disclosed and modifications and other embodiments are intended to be included within the scope of the invention. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation. As used throughout this specification and in the claims which follow, the singular forms "a", "an" and "the" include the plural form, unless the context clearly indicates otherwise.

The terminology and phraseology used herein is for the purpose of description and should not be regarded as limiting. The use of the terms "comprising", "containing", "having" and "including" and variations thereof used herein, are meant to encompass the items listed thereafter and equivaients thereof as well as additional items.

This invention relates to the production of a transgenic plant incorporating three genes separated by a nucleic acid sequence encoding the FMDV 2A peptide and regulated by a stress-inducible promoter, hereinafter referred to as the polygenic DNA construct (Figure 1 , (SEQ ID NO:1)). The FMDV 2A sequence contains a cleavage site so that upon translation each protein is expressed individually and not as one continuous fusion polypeptide. This is advantageous as a single polygenic nucleic acid construct is provided which encodes each of the three polypeptides of interest. Accordingly, each protein encoded by the polygenic DNA construct will be expressed as an individual polypeptide and functions separately and provides the desired protection to the cells upon exposure to an abiotic stress. The transgenic plant displays enhanced tolerance to drought stress due to the concerted expression of the transgenes under water limiting conditions. Furthermore, the transgenic plant does not demonstrate any adverse growth characteristics due to the fact that the promoter is stress-inducible, which ensures that the heterologous polypeptides are only produced when required.

it will be appreciated that the polynucleotides for minimal promoter (PsapI D), the stacked genes namely, XvSapl, XvPn<2, XvAtdmut2, the FMDV 2A element and the nos terminator (nosT) may be artificially synthesised.

The polynucleotide of the present invention comprises a polygenic expression cassette which includes an abiotic stress-inducible promoter, a first gene of interest, a 2A element, a second gene of interest, a second 2A element, a third gene of interest and a terminator.

The terms "nucleic acid", "nucleic acid moiecule" or "polynucleotide" encompass both ribonucleotides (RNA) and deoxyribonucieotides (DNA), including cDNA, genomic DNA, and synthetic DNA. The nucleic acid may be double-stranded or single-stranded. Where the nucleic acid is single-stranded, the nucleic acid may be the sense strand or the antisense strand. A nucleic acid moiecule may be any chain of two or more covaiently bonded nucleotides, including naturally occurrtng or non-naturally occurring nucleotides, or nucleotide analogs or derivatives. By "RNA" is meant a sequence of two or more covaiently bonded, naturally occurring or modified ribonucleotides. The term "DNA" refers to a sequence of two or more covaiently bonded, naturally occurring or modified deoxyribonucleottdes.

A "polycistronic expression cassette" as used herein refers to a polycistronic expression unit comprising nucleic acid molecules encoding more than one polypeptide of interest for the simultaneous and coordinated expression of the more than one polypeptide of interest in response to an abiotic stress. The abiotic-stress inducible promoter allows for the simultaneous transcription of the more than one gene of interest, as well as the FMDV 2A peptide. The cleavage of the more than one polypeptide of interest occurs at the 2A peptide sequence during translation.

The promoter of the polycistronic expression cassette includes signals for DNA or RNA dependent RNA polymerase binding and transcription initiation. The promoter is an abiotic stress inducible promoter. Those of skill in the art will appreciate that the activity on an inducible promoter increases or decreases in response to a signal, which in the present invention is an abiotic stress, such as, osmotic stress, dehydration stress, temperature stress, drought, salinity or desiccation.

The term "gene of interest" refers to a polynucleotide sequence, of any, length that encodes a gene product of interest, i.e. the protein, polypeptide or polynucleotide of interest. The gene of interest is a heterologous gene with respect to the host cell. The selected sequence can be a full length or a truncated gene, a fusion or tagged gene and can be a cDNA, a genomic DNA, or a DNA fragment, preferably, a cDNA.

A "protein," "peptide" or "polypeptide" is any chain of two or more amino acids, including naturally occurring or non-naturally occurring amino acids or amino acid analogues, irrespective of post-translational modification (e.g., g!ycosylation or phosphorylation).

A "host cell" refers to a cell into which the polycistronic expression cassette and its promoter are introduced. The term host cell includes both prokaryotic cells used for propagation of the construct to prepare vector stocks, and eukaryotic cells for expression of the polypeptides of interest, such as plant cells.

The term "complementary" refers to two nucleic acids molecules, e.g., DNA or RNA, which are capable of forming Watson-Crick base pairs to produce a region of double-strandedness between the two nucleic acid molecules. It will be appreciated by those of skill in the art that each nucleotide in a nucleic acid molecule need not form a matched Watson-Crick base pair with a nucleotide in an opposing complementary strand to form a duplex. One nucleic acid molecule is thus "complementary" to a second nucleic acid molecule if it hybridizes, under conditions of high stringency, with the second nucleic acid molecule. A nucleic acid molecule according to the invention includes both complementary molecules.

As used herein a "substantially identical" sequence is an amino acid or nucleotide sequence that differs from a reference sequence only by one or more conservative substitutions, or by one or more non-conservative substitutions, deletions, or insertions located at positions of the sequence that do not destroy or substantially reduce the antigenicity of one or more of the expressed polypeptides or of the polypeptides encoded by the nucleic acid molecules. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the knowledge of those with skill in the art. These include using, for instance, computer software such as ALIGN, Megaiign (DNASTAR), CLUSTALW or BLAST software. Those skilled in the art can readily determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. In one embodiment of the invention there is provided for a polypeptide or polynucleotide sequence that has at least about 80% sequence identity, at least about 90% sequence identity, or even greater sequence identity, such as about 95%, about 96%, about 97%, about 98% or about 99% sequence identity to the sequences described herein.

Alternatively, or additionally, two nucleic acid sequences may be "substantially identical" if they hybridize under high stringency conditions. The "stringency" of a hybridisation reaction is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation which depends upon probe length, washing temperature, and salt concentration. In general, longer probes required higher temperatures for proper annealing, while shorter probes require lower temperatures. Hybridisation generally depends on the ability of denatured DNA to re- anneal when complementary strands are present in an environment below their melting temperature. A typical example of such "stringent" hybridisation conditions would be hybridisation carried out for 18 hours at 65 °C with gentle shaking, a first wash for 12 min at 65 °C in Wash Buffer A (0.5% SDS; 2XSSC), and a second wash for 10 min at 65 °C in Wash Buffer B (0.1% SDS; 0.5% SSC). In some embodiments, the nucleic acid molecules of the invention are operably linked to other sequences. By "operably linked" is meant that the nucleic acid molecules encoding the XvSapl (SEQ ID NO:9), XvPrx2 (SEQ ID NO: 10} and XvA!dmut2 (SEQ ID NO:1 1) polypeptides or FMDV 2A (SEQ ID NO:12) peptides described herein and regulatory sequences are connected in such a way as to permit expression of the proteins of interest when the appropriate molecules are bound to the regulatory sequences. Such operably linked sequences may be contained in vectors or expression constructs which can be transformed or transfected into host ceils for expression, it will be appreciated that expression of the proteins of interest will occur in response to an abiotic stress.

The nucleic acid molecules of the invention include the XvSapl gene (SEQ ID NO:3), the XvPrx2 gene (SEQ ID NO:4) and the XvAldmut2 gene (SEQ ID NO:5), the aforementioned genes are separated by a FMDV 2A linker (SEQ ID NO:6).

XvSapl is a stress associated protein isolated from a cDNA library synthesized from dehydration stressed Xerophytha viscosa leaves. The XvSapl protein (SEQ ID NO:9) is highly hydrophobic and possesses two membrane lipoprotein lipid attachment sites. These features suggest that XvSapl is anchored within the plasma membrane. It displays high sequence similarity with G-protein coupled receptors and consequently is postulated to play a signalling role during abiotic stress. The amino acid sequence of XvSapl displayed 49% identity to WCOR413 from wheat (renamed TacCOR413-PM). This family of stress inducible proteins consist of two distinct groups based on their intracellular localisation, COR413 plasma membrane (COR413-PM) proteins and COR413 thylakoid (COR413-TM) proteins. The COR413 protein family has not been identified in other eukaryotic or prokaryotic databases suggesting that it is unique to the plant kingdom. Expression of XvSapl in Escherichia coli cells conferred osmotic stress tolerance when the cells were grown in 1 M sorbitol. Transgenic Arabidopsis thaliana and Nicoiiana tabacum plants consitutively expressing XvSapl showed increase tolerance to osmotic, salt, heat and dehydration stress. These features suggest that XvSapl could play an important role in repair of damage to the cell membrane.

XvPrx2 _s encodes a type H Prx, the XvPrx2 protein (SEQ ID NO:10) was isolated from Xerophyta viscosa from a low temperature stress library. The Prx's are a family of multifunctional antioxidant thioi-dependent peroxidases that have been identified to be ubiquitous in most organisms. This diversity is reflected in slight evolutionary modifications in sequence and structure built around a common peroxidatic active site. The major functions of Prx's comprise cellular protection against oxidative stress, modulation of intracellular signalling cascades that apply H ₂ 0 ₂ as a second messenger molecule and regulation of cell proliferation. The accumulation of these toxic compounds in plant ceils especially reactive oxygen/nitrogen species can cause cell death which is detrimental to the plant. The XvPrx2 protein was determined to be a cytosoi localised, stress inducible antioxidant enzyme involved in the protection of nucleic acids by scavenging reactive oxygen species. Besides these characteristics, two further characteristics of this protein are of significance. The first is the discovery that multiple XvPrx2 homologues exist in X. viscosa. The second is that the XvPrx2 protein is atypical in that it possesses a single cysteine only.

The XvAldmut2 gene was isolated from a Xerophyta viscosa dehydration library and encodes for an aldose reductase, which catalyses the reduction of sugars to their analogous alcohol. It has also been demonstrated that plant aldose reductase can detoxify cytotoxic aldehydes, such as 4-hydroxynon-2-enal that is a product of ROS-induced lipid peroxidation. Transcript and protein levels of XvAldmut2 (SEQ ID NO:1 1) have been shown to increase within leaves in response to water deficit.

The term "recombinant" means that something has been recombined. When used with reference to a nucleic acid construct the term refers to a molecule that comprises nucleic acid sequences that are joined together or produced by means of molecular biological techniques. Recombinant nucleic acid constructs may include a nucleotide sequence which is ligated to, or is manipulated to become ligated to, a nucleic acid sequence to which it is not ligated in nature, or to which it is ligated at a different location in nature. Accordingly, a recombinant nucleic acid construct indicates that the nucleic acid molecule has been manipulated using genetic engineering, i.e. by human intervention. Recombinant nucleic acid constructs may be introduced into a host ceil by transformation.

The term "2A" or "2A element" refers to an about 18-24 amino acid sequence, which can be found in picornaviruses, such as Foot-and-Mouth Disease Virus. A highly conserved consensus motif at the C-terminus of the 2A element mediates cleavage between the C-terminal glycine and the N-terminal proline. This "cleavage" does not require any additional factors like proteases (Szymczak et al (2005)} and allows for polycistronic transcription of genes of interest with resultant cleavage of the translated polypeptides into separate proteins. Those of skill in the art will appreciate that when a FMDV 2A element is operably linked to a first gene and a second gene is operab!y finked to the 2A element, then the 2A element facilitates the co-trans!ational "cleavage" of the expressed polypeptides into separate proteins. The 2A element thus allows multiple proteins to be encoded as a single po!yprotein, which dissociate into component proteins on translation. The 2A peptide sequence impairs normal peptide bond formation through a mechanism of ribosomal skipping.

The term "vector" refers to a means by which polynucleotides or gene sequences can be introduced into a celt. There are various types of vectors known in the art including p!asmids, viruses, bacteriophages and cosmids. Generally polynucleotides or gene sequences are introduced into a vector by means of a cassette. The term "cassette" refers to a polynucleotide or gene sequence that is expressed from a vector, for example, the polynucleotide or gene sequence encoding the XvSapl , XvPrx2 and XvAld polypeptides and the FMDV 2A peptides. It wiil be appreciated in the present invention that the cassette provides regulatory sequences in the form of an abiotic stress inducible promoter and a terminator. "Regulatory sequences" include but are not limited to promoters, transcription termination sequences, enhancers, splice acceptors, donor sequences, introns, ribosome binding sequences, poly(A) addition sequences, and/or origins of replication.

The current invention provides a specific combination of a stress-inductble promoter and three stress inducible genes isolated from Xerophyta viscosa, where each gene is separated by a 69 bp FMDV 2A nucleotide sequence.

Various combinations of stress inducible genes, operably linked to the XvPsapI D promoter (SEQ ID NO:2) and nosT terminator (SEQ ID NO:7) and individually separated by the FMDV 2A linker sequence were evaluated under drought stress conditions. The combination XvSap\::FMDV2A::XvPrx2:: FMDV2A::XvAldmut2::nosJ under control of the minimal stress inducible promoter (XvPsapI D (SEQ ID NO:2); as described in WO 2014/037919) displayed the highest levels of tolerance to drought.

The following example is offered by way of illustration and not by way of limitation.

EXAMPLE

Cloning of the polygenic DNA construct into pTF101.1::Psap1D recombinant vector

The XvSapl::FMDV2A::XvPrx2::FMDV2A::XvAldmut2::nosT construct was synthesised de novo incorporating a Bcl\ site at the 5 ^! end as well as a H/ndlll site at the 3' site of the construct. Plasmid isolation was carried out on the synthesised construct from the pUC57 plasmtd. The synthesised product was then digested, eiectrophoresed and excised from the gei and purified.

The polygenic DNA construct (SEQ ID NO:1) without the minimal promoter was cloned into the pDrive (Qiagen, USA) vector by digesting both the vector as well as the polygenic DNA construct with Bc/f and H/ndill restriction enzymes at 37 °C for 1 hour. The digested products where ligated to form the recombinant pDrive p!asmid (pDf\ve:-.XvSapl::FMDV2A::XvPrx2:\FMDV2A::XvAldmut2::nosT). The recombinant plasmid was transformed into £ coli DH5a cells and colony PCR was performed to identify positively transformed clones. Colony PCR was performed using two primer sets M13 F (-20) having the sequence GTA AAA CGA CGG CCA GT (SEQ ID NO:17) and M13 R (-20) having the sequence AAC AGC TAT GAC CAT G (SEQ ID NO: 18) as well as XvSap! fic/l F having the sequence ATG ATC AAT GAG GAA CGA GGG TTT TCT G (SEQ ID NO:19) and XvPrx2 C/al R having the sequence TAT CGA TGA CTG CCT TCA AGA TCT C (SEQ !D NO:20). These primer sets amplified a 2500 bp as well as 1401 bp fragment, respectively. The following PCR conditions were used for the amplifications for both primer sets: 94 °C for 5 min; 30 cycles of 94 °C for 30 s, 54 °C for 45 s, 72 °C for 90 s; and a final extension of 72 °C for 10 min.

The polygenic DNA construct was then cloned into pTF101.1 vector (SEQ ID NO:8) containing the minimal PsapI D promoter by digesting the pDrive polygenic DNA construct with Bell and H/ndltl restriction enzymes and the pTF101.1 vector containing the minimal PsapI D promoter with SamHl and ΗΊηύ\\\. It should be noted that Sc/I and SamHl have compatibie cohesive ends and can therefore iigate. The polygenic DNA construct was ligated to pTF101.1 ::Psap1 D digested ends to form the recombinant pTF101.1 plasmid (pTF101.1 ::Psap1 D::XvSap/::F/WDV2A::XvP/x2:: FMDV2A::XvAldmut2::nosT) (Figure 1). The recombinant plasmid was transformed into E. coii DH5a ceils and colony PCR was performed using XvSapl specific primers (XvSapi Sc/I F (SEQ ID NO: 19) and XvSapl C/al R having the sequence TAT CGA TAA ACT CAG CCT CAT AGA TGA AGA C (SEQ !D NO:21).under the following conditions: 95 °C for 10 min; 30 cycles of 95 °C for 30 s, 54 °C for 45 s, 72 °C for 60 s and a final extension of 72 °C for 10 min in order to identify positively transformed clones. An EcoRI and Ηϊηύ\\\ restriction enzyme digest was performed to release the entire insert {Psap1 D::XvSapl::FMDV2A::XvPrx2::FMDV2A::XvA!dmut:nos^ and the size was verified on an ethidium bromide stained agarose gei. Transformation of Agrobacterium tumefaciens

The recombinant pTF101.1 construct was transformed into competent A. tumefaciens EHA101 ceits. Following transformation and growth of A. tumefaciens ceils, colony PCR was performed to identify positively transformed clones. The presence of the polygenic DNA construct was determined by PCR amplification of part of the minimal PsapI D promoter fragment as well as part of the T-DNA RB region using a promoter-specific forward primer: pSap1-RB F5 having the sequence TCG AAT GCT ATT GAT CCT GTC GT (SEQ ID NO: 13) and a T-DNA right border specific reverse primer: pSap1-RB R5 having the sequence AGC TCA AGC TCC AAT ACG CAA (SEQ ID NO: 14). Amplification of the 449 bp fragment was carried out under the following PCR conditions: 95 °C for 5 min; 35 cycles of 95 °C for 30 s, 54 °C for 30 s, 72 °C for 30 s; and a final extension of 72 °C for 10 min.

Plant transformation and selection

Wild type N. tabacum (SR1 ecotype) seed was sterilised and germinated on sterile potting soil mix. Plants were cultured in the growth room with set conditions (24 °C; 16 h light, 8 h dark).

Four to six inch leaves were selected from 2 month old plants. Leaves were soaked in sterile water for 30 min and sterilised. Sterile leaves were sliced into uniform segments of 5 mm avoiding the leaf margins and mid vein. Leaf explants were placed adaxial side up onto pre-cu!ture media.

One millilitre of a 16 h culture of A. tumefaciens carrying the polycistronic multigene construct was inoculated into 200 ml of YEP media supplemented with the appropriate antibiotics. The cultures were incubated at 30 °C until an absorbance reading of approximately 0.8 at 600 nm was obtained. The cultures were centrifuged for 20 min at 6000 x g at 4 °C. The supernatant was discarded and the bacterial pellets re-suspended in 50 ml of liquid co-cultivation media.

Leaf discs were infected for 30 min in the dark with the Agrobacterium inoculum containing the polygenic DNA construct in sterile petri dishes. The petri dish was agitated once every 10 min. Thereafter, infected leaf discs were blot dried on sterile filter paper. Each infected explant was transferred to co-cultivation medium and incubated for 3 days at 23 °C (18 h light, 6 h dark; light intensity of 140 pmol/m ² /s). The adaxial part of the leaf was kept in contact with the medium. Fo!iowing the 3 day co-cultivation period, leaf discs were selected on shooting medium. Leaf explants were placed under an 18 h light regime with light intensity of 140 prno!/m ² /s at 28 °C. Putative transformants were sub-cultured fortnightly onto fresh media until sizable shoots were formed. BASTA resistant shoots were selected, excised and transferred to rooting media.

Putative transformants with well-established root systems were transferred to pots containing sterile potting soil and cultured with set conditions. The plants were covered with Saran Wrap® for 8 days to assist acclimatisation and minimise dehydration. Once acclimatised, the putative transformants were transferred to 6 inch pots containing potting soil under normal growth conditions. Mature plants were self- pollinated and seed was harvested from mature dry pods.

Putative transgenic tobacco seed was sterilised and germinated on MS media supplemented with 8 g/l agar and 3 mg/ml BASTA. Plants were cultured in the growth room with set conditions (24 °C; 16 h day, 8 h night). The surviving BASTA resistant plants with well-established root systems were transferred to trays containing 0.1 g/l Gaucho SW treated potting soil and covered with Saran Wrap® for 1 week. Three weeks later, BASTA resistant transgenic plants were transferred to pots containing potting soil.

Leaves were sampled from putative transgenic plants and were flash frozen in liquid nitrogen. Genomic DNA was extracted using the De!laporta extraction protocol. The presence of the polygenic DNA construct was determined by PCR amplification with the Psap1-RB F5 (SEQ ID NO: 13) and Psap1-RB R5 (SEQ ID NO:14) primer pair. The bar gene (421 bp) was also amplified using the following PCR conditions (95 °C for 10 min; 35 cycles of 95 °C for 30 s, 54 °C for 30 s, 72 °C for 30 s; and a final extension of 72 °C for 5 min) and using the bar gene specific primers: Bar !-F GGT CTG CAC CAT CGT CAA CC (SEQ ID NO: 15) and Bar l-R GTC ATG CCA GTT CCC GTG CT (SEQ ID NO: 16).

Dehydration and rehydration of transgenic plants

The first dehydration/rehydration treatments were carried out in a Percival® chamber and the second dehydration/rehydration treatment was carried out in a Conviron® chamber.

Prior to the first dehydration/rehydration treatment, plants were transferred to pots containing set amounts of soil and water. Plants were moved to the Percival® chamber (Percival® Iniellus control system) and incubated under set conditions (26 °C; 16 h day, 8 h night; 60% humidity; light intensity of 100 pmol/m ² /s) for 1 week to acclimatise. Dehydration stress was carried out on whole plants and achieved by withholding water for a period of seven days and then rehydrating the plants for a further five days.

Prior to the second dehydration/rehydration treatment, whole mature plants were transferred to pots containing set amounts of soil and water. Plants were moved to the Conviron® chamber and incubated under set conditions (26 °C; 16 h day, 8 h night; 60% humidity; light intensity of 100 _M mol/m ^z /s) for 1 week to acclimatise. Dehydration stress was carried out on whole plants and achieved by withholding water for a period of ten days and rehydrating the plants for a further four days. Throughout the dehydration period, flowers and seed pods were counted on day one and day ten of dehydration, prior to rehydration. Rehydration was initiated on day ten after the flowers and pods were counted. Rehydration lasted four days and the total biomass of the transgenic plants was measured on day four of the rehydration.

Dehydration and rehydration treatment of transgenic N. tabacum plants The first dehydration and rehydration treatments on wild type (WT) tobacco had a major impact on the overall morphology of the plant. In the absence of the polygenic DNA construct the leaves look withered and close to dying when compared to the transgenic lines containing the polygenic DNA construct (Figure 2). The transgenic plants also had leaves that looked fuller and bigger and appeared to have a minor response to the stress. On the other hand the wild type control plants were severely affected following the stress treatment.

The effect of dehydration on seed pods and flowers of wild type control and transgenic tobacco plants

Transgenic plants (fully hydrated) reached maturity significantly earlier than the wild type counterparts as evidenced by the higher number of pods (Figure 3) and flowers (Figure 4) observed. For those transgenic plants that were dehydrated there was also an observable difference in the number of pods and flowers compared to wild type plants (Figure 3 and 4).

REFERENCES

Szymczak A.L et al (2005) Expert Opin. Biol. Ther. 5: 627-638