WO2004087928A2 - Transgenic plants expressing a gene encoding a xyloglucan endotransglycosylase - Google Patents

Abstract

Broad leaf plants comprising novel XET sequences, XET nucleotide sequences for use in the invention, and methods for preparing such plants.

Description

Transgenic Plant

Background

The present invention relates to plant cell material of broad-leafed plants that has improved physical properties for food processing than plant cell material from conventionally available broad-leafed plants, broad- leafed plants comprising such improved plant cell material, and methods for producing such broad-leafed plants. In particular, the invention relates to plant cell material of broad-leafed plants comprising an exogenous nucleic acid sequence of a xyloglucan endotransglycosylase, broad-leafed plants comprising said exogenous nucleic acid sequence, and the genetic material required therefore, such as DNA and RNA, vectors, host cells, methods of introduction of genetic material into plant cells, and uses thereof.

One example of the broad-leafed plant market is that of the pre-packed baby leaf salad market. This market was estimated at $1.6 billion worldwide during 1999, an increase of 15.9% on the previous year (International Fresh-Produce Association) . It seems likely that this growth will continue for some time, given the year-round availability of lettuce, the improved quality and variety available in the supermarkets, and a general movement towards both healthier eating and convenience foods.

In post-harvest processing, salad leaves typically undergo transportation, washing, sanitisation, and de- watering all of which inter alia place the leaves under physical and physiological stresses which they are required to endure upto and including being placed on the supermarket shelf. Once placed on the supermarket shelf the leaves must then have a long enough shelf life to appear desirable to the consumer. One of the most important factors for the producer is to produce salad leaves that are capable of undergoing rigorous processing without substantial detriment to quality and thus to supply the supermarkets with the quality of produce that is required. Salad leaves that are not highly processable, that is to say, leaves that are readily damaged during post-harvest processing may result in actual or potential sales losses to both the producer and vendor.

Existing methods to improve post-harvest processability of broad-leafed plants have focused on the method of crop production, that is, the growing environment for the crop and/or on the processing and/or on packaging methods. For example, farm sites have been specifically selected for environments conducive to producing plant phenotypes that are better able to withstand post-harvest processing. However, such farm sites will become limited in future years as population pressures demand that farmland is taken over, for example, for building purposes.

Farming methods have also been refined to improve the processability phenotype of broad-leaf crops. Conventional methods that have brought about improvement in processability include the optimisation of seed density, the use of broad-leaf plant water-stressing methods to improve a crop's processability (inter alia by limiting water usage) , and harvesting by hand where possible in order to minimise the risk of physically damaging leaves. However, much of the improvement to processability that may be possible today is crop- specific in relation to the use of water and machinery.

Conventional processing methods comprising the machinery, packing materials, and the cold-chain are all under constant improvement. However, conventional processing methods are typically crop specific, and require substantial capital investment. Furthermore, processing regulations demand that certain standards are met. There are limits to the modifications that can be made to conventional processing methods, for example, the leaves must be washed, dried and packaged to give the added value to the retailer and convenience to the consumer. Such improvements typically bring about but a small improvement to the shelf-life of the crop through the reduction of losses through processing damage.

Clarkson et al., Postharvest Biology and Technology 30 (2003) 287 - 298 identify key traits for improving post harvest processability of baby leaf salad. Baby leaf salad plant material was subjected to certain stress treatments and then leaves of selected ages were taken and assessed for various physical and physiological parameters. However, Clarkson et al do not describe how such key traits, or indeed, which traits could be genetically engineered into plant cell material.

Herbers et al(2001) Planta 212: 279-287 describe the functional characterisation of a tobacco xyloglucan endotransglycosylase through antisense expression of a XET-1 nucleic acid sequence in tobacco. However, there is no suggestion or allusion to using XET sequences to produce broad leaf edible plants having improved characteristics for food processing. In the prior art, transgenic approaches to plant improvement (including lettuce) using Agrobacteriυm tumefaciens have been used previously, for example, in lettuce: Michelmore et al . (1987) Plant Cell Reports 6:439-442 and Curtis et ai. (1994) Journal of Experimental Botany 45:1441-1449. Transformation of lettuce has been used to try and improve shelf-life by manipulating the levels of cytokinin (Curtis et al . (1999b) Plant Cell Reportslδ : 889-896; Frugis et al .

(1999) Plant Physiology 119: 371-373; Frugis et al (2001) Plant Physiology 126:1370-1380; McCabe et al . (2001)

Plant Physiology 127:505-516). However, the prior art appears to be silent on the genetic transformation of broad leaf edible plants such as lettuce with a view to improving the processability thereof.

XETs cleave xyloglucan polymers in the cell wall and re- tether the cut end to another xyloglucan chain. Xyloglucan is a long chain polysaccharide and is the most abundant hemicellulose in the plant cell wall, forming hydrogen bonds with cellulose microfibrils, giving increased strength to the cell wall. XETs are proposed to have involvement in three physiological functions (Campbell and Braam, (1999) Trends in Plant Science 4:361-366: cell wall loosening during turgor driven cell expansion; cell wall biosynthesis and wall strengthening; and cell wall degradation. However, their exact physiological function is not known. The activity of XETs is diverse and of fundamental importance to the plant though their function as alluded to above requires further investigation for understanding. The targeting of XETs in broad leaf edible plant species represents a novel approach to improving the processability thereof, and by extension shelf-life, of edible tissues (leaf tissue) of such species.

The present inventors have found that the expression of an introduced heterologous or exogenous nucleic acid sequence derived from a xyloglucan endotransglycosylase (XET) (also known as xyloglucan endotranshydrolase (XTH) after a recent re-classification (Rose et al (2002) Plant Cell Physiol. 43:1421-1435)), can enhance the processability of broad-leaf plant material, such as baby leaf salad species. Thus, for the purposes of the present invention "XET" will be used to refer to XET and/or XTH sequences taking into account the recent change in nomenclature for the xyloglucan endotransglycosylase/ xyloglucan endotranshydrolase enzyme, so as to minimise confusion over nomenclature to the skilled addressee.

Thus, there exists a need for improving the physical and physiological properties of broad leaf edible plant material, such as baby leaf salad material, in order for such broad lead edible plant material to better withstand conventional broad leaf salad processing methods.

Detailed description

According to the present invention there is provided a method of altering the xyloglucan polymer content of a plant cell wall in a plant cell of an edible, broad leaf plant that comprises introducing into said plant cell an isolated nucleic acid that comprises a XET nucleic acid sequence that is operably linked to an exogenous promoter that drives expression in the said plant cell. The altering of xyloglucan polymer content is achieved by altering XET activity associated with the cell wall. Thus, as a further aspect of the present invention there is provided a method of altering XET activity of a plant cell wall in a plant cell of an edible, broad leaf plant that comprises introducing into said plant cell an isolated nucleic acid that comprises a XET nucleic acid sequence that is operably linked to an exogenous promoter that drives expression in the said plant cell.

The isolated nucleic acid used herein is typically derived from a XET nucleic acid sequence selected from plant tissue from a broad leaf edible plant. Suitable broad leaf edible plants harbouring XET nucleic acids for use in the method of the invention are selected from the group lactuca sativa, Mizuna, lollo rosso, Frisee, rocket, wild rocket, lambs lettuce, little gem, cos, spinach, chard, ruby chard, watercress, red oak leaf (salad leaf), green oak leaf (salad leaf), and Apollo. Preferably, the isolated nucleic acid for use in the method of the invention is derived from a XET nucleic acid sequence from a leaf from a plant selected from lactuca sativa such as lollo rosso, cos, iceberg, little gem, Frisee, lambs lettuce, red oak leaf (salad leaf) , green oak leaf (salad leaf), and Apollo. Most preferably, the isolated nucleic acid sequence is from a lollo rosso

(lactuca sativa) plant and is that of Figure 1 (SEQ ID

No.l) .

The introduced isolated nucleic acid sequence may comprise a XET nucleic acid sequence in the sense orientation that is operably linked to an exogenous promoter that drives expression in the said plant cell causing a reduction or down-regulation in the expression of the XET target gene by co-suppression. In the alternative, the introduced isolated nucleic acid may comprise a XET nucleic acid sequence in the anti-sense orientation that is operably linked to an exogenous promoter that drives expression in the said plant cell causing a down-regulation in the expression of the XET target gene. Preferably, the XET nucleic acid sequence used in a method of the present invention is in the anti- sense orientation. Preferably, the XET nucleic acid sequence used in the method of the invention comprises the nucleic acid sequence shown in Figure 1 (Seq Id no. 1) and is in the anti-sense (reverse) orientation. Thus, as an aspect of the invention there is provided a nucleotide sequence from a broad leaf, edible plant comprising a DNA sequence encoding an antisense RNA molecule operably linked to a promoter and a terminator, said promoter and terminator functioning in a plant cell, wherein said antisense RNA molecule is complementary to a portion of the coding sequence for a protein having XET activity associated with the plant cell wall wherein said protein is a XET. Preferably, the antisense RNA molecule is complementary to a sense mRNA molecule encoding for a XET or a fragment thereof of a lactuca sativa plant, such as the sense mRNA molecule encoding for a XET sequence of Figure 1.

In using anti-sense genes or partial gene sequences to down-regulate gene expression, a nucleotide sequence is placed under the control of a promoter in a "reverse orientation" such that transcription yields RNA which is complementary to normal or native mRNA transcribed from the "sense" strand of the target gene, for example, the endogenous target gene. See, for example, Rothstein et al, 1987; Smith et al, (1988) Nature 334, 724-726; Zhang et al, (1992) The Plant Cell 4, 1575-1588, English et al . ,

(1996) The Plant Cell 8, 179-188. Antisense technology is also reviewed in Bourque, (1995) , Plant Science 105, 125-149, and Flavell, (1994) PNAS USA 91, 3490-3496.

Thus, as described above, a nucleotide sequence which is complementary to any of those coding sequences disclosed herein, and especially the coding sequence shown in Figure 1, forms a further aspect of the present invention.

An alternative to anti-sense is to use a copy of all or part of the target gene inserted in the sense orientation, to achieve reduction in expression of the target gene by co-suppression. See, for example, van der Krol et al., (1990) The Plant Cell 2, 291-299; Napoli et al., (1990) The Plant Cell 2, 279-289; Zhang et al., (1992) The Plant Cell 4, 1575-1588, and US-A-5, 231, 020. Further refinements of the co-suppression technology may be found in W095/34668 (Biosource) ; Angell & Baulcombe

(1997) The EMBO Journal 16,12:3675-3684; and Voinnet & Baulcombe (1997) Nature 389: pg 553.

Double stranded RNA (dsRNA) has been found to be even more effective in gene silencing than both sense or antisense strands alone (Fire A. et al Nature, Vol 391,

(1998)). dsRNA mediated silencing is gene specific and is often termed RNA interference (RNAi) (See also Fire (1999) Trends Genet . 15: 358-363, Sharp (2001) Genes Dev. 15: 485-490, Hammond et al . (2001) Nature Rev. Genes 2: 1110-1119 and Tuschl (2001) Chem . Biochem . 2: 239-245). RNA interference is a two step process. First, dsRNA is cleaved within the cell to yield short interfering RNAs (siRNAs) of about 21-23nt length with 5' terminal phosphate and 3' short overhangs (~2nt) . The siRNAs target the corresponding mRNA sequence specifically for destruction (Zamore P.D. Nature Structural Biology, 8, 9, 746-750, (2001) .

Thus in one embodiment, the invention provides double stranded RNA comprising a XET-encoding sequence, which may for example be a "long" double stranded RNA (which will be processed to siRNA, e.g., as described above). These RNA products may be synthesised in vitro, e.g., by conventional chemical synthesis methods.

RNAi may be also be efficiently induced using chemically synthesized siRNA duplexes of the same structure with 3'- overhang ends (Zamore PD et al, Cell, 101, 25-33, (2000) ) .

Thus siRNA duplexes containing between 20 and 25 bps, more preferably between 21 and 23 bps, of a XET sequence form one aspect of the invention e.g. as produced synthetically, optionally in protected form to prevent degradation.

Alternatively siRNA may be produced from a vector, in vitro (for recovery and use) or in vivo.

Accordingly, the vector may comprise a nucleic acid sequence encoding all or part of SEQ ID NO.2 (or a variant thereof) , suitable for introducing an siRNA into the cell in any of the ways known in the art, for example, as described in any of references cited herein, which references are specifically incorporated herein by reference.

In one embodiment, the vector may comprise a nucleic acid sequence according to the invention in both the sense and anti-sense orientation, such that when expressed as RNA the sense and antisense sections will associate to form a double stranded RNA. This may for example be a long double stranded RNA (e.g., more than 23nts) which may be processed in the cell to produce siRNAs (see for example Myers (2003) Nature Biotechnology 21 : 324-328) .

Alternatively, the double stranded RNA may directly encode the sequences which form the siRNA duplex, as described above. In another embodiment, the sense and antisense sequences are provided on different vectors.

These vectors and RNA products may be useful for example to inhibit de novo production of the XET polypeptide in a cell. They may be used analogously to the expression vectors in the various embodiments of the invention discussed herein. For example dsRNA comprising the sequence of the XET gene or fragments thereof (eg SEQ ID NO.1) can be used to prevent expression of that gene (e.g. by cloning parts of the XET sequence into an iRNA binary vector using the GATEWAY™ system available from Invitrogen.

Further options for down regulation of gene expression include the use of ribozymes, e.g. hammerhead ribozymes, which can catalyse the site-specific cleavage of RNA, such as mRNA (see e.g. Jaeger (1997) Curr Opin Struct Biol 7:324-335, or Gibson & Shillitoe (1997) Mol Biotechnol 7: 242-251). The complete XET nucleic acid sequence corresponding to the coding sequence (in reverse orientation for anti- sense) need not be used. For example fragments of sufficient length may be used, such as the sequence shown in Figure 1. It is a routine matter for the person skilled in the art to screen fragments of various sizes and from various parts of the coding sequence to optimise the level of anti-sense inhibition. It may be advantageous to include the initiating methionine ATG codon, and perhaps one or more nucleotides upstream of the initiating codon. A further possibility is to target a conserved sequence of a gene, e.g. a sequence that is characteristic of one or more genes, such as a regulatory sequence.

The sequence employed may be about 500 nucleotides or less, possibly about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, or about 100 nucleotides. It may be possible to use oligonucleotides of much shorter lengths, 14-23 nucleotides, although longer fragments, and generally even longer than about 500 nucleotides are preferable where possible, such as longer than about 600 nucleotides, than about 700 nucleotides, than about 800 nucleotides, than about 1000 nucleotides or more.

It may be preferable that there is complete sequence identity in the sequence used for down-regulation of expression of a target sequence, and the target sequence, although total complementarity or similarity of sequence is not essential. One or more nucleotides may differ in the sequence used from the target gene. Thus, a sequence employed in a down-regulation of gene expression in accordance with the present invention may be a wild-type sequence (e.g. gene) selected from those available, or a variant of such a sequence.

The sequence need not include an open reading frame or specify an RNA that would be translatable. It may be preferred for there to be sufficient homology for the respective anti-sense and sense RNA molecules to hybridise. There may be down regulation of gene expression even where there is about 5%, 10%, 15% or 20% or more mismatch between the sequence used and the target gene. Effectively, the homology should be sufficient for the down-regulation of gene expression to take place.

Thus the present invention further provides the use of a XET nucleotide sequence (e.g. SEQ ID No 1), or its complement, or a variant of either for down-regulation of gene expression, particularly down-regulation of expression of the XET gene or a homologue thereof, preferably in order to influence cell wall properties such as physical (eg % plasticity) properties in edible broad leaf plant tissue.

Anti-sense or sense regulation may itself be regulated by employing an inducible promoter in an appropriate construct.

Thus examples of processes by which XET activity in edible broad leaf plant tissue may be influenced or affected (especially inhibited) include any of:

(i) causing or allowing transcription from a nucleic acid which comprises a sequence which is the complement of a XET-encoding nucleotide sequence in the plant, such as to reduce XET expression by an antisense mechanism; (ii) causing or allowing transcription from a XET nucleic acid, or a part thereof, such as to reduce XET expression by co-suppression;

(iii) use of a nucleic acid encoding a ribozyme specific for a XET nucleic acid.

The exogenous promoter may be selected from inducible, chemical-regulated, constitutive, developmental and tissue specific promoters.

An exogenous promoter is one that denotes a promoter that is introduced in front of a nucleic acid sequence of interest and is operably associated therewith. Thus an exogenous promoter is one that has been placed in front of a selected XET nucleic acid component as herein defined and does not consist of the natural or native promoter usually associated with the nucleic acid component of interest as found in wild type circumstances. Thus a promoter may be native to a plant cell of interest but may not be operably associated with the nucleic acid of interest in front in wild-type plan cells. Typically, an exogenous promoter is one that is transferred to a host cell or host plant from a source other than the host cell or host plant.

The cDNA' s encoding the XET sequences (either in the sense orientation for co-suppression, or in the anti- sense orientation) of the invention contain at least one type of promoter that is operable in a plant cell, for example, an inducible or a constitutive promoter operatively linked to a nucleic acid sequence or nucleic acid sequence component as herein defined and as provided by the present invention. As discussed, this enables control of expression of the gene. The invention also provides plants transformed with said XET nucleic acid sequence or construct and methods including introduction of such a nucleic acid sequence or construct into a plant cell and/or induction of expression of said nucleic acid sequence or construct within a plant cell, e.g. by application of a suitable stimulus, such as an effective exogenous inducer.

The term "inducible" as applied to a promoter is well understood by those skilled in the art. In essence, expression under the control of an inducible promoter is "switched on" or increased in response to an applied stimulus (which may be generated within a cell or provided exogenously) . The nature of the stimulus varies between promoters. Some inducible promoters cause little or undetectable levels of expression (or no expression) in the absence of the appropriate stimulus . Other inducible promoters cause detectable constitutive expression in the absence of the stimulus. Whatever the level of expression is in the absence of the stimulus, expression from any inducible promoter is increased in the presence of the correct stimulus. The preferable situation is where the level of expression increases upon application of the relevant stimulus by an amount effective to alter a phenotypic characteristic. Thus an inducible (or "switchable") promoter may be used which causes a basic level of expression in the absence of the stimulus which level is too low to bring about a desired phenotype (and may in fact be zero) . Upon application of the stimulus, expression is increased (or switched on) to a level, which brings about the desired phenotype. One example of an inducible promoter is the ethanol inducible gene switch disclosed in Caddick et al (1998) Nature Biotechnology 16: 177-180. A number of inducible promoters are known in the art.

Chemical-regulated promoters can be used to modulate the expression of a gene in a plant through the application of an exogenous chemical regulator. Depending upon the objective, the promoter may be a chemical-inducible promoter, where application of the chemical induces gene expression, or a chemical-repressible promoter, where application of the chemical represses gene expression. Chemical-inducible promoters are known in the art and include, but are not limited to, the maize In2-2 promoter, which is activated by benzenesulfonamide herbicide safeners, the maize GST promoter, which is activated by hydrophobic electrophilic compounds that are used as pre-emergent herbicides, and the tobacco PR-la promoter, which is activated by salicylic acid. Other chemical-regulated promoters of interest include steroid- responsive promoters (see, for example, the glucocorticoid-inducible promoter in Schena et al . (1991) Proc. Natl . Acad. Sci . USA 88:10421-10425 and McNellis et al . (1998) Plant J. 14 (2) : 247-257) and tetracycline- inducible and tetracycline-repressible promoters (see, for example, Gatz et al . (1991) Mol . Gen . Genet . 227:229- 237, and U.S. Patent Nos. 5,814,618 and 5,789,156), herein incorporated by reference.

So-called constitutive promoters may also be used in the methods of the present invention. Constitutive promoters include, for example, CaMV 35S promoter (Odell et al .

(1985) Nature 313:810-812); rice actin (McElroy et al .

(1990) Plant Cell 2:163-171); ubiquitin (Christensen et al . (1989) Plant Mol . Biol . 12:619-632 and Christensen et al . (1992) Plant Mol . Biol . 18:675-689); pEMU (Last et al . (1991) Theor. Appl . Genet . 81:581-588); MAS (Velten et al . (1984) EMBO J. 3:2723-2730) ; ALS promoter (U.S.

Application Serial No. 08/409,297), and the like. Other constitutive promoters include those in U.S. Patent Nos.

5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785;

5,399,680; 5,268,463; and 5,608,142.

Naturally, the man skilled in the art will appreciate that terminator DNA sequences may also be present in constructs used in the invention. A terminator is contemplated as a DNA sequence at the end of a transcriptional unit which signals termination of transcription. These elements are 3' -non-translated sequences containing polyadenylation signals, which act to cause the addition of polyadenylate sequences to the

3' end of primary transcripts. For expression in plant cells the nopaline synthase transcriptional terminator

(A. Depicker et al., 1982, J. of Mol. & Applied Gen. 1:561-573) sequence serves as a transcriptional termination signal.

Those skilled in the art are well able to construct vectors and design protocols for recombinant nucleic acid sequence or gene expression. Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. For further details see, for example, Molecular Cloning: a Laboratory Manual : 2nd edition, Sambrook et al , 1989, Cold Spring Harbor Laboratory Press. Many known techniques and protocols for manipulation of nucleic acid, for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Current Protocols in Molecular Biology, Second Edition, Ausubel et al. eds., John Wiley & Sons, 1992. The disclosures of Sambrook et al. and Ausubel et al. are incorporated herein by reference. Specific procedures and vectors previously used with wide success upon plants are described by Bevan (Nucl. Acids Res. 12, 8711-8721 (1984)) and Guerineau and Mullineaux (1993) (Plant transformation and expression vectors. In: Plant Molecular Biology Labfax (Croy RRD ed. ) Oxford, BIOS Scientific Publishers, pp 121-148) .

Naturally, the skilled addressee will appreciate that each nucleic acid sequence (eg XET sequence; marker sequence, if present) will generally be under regulatory control of its own exogenous promoter and terminator.

Selectable genetic markers may facilitate the selection of transgenic plants and these may consist of chimaeric genes that confer selectable phenotypes such as resistance to antibiotics such as kanamycin (eg nptll) , neomycin, hygromycin, puramycin, phosphinotricin, chlorsulfuron, methotrexate, gentamycin, spectinomycin, imidazolinones and glyphosate.

When introducing selected XET nucleic acid sequences according to the present invention into a cell, certain considerations must be taken into account, being well known to those skilled in the art. The nucleic acid to be inserted should be assembled within a construct, which contains effective regulatory elements, which will drive transcription. There must be available a method of transporting the construct into the cell. Once the construct is within the cell membrane, integration into the endogenous chromosomal material either will or will not occur. Finally, as far as plants are concerned the target cell type must be such that cells can be regenerated into whole plants.

Plants transformed with DNA segments containing sequences of interest as provided herein may be produced by standard techniques, which are already known for the genetic manipulation of plants. DNA can be transformed into plant cells using any suitable technology, such as a disarmed Ti-plasmid vector carried by Agrobacterium exploiting its natural gene transfer ability (EP-A- 270355, EP-A-0116718, NAR 12(22) 8711 -87215 1984), particle or micro projectile bombardment (US 5100792, EP-

A-444882, EP-A-434616) microinjection (WO 92/09696, WO

94/00583, EP 331083, EP 175966, Green et al . (1987) Plant

Tissue and Cell Culture, Academic Press), electroporation

(EP 290395, WO 8706614) other forms of direct DNA uptake (DE 4005152, WO 9012096, US 4684611), liposome mediated DNA uptake (e.g. Freeman et al. Plant Cell Physiol . 29: 1353 (1984)), or the vortexing method (e.g. Kindle, PNAS U. S. A. 87: 1228 (1990d) Physical methods for the transformation of plant cells are reviewed in Oard, 1991, Biotech . Adv. 9: 1-11.

Thus once a nucleic acid sequence or gene has been identified, it may be reintroduced into plant cells using techniques well known to those skilled in the art to produce transgenic plants of the appropriate phenotype.

Agrobacterium transformation is widely used by those skilled in the art to transform dicotyledonous species. Micro projectile bombardment, electroporation and direct

DNA uptake are preferred where Agrobacterium is inefficient or ineffective. Alternatively, a combination of different techniques may be employed to enhance the efficiency of the transformation process, e.g. bombardment with Agrobacterium coated micro particles

(EP-A-486234) or micro projectile bombardment to induce wounding followed by co-cultivation with Agrobacterium

(EP-A-486233) .

Following transformation, a plant may be regenerated, e.g. from single cells, callus tissue or leaf discs, as is standard in the art. Almost any plant can be entirely regenerated from cells, tissues and organs of the plant. Available techniques are reviewed in Vasil et al., Cell Culture and Somatic Cell Genetics of Plants, Vol . I, II and III, Laboratory Procedures and Their Applications, Academic Press, 1984, and Weiss Bach and Weiss Bach, Methods for Plant Molecular Biology, Academic Press, 1989.

The particular choice of a transformation technology will be determined by its efficiency to transform certain plant species as well as the experience and preference of the person practising the invention with a particular methodology of choice. It will be apparent to the skilled person that the particular choice of a transformation system to introduce nucleic acid into plant cells is not essential to or a limitation of the invention, nor is the choice of technique for plant regeneration.

The invention further encompasses a host cell transformed with vectors or constructs as set forth above, especially a plant or a microbial cell. Thus, a host cell, such as a plant cell, including nucleotide sequences of the invention as herein indicated is provided. Within the cell, the nucleotide sequence may be incorporated within the chromosome.

Also according to the invention there is provided a plant cell having incorporated into its genome at least a nucleotide sequence, particularly heterologous nucleotide sequences, as provided by the present invention under operative control of regulatory sequences for control of expression as herein described. The coding sequence may be operably linked to one or more regulatory sequences which may be heterologous or foreign to the nucleic acid sequences employed in the invention, such as not naturally associated with the nucleic acid sequence (s) for its (their) expression. The nucleotide sequence according to the invention may be placed under the control of an externally inducible promoter to place expression under the control of the user. A further aspect of the present invention provides a method of making such a plant cell involving introduction of nucleic acid sequence (s) contemplated for use in the invention or a suitable vector including the sequence (s) contemplated for use in the invention into a plant cell and causing or allowing recombination between the vector and the plant cell genome to introduce the said sequences into the genome. The invention extends to plant cells containing a nucleotide sequence according to the invention as a result of introduction of the nucleotide sequence into an ancestor cell.

The term "heterologous" may be used to indicate that the gene/sequence of nucleotides in question have been introduced into said cells of the plant or an ancestor thereof, using genetic engineering, ie by human intervention. A transgenic plant cell, i.e. transgenic for the nucleotide sequence in question, may be provided. The transgene may be on an extra-genomic vector or incorporated, preferably stably, into the genome. A heterologous gene may replace an endogenous equivalent gene, ie one that normally performs the same or a similar function, or the inserted sequence may be additional to the endogenous gene or other sequence. An advantage of introduction of a heterologous gene is the ability to place expression of a sequence under the control of a promoter of choice, in order to be able to influence expression according to preference. Furthermore, mutants, variants and derivatives of the wild-type gene, e.g. with higher activity than wild type, may be used in place of the endogenous gene. Nucleotide sequences heterologous, or exogenous or foreign, to a plant cell may be non- naturally occurring in cells of that type, variety or species. Thus, a nucleotide sequence may include a coding sequence of or derived from a particular type of plant cell or species or variety of plant, placed within the context of a plant cell of a different type or species or variety of plant. A further possibility is for a nucleotide sequence to be placed within a cell in which it or a homologue is found naturally, but wherein the nucleotide sequence is linked and/or adjacent to nucleic acid which does not occur naturally within the cell, or cells of that type or species or variety of plant, such as operably linked to one or more regulatory sequences, such as a promoter sequence, for control of expression. A sequence within a plant or other host cell may be identifiably heterologous, exogenous or foreign. Thus, nucleic acids of the invention may comprise, consist or consist essentially of any of the XET sequences of a broad leaf edible plant as disclosed herein (which may be a gene, a genomic clone or other sequence, a cDNA, or an ORF or exon of any of these etc.). For example, where gDNA is disclosed, nucleic acids comprising any one or more introns or exons from any of the gDNA are also embraced. Likewise, where cDNA is disclosed, nucleic acids comprising only the translated region (from initiation to termination codons) are also embraced.

Likewise, DNA is generally found in double-stranded form, and the complementary strand of such DNA sequences is also included in the ambit of the invention. A nucleic acid is ^Λthe complement' of another nucleic acid to which it is complementary. ^ΛComplementary to' means that the sequence is capable of base pairing with the coding sequence whereby A is the complement of T (and U) ; G is the complement of C; and may be of equal length to, or of a portion of, said DNA sequence.

Nucleic acid molecules according to the present invention may be provided in isolated and/or purified form from their natural environment, in substantially pure or homogeneous form, or free or substantially free of other nucleic acids of the species of origin. Where used herein, the term isolated' encompasses all of these possibilities. The nucleic acid molecules may be wholly or partially synthetic. In particular they may be recombinant in that nucleic acid sequences which are not found together in nature (do not run contiguously) have been ligated or otherwise combined artificially. Alternatively they may have been synthesised directly e.g. using an automated synthesiser.

In this aspect of the present invention there are also provided XET nucleic acids which are variants of any of the XET sequences provided herein. A variant nucleic acid molecule shares homology with, or is identical to, at least all or part of a coding sequence (e.g. SEQ ID

No.l) discussed above, and preferably shares homology with all or part of the XET cDNA sequences shown herein

(eg SEQ ID No 1) .

Generally variants may encode, or be used to isolate or amplify nucleic acids which encode, polypeptides which have XET activity as alluded to above. Such variants may encode polypeptides having XET activity.

Sequence variants which occur naturally may include XET alleles (which typically include polymorphisms or mutations at one or more bases) or pseudoalleles (which may occur at closely linked loci to the XET gene) .

Variants which do not encode polypeptides having XET activity are useful, for example, for use in probing or silencing. Also included within the scope of the present invention are isogenes, or other genes or fragments thereof homologous to the XET sequences of the invention (eg of SEQ ID No.l) and belonging to the same family as the XET gene. Although these may occur at different genomic loci to the XET gene, they typically share conserved regions with it, such as the conserved nucleic acid sequence giving rise to the sense mRNA molecule encoding for a XET amino acid sequence comprising DEIDFEFLG (SEQ ID NO.3). Thus the nucleic acid variant sequence may be an XET orthologue obtainable from a broad leaf edible plant species other than Lactuca sativa (eg lollo rosso) .

Artificial variants (derivatives) may be prepared by those skilled in the art, for instance by site directed or random mutagenesis, or by direct synthesis. Preferably the variant nucleic acid is generated either directly or indirectly (e.g. via one or more amplification or replication steps) from an original nucleic acid having all or part of the sequence shown in any of the XET sequences described herein.

Thus a variant may be a distinctive part or fragment (however produced) corresponding to a portion of the sequence provided. The fragments may encode particular functional parts of the polypeptide. Alternatively, the fragments may have utility in probing for, or amplifying, the sequence provided or closely related ones. Suitable lengths of fragment, and conditions, for such processes are discussed in more detail below.

Also included are nucleic acids corresponding to those above, but which have been extended at the 3' or 5' terminus.

The term ^variant' nucleic acid as used herein encompasses all of these possibilities. When used in the context of the present invention with regard to polypeptides or proteins, ariant' indicates the encoded expression product of the variant nucleic acid.

Some of the aspects of the present invention relating to variants will now be discussed in more detail. Similarity or homology (the terms are used interchangeably) or identity may be as defined and determined by the TBLASTN program, of Altschul et al . (1990) J. Mol. Biol. 215: 403-10, or BestFit, which is part of the Wisconsin Package, Version 8, September 1994,

(Genetics Computer Group, 575 Science Drive, Madison,

Wisconsin, USA, Wisconsin 53711) . Preferably sequence comparisons are made using MultAlin (see Corpet (1988), Nucleic Acids Research 16 10881-10890) . Default parameters are preferably set as follows:

Gap value (penalty for the first residue in a gap) : -12 Gap length weight (penalty for additional residues in a gap) : -2

Alternatively, sequence comparisons may be made using FASTA and FASTP (see Pearson & Lipman (1988) Methods in Enzymology 183: 63-98). Parameters may be set, using the default matrix, as follows:

Gapopen (penalty for the first residue in a gap) : -12 for proteins / -16 for DNA

Gapext (penalty for additional residues in a gap) : -2 for proteins / -4 for DNA

KTUP word length: 2 for proteins / 6 for DNA.

Homology may be at the nucleotide sequence and/or encoded amino acid sequence level. Preferably, the nucleic acid and/or amino acid sequence shares at least about 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% homology or identity with a XET nucleic acid sequence (e.g. with SEQ ID No 1). Homology may be over the full length of the relevant sequence shown herein, or may be over a part of it, preferably over a contiguous sequence of about or greater than about 20, 25, 30, 33, 40, 50, 67, 133, 167, 200, 233, 267, 300, 333, 400 or more amino acids or codons, compared with XET (e.g. with SEQ ID No.l).

Thus a variant polypeptide in accordance with the present invention may include within the amino acid sequence shown in Figure 1 (SEQ ID No 2) , a single amino acid or 2, 3, 4, 5, 6, 7, 8, or 9 changes, about 10, 15, 20, 30, 40 or 50 changes, or greater than about 50, 60, 70, 80 or 90 changes. In addition to one or more changes within the amino acid sequence shown, a variant polypeptide may include additional amino acids at the C-terminus and/or N-terminus .

In a further aspect of the present invention there is provided a method of identifying and/or cloning a nucleic acid variant from a plant which method employs a XET sequence as described herein.

For instance, nucleotide sequence information provided herein may be used in a data-base (e.g. of ESTs, or STSs) search to find homologous sequences.

Nucleotide sequence information provided herein may be used to design probes and primers for probing or amplification of XET ox variants thereof. An oligonucleotide for use in probing or PCR may be about 32 or fewer nucleotides in length (e.g. 16, 18, 21 or 24) . Generally specific primers are upwards of 14 nucleotides in length. For optimum specificity, primers of 16-32 nucleotides in length may be preferred, for example GATGAAATTGACTTTGAGTT (SEQ ID No. 4) and CATGATATACAATTATTGTac (SEQ ID NO.5) of Figure 1). Those skilled in the art are well versed in the design of primers for use processes such as PCR.

If required, probing can be done with entire restriction fragments of the cDNA sequences disclosed herein, or the full-length cDNAs themselves. Fragments may be used e.g. the sequence employed may be about 100 nucleotides or more, about 200 nucleotides or more, about 300 nucleotides or more, or about 400 nucleotides or more, in each case the sequence may be a contiguous sequence selected from those nucleic acids disclosed herein.

Naturally sequences may be based on either a XET sequence, or the complement thereof. Small variations may be introduced into the sequence to produce 'consensus' or 'degenerate' primers if required.

Such probes and primers form one aspect of the present invention.

Probing may employ the standard Southern blotting technique. For instance DNA may be extracted from cells and digested with different restriction enzymes. Restriction fragments may then be separated by electrophoresis on an agarose gel, before denaturation and transfer to a nitrocellulose filter. Labelled probe may be hybridised to the DNA fragments on the filter and binding determined. DNA for probing may be prepared from RNA preparations from cells. Probing may optionally be done by means of so-called 'nucleic acid chips' (see Marshall & Hodgson (1998) Nature Biotechnology 16: 27-31, for a review) . In one aspect, there is provided a method of obtaining a XET nucleic acid molecule which comprises:

(a) providing a preparation of nucleic acid from a cell, e.g. from plant cells,

(b) providing a nucleic acid molecule which is a nucleic acid of the invention or probe or primer therefore,

(c) contacting nucleic acid in said preparation with said probe or primer under conditions for hybridisation, and, (d) identifying said nucleic acid if present by its hybridisation with said nucleic acid molecule.

Test nucleic acid may be provided from a cell as genomic DNA, cDNA or RNA, or a mixture of any of these, preferably as a library in a suitable vector. If genomic DNA is used the probe may be used to identify untranscribed regions of the gene (e.g. promoters etc) .

Binding of a probe to target nucleic acid (e.g. DNA) may be measured using any of a variety of techniques at the disposal of those skilled in the art. For instance, probes may be radioactively, fluorescentiy or enzymatically labelled. Other methods not employing labelling of probe include amplification using PCR (see below) , RNase cleavage and allele specific oligonucleotide probing. The identification of successful hybridisation is followed by isolation of the nucleic acid which has hybridised, which may involve one or more steps of PCR or amplification of a vector in a suitable host .

Preliminary experiments may be performed by hybridising under low stringency conditions. For probing, preferred conditions are those which are stringent enough for there to be a simple pattern with a small number of hybridisations identified as positive which can be investigated further.

For example, hybridizations may be performed, according to the method of Sambrook et al. (1989; below) using a hybridization solution comprising: 5X SSC (wherein 'SSC = 0.15 M sodium chloride; 0.15 M sodium citrate; pH 7), 5X Denhardt's reagent, 0.5-1.0% SDS, 100 μg/ml denatured, fragmented salmon sperm DNA, 0.05% sodium pyrophosphate and up to 50% formamide. Hybridization is carried out at 37-42°C for at least six hours. Following hybridization, filters are washed as follows: (1) 5 minutes at room temperature in 2X SSC and 1% SDS; (2) 15 minutes at room temperature in 2X SSC and 0.1% SDS; (3) 30 minutes - 1 hour at 37°C in IX SSC and 1% SDS; (4) 2 hours at 42-65°C in IX SSC and 1% SDS, changing the solution every 30 minutes .

One common formula for calculating the stringency conditions required to achieve hybridization between nucleic acid molecules of a specified sequence homology is (Sambrook et al., 1989): T_m = 81.5°C + 16.6Log [Na+] + 0.41 (% G+C) - 0.63 (% formamide) - 600/#bp in duplex.

As an illustration of the above formula, using [Na+] = [0.368] and 50-% formamide, with GC content of 42% and an average probe size of 200 bases, the T_m is 57°C. The T_m of a DNA duplex decreases by 1 - 1.5°C with every 1% decrease in homology. Thus, targets with greater than about 75% sequence identity would be observed using a hybridization temperature of 42°C. Such a sequence would be considered substantially homologous to the nucleic acid sequence of the present invention. It is well known in the art to increase stringency of hybridisation gradually until only a few positive clones remain. For detection of sequences that are greater than about 90% identical, suitable conditions include hybridization overnight at 65 °C in 0.25M Na₂HP0₄, pH 7.2, 6.5% SDS, 10% dextran sulfate and a final wash at 60 °C in 0.1X SSC, 0.1% SDS.

An alternative, which may be particularly appropriate with plant nucleic acid preparations, is a solution of 5x SSPE (final 0.9 M NaCl, 0.05M sodium phosphate, 0.005M EDTA pH 7.7), 5X Denhardt's solution, 0.5% SDS, at 65 °C overnight, (for high stringency, highly similar sequences). Washes in 0.2x SSC/0.1% SDS at 65 °C for high stringency.

In a further embodiment, hybridisation of a nucleic acid molecule to a variant may be determined or identified indirectly, e.g. using a nucleic acid amplification reaction, particularly the polymerase chain reaction (PCR) . PCR requires the use of two primers to specifically amplify target nucleic acid, so preferably two nucleic acid molecules with sequences characteristic of XET are employed. Using RACE PCR, only one such primer may be needed (see "PCR protocols; A Guide to Methods and Applications", Eds. Innis et al, Academic Press, New York, (1990)).

Thus a method involving use of PCR in obtaining nucleic acid according to the present invention may include: (a) providing a preparation of nucleic acid, e.g. from a plant cell, (b) providing a pair of nucleic acid molecule primers for PCR, at least one of said primers being a primer according to the present invention as discussed above,

(c) contacting nucleic acid in said preparation with said primers under conditions for performance of PCR,

(d) performing PCR and determining the presence or absence of an amplified PCR product

The presence of an amplified PCR product may indicate identification of a XET sequence.

In all cases above, if need be, clones or fragments identified in the search may be extended. For instance if it is suspected that they are incomplete, the original DNA source (e.g. a clone library, mRNA preparation etc.) can be revisited to isolate missing portions e.g. using sequences, probes or primers based on that portion which has already been obtained to identify other clones containing overlapping sequence.

As used herein, nucleotide sequences discussed or obtainable as described above (including XET and variants thereof) will be referred to as "XET nucleotide sequences" or "XET nucleic acids" (and correspondingly "XET" polypeptide) unless context demands otherwise.

The methods described above may also be used to determine the presence of one of the nucleotide sequences of the present invention within the genetic context of an individual plant, optionally a transgenic plant such as may be produced as described in more detail below. This may be useful in plant breeding programmes e.g. to directly select plants containing alleles which are responsible for desirable traits in that plant species, either in parent plants or in progeny (e.g hybrids, FI, F2 etc.). Thus use of markers which can be designed by those skilled in the art on the basis of the nucleotide sequence information disclosed herein, for selection of a gene encoding a polypeptide with XET activity, forms one aspect of the present invention.

In a further aspect of the present invention, XET nucleotide sequences are in the form of a recombinant and preferably replicable vector.

'Vector' is defined to include, inter alia, any plasmid, cosmid, phage or Agrobacterium binary vector in double or single stranded linear or circular form which may or may not be self transmissible or mobilizable, and which can transform a prokaryotic or eukaryotic host either by integration into the cellular genome or exist extrachromosomally (e.g. autonomous replicating plasmid with an origin of replication) .

Generally speaking, those skilled in the art are well able to construct vectors and design protocols for recombinant gene expression. Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. For further details see, for example, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrook et al, 1989, Cold Spring Harbor Laboratory Press or Current Protocols in Molecular Biology, Second Edition, Ausubel et al. eds., John Wiley & Sons, 1992.

Specifically included are shuttle vectors by which is meant a DNA vehicle capable, naturally or by design, of replication in two different host organisms, which may be selected from actinomycetes and related species, bacteria and eucaryotic (e.g. higher plant, mammalian, yeast or fungal cells) .

A vector including nucleic acid according to the present invention need not necessarily include a promoter or other regulatory sequence, particularly if the vector is to be used to introduce the nucleic acid into cells for recombination into the genome.

Preferably the nucleic acid in the vector is under the control of, and operably linked to, an appropriate promoter or other regulatory elements for transcription in a host cell such as a microbial, e.g. bacterial, or plant cell. The vector may be a bi-functional expression vector which functions in multiple hosts. In the case of genomic DNA, this may contain its own promoter or other regulatory elements and in the case of cDNA this may be under the control of an appropriate promoter or other regulatory elements for expression in the host cell.

Plants which include a plant cell according to the invention are also provided, along with any part or propagule thereof, seed, selfed or hybrid progeny and descendants. Particularly provided are transgenic crop plants, which have been engineered to carry genes identified as stated above. Examples of suitable plants include broad leaf edible plants, including but not limited, to those selected from the group lactuca sativa, including Mizuna, lollo rosso, Frisee, rocket, wild rocket, lambs lettuce, little gem, cos, red oak leaf (salad leaf) , green oak leaf (salad leaf) , Apollo, and spinach, chard, ruby chard, and watercress, . Preferably, the broad leaf edible plant is selected from the group lactuca sativa, such as lollo rosso, cos, iceberg, little gem, Frisee, lambs lettuce, red oak leaf (salad leaf) , green oak leaf (salad leaf), and Apollo.

In a preferment, there is provided a plant comprising a plant cell according to the invention. In a further preferment, there is provided a plant comprising a plant cell of the invention wherein the percentage plasticity of plant tissue comprised of said plant cell is reduced when compared to the percentage plasticity (as herein calculated in the accompanying examples) of plant tissue of a wild type or control plant of the same type grown under similar growth conditions to that of the plant comprised of said plant cell. Typically, the percentage plasticity of plant tissue of such a plant lies in the range of from 0.50% to about 4.00%, preferably from about 0.60% to about 3.50%, and most preferably from about 0.60% to about 1.50%, when compared with a suitable control plant, eg a wild type plant and depending on the broad leaf edible plant species, growing environment, and the like.

In addition to a plant, the present invention provides any clone of such a plant, seed, selfed or hybrid progeny and descendants, and any part of any of these, such as cuttings, seed. The invention provides any plant propagule, that is any part which may be used in reproduction or propagation, sexual or asexual, including cuttings, seed and so on. Also encompassed by the invention is a plant which is a sexually or asexually propagated off-spring, clone or descendant of such a plant, or any part or propagule of said plant, off^¬ spring, clone or descendant. "Homology" in relation to an amino acid sequence of the invention may be used to refer to identity or similarity, preferably identity. As noted already above, high level of amino acid identity may be limited to functionally significant domains or regions, e.g. any of the domains identified herein.

The invention will now be described with reference to the following figures and examples. It is to be understood that the figures and examples are not to be construed as limiting the scope of the invention in any way.

FIGURES Figure 1: The genetic sequence and amino acid translation for the 500 base pair XET fragment 3B1, cloned from L. sativa cv Ravita using degenerate primers to conserved domains of known XETs . Primer sequences are shown underlined and their translated amino acids in bold type. Fragment was isolated through PCR, cloned into a vector and sequenced using BigDye terminators.

Figure 2: Degenerate primers XETf2 (SEQ ID NO. ) and XETr2(SEQ ID NO.5). Degenerate primers designed to isolate XET fragments between 500 and 600 bp in L. sativa using conserved domains in XET proteins from known XET and XTR sequences in the public domain. Restriction enzyme sites are shown underlined, the arrow indicates cleavage point of enzyme. Figure 3: A schematic section through a northern blot transfer set up. Capillary action draws the high ionic content transfer buffer through the gel transferring the RNA onto the membrane for further analysis.

Figure 4: The vector SLJ732, showing the GUS and Kanamycin resistance (nptll) genes, and location of restriction endonuclease sites used. (Jones et al . , (1992) Transgenic

Research 1:285-297.) Arrows in shaded boxes show direction of open reading frame. LB and RB: left border and right border respectively of T-DNA. HIIIK and RIK are restriction endonuclease sites of Hin dill and Eco RI respectively that have been filled in with Klenow polymerase and dNTPs.

Figure 5: Amino acid alignment for selected Arabidopsis partial XET sequences from GenBank. Meri-5 (SEQ ID NO. 9) , XTR3 (SEQ ID NO. 10) and XTR18 (SEQ ID NO.11) are group 2 XETs (Campbell & Braam, 1999) ; EXGT-A1 (SEQ ID NO. 12) , XTR12 (SEQ ID NO. 13) and XTR15 (SEQ ID NO.14) are group 1 XETs and ATXG (SEQ ID NO. 15) and EXGT-A3 (SEQ ID NO.16) are group 3 XETs. The DEIDFEFLG (SEQ ID NO.3) motif is a conserved domain across all XETs and XTRs and the cysteine residues at the C-terminal end are also highly conserved. The alignment was performed using ClustalW software. Regions to which primers XETf2 and XETr2 were designed are shown underlined in the first line only.

Figure 6: Proposed amino acid sequence alignment for XET fragments from L. sativa cv Ravita (a leaf type, lollo rosso lettuce) with XETs from the GenBank database. R31 (SEQ ID NO 20) and 3B1 (SEQ ID NO. 22) from lollo rosso, with group 2 XETs Bru~l (SEQ ID NO.21) and Meri -5 (SEQ ID NO. 17) , (that also have a pair of cysteine (C) residues absent) and XTR1 (SEQ ID NO. 18) and XTRS (SEQ ID NO. 19) , to show homology and likelihood of the cloned sequences to be XET genes.

Figure 7: Vector SLJ732-3B1. The vector SLJ732-3B1 was constructed by replacing the GUS open reading frame in SLJ732 with the XET fragment 3B1 (isolated from L. sativa cv Ravita) in an antisense orientation. The BamHI and Sacl sites (in bold) were used to facilitate this sequence exchange.

Figure 8: Cell wall plasticity and elasticity values were obtained from the chart output of an instron device. The example is a trace from a Lactuca sativa, lollo rosso type, leaf harvested into methanol, re-hydrated and subjected to consecutive loads of 20 g, A and B in the Instron. Extensibility values were calculated from the gradient of the slope and tangents to the curve at the load peak, using the equations shown.

Figure 9: XET activity was assayed using the method of Fry et al (1992), supra. Enzyme activity was reduced in plants 19_3 and 19_5 that have been shown to express the anti-sense XET-like fragment. Thus there has been a reduction in enzyme activity directly associated with the expression of the anti-sense 3B1 fragment.

Figure 10: Epidermal cell size was determined from a series of imprints made from the leaf adaxial surface. The effect of reduced XET enzyme activity in plants 19 3 and 19__5 is a reduced cell area. This is most likely to be a direct consequence of the enzymes inhibition, reducing the ability of cells to enlarge through the loosening of their cell wall.

Figure 11: Using a homemade Instron device the biophysical properties of the leaves were assayed.

Irreversible extensibility was reduced in plants 19_3 and

19_5 that had also shown a reduced XET enzyme activity. Reduced XET activity results in a less extensible and

"stiffer" cell wall.

Examples

Outline of Methodology Employed

It is known that antisense technology can be used to knock out or down- regulate a gene or a group of genes.

The antisense approach was used to reduce cell expansion by expressing an antisense XET gene fragment (see Figure 1) from lollo rosso to knock out XET gene expression involved in the loosening of the cell wall. Sense and antisense copies of the XET gene are present in the cell and double stranded RNA (dsRNA) is anticipated to form between the complementary single stranded mRNA and the single stranded antisense RNA fragment. The dsRNA complex should play a role in reducing translation of sense XET sequences to protein thereby down-regulating XET gene expression.

A cloned XET fragment from young expanding lettuce leaf tissue (lollo rosso) , grown carefully to avoid mechanical stress, in order to specifically clone a fragment from XET involved in cell expansion through cell wall loosening. XET is thought to be highly expressed at this young and expanding stage in leaf development.

RNA was extracted from lollo rosso plant leaves using a commercially available kit. An RT (reverse transcriptase) reaction was used to generate cDNA from the total RNA, again using a commercially available kit. PCR was performed on the cDNA with degenerate primers. Sequences designed to known XET sequences containing specific restriction enzyme sites to allow future handling of the sequence. These primers are novel to our design and specification, using our PCR conditions. The PCR products were cloned in a high copy number vector ligated in using commercially available ligase and sequenced using BigDye Terminator methods .

The novel lollo rosso XET-like fragment (Figure 1) will be inserted into the leaf crops of interest using a transformation protocol using the Agrobacterium tumefaciens vector. The gene is inserted into the vector in an antisense orientation under the control of constitutive promoter CaMV35S.

The vector SLJ732 (Jones et al . 1992 ) was prepared by removing the GUS open reading frame with specific restriction enzymes and the XET fragment was ligated in, in an antisense orientation. The vector was grown in E. coli, purified using a commercially available kit and transformed into A. tumefaciens by electroporation.

Agrobacterium is then used to transfer the gene into the plant's genome and successful transformants selected, screened for phenotype and genotype and a succession of generations maintain to develop a stable transgenic line.

Cotyledons of lettuce plants can be transformed using the method of Curtis et al . (1994) and Ti plants regenerated and seed collected. Standard transformation protocols may be used to transform most leafy crops.

Transformation of lollo rosso

The method of Curtis et al . (1994) was followed with the following modifications as outlined below and detailed further hereunder. Selection was on 50 mg l^-1 kanamycin media. The transformation vector was cultured for 48 hours in minimal A media supplemented with tetracycline

(5 mg 1^_1) . The Agrobacterium was pelleted and resuspended in a small volume of liquid UM media and the wounded cotyledons were dipped in this suspension for 3 seconds before continuing with the method of Curtis et al . (1994) . Growth room conditions were: 26°C ±1°C, 16 hour photoperiod, white fluorescent light at 40-80 μmol πf² s^"1. Glasshouse conditions were: 16 hour day, supplementary lighting, set to vent at 20°C at day and 14°C at night, good air circulation and regular watering from the base.

Plants were transferred to the glasshouse as soon as possible once in soil to minimise early bolting. Transferred plants were maintained in a propagator for 3 weeks opening the vents sequentially during that time. A unique soil mix of 2 parts vermiculite and 1 part Levingtons F2 was used.

The T₂ generation was screened by germinating the seed on soil and at the first true leaf stage selected for kanamycin resistance (and thus the XET fragment) by application of the antibiotic in a daily spray. We increased the dose of the antibiotic over the initial growth period of 4 weeks. The resistant plants grew faster than the non-resistant, non-transgenic plants that displayed a visible phenotype where they lost all their pigmentation.

Results

The following are results from the T₂ generation showing a phenotype and genotype as anticipated.

Indications that the T2 generation will have improved processability include evidence that the plants will have reduced XET activity, resulting in reduced epidermal cell area and reduced extensibility.

It is known from previous work on non-transgenic plants as described in Clarkson et al (2003) that a 50% increase in shelf-life was linked to a 25% reduction in cell area. In the present XET work cell area was reduced by approximately 20% so we should expect that there should be an increase in shelf-life as a result of improved processability.

In future generations it is expected to be able to breed a true type plant from line 19_3 or line 19_5 with an increased processability. Such plants will show the characteristics of the parents 19_3 and 19_5: a reduced XET activity resulting in smaller cells, less plastic leaves and as a result an increased shelf-life post processing. Molecular biology methodology and transformation techniques

1 Preparation of competent Escherichia coli cells

E. coli DH5α cells, were streaked out on an L broth plate and incubated overnight at 37 °C to produce single colonies. The plate was wrapped in Parafilm M (American National Can, Chicago, USA) and stored at 4 °C.

A single colony was transferred to 5 ml of liquid L broth and cultured overnight in a 20 ml tube, at 37 °C, on a shaker. In a sterile 500 ml conical flask, 100 ml of fresh L broth was diluted with 1 % (v/v) of the overnight culture and incubated at 37 °C until the OD₆oo was approximately 0.7. The culture was cooled on ice in polypropylene tubes, and transferred into a cold room (4 °C) to complete the preparation. The bacteria cells were recovered by centrifugation at 4 °C in a pre-cooled Sorvall SLA600-TC rotor, for 10 min at 2600 g. The supernatant was removed and the pellet resuspended in 20 ml of ice cold TFB (transformation buffer) by gentle vortexing, and recovered as previously, before finally being resuspended in 4 ml of TFB containing 10 % (v/v) final cone, glycerol. The competent cells were snap frozen as 100 μl aliquots and stored at -80 °C in preparation for transformation.

2 Heat shock transformation of competent E. coli cells

Transformation vectors were resuspended in 10 μl of lx TE buffer. An aliquot of prepared competent cells (2.3.1) or 50 μl of competent cells (GibcoBRL, GibcoBRL Life Technologies Ltd., Paisley) were defrosted on wet ice. 2 μl of the resuspended vector was added and the tube held on ice for 30 min. A heat shock of 42 °C for 90 sec was applied, 1 ml of L broth was added and placed on a shaker at 37 °C for 1 h. The cells were recovered by a flick spin, the supernatant poured off and the cells resuspended in the remaining supernatant with a pipette.

This suspension was spread on an L broth plate supplemented with either 5 mg l^"1 tetracycline or ampicillin 50 mg 1^_1 dependent on the antibiotic resistance markers present on the vector. Plates were incubated overnight in the dark at 37 °C and when single colonies were formed the plates were stored in the dark at 4 °C.

3 Purification of plasmids from E. coli

Plasmids were purified from mega preps of E. coli cells (100 ml L broth supplemented with 5 mg l^"1 tetracycline in a 500 ml flask, shaken overnight at 37 °C) using NUCLEOBOND^® AX100 cartridges (ABgene, Epsom) following the manufacturer's instructions, by filtration to clarify the bacterial lysate. Purified plasmid DNA was redissolved in 100 μl of lx TE buffer and determined on an agarose gel (2.3.4) following restriction enzyme digests (2.3.5).

4 Agarose gel electrophoresis

DNA and RNA products were determined on agarose gels. 0.7 - 1.5 % (w/v) agarose (Sigma) was melted in lx TAE buffer and cooled to 65 °C, 2 μl of ethidium bromide stock solution was added, the gel cast and allowed to set. lx TAE was used as the running buffer, samples in lx loading dye were "wet" loaded and the gel run until the loading dye had moved sufficiently to separate the fragments. 1 Kb DNA ladder (GibcoBRL) and known concentration standards were run when needed. Images were captured using an Alpha Imager System (Flowgen, Shenstone) .

5 Restriction ensyne digest

DNA was cut with restriction enzymes (Promega UK Ltd., Southampton) , in the appropriate buffer at the optimal temperature, following the manufacturer's guidelines. Sac I and BamE I were used in the supplied multi-core enzyme buffer at 37 °C.

6 Transformation of Agrόbacterivαn tvααefaciens by electroporation

Electrocompetent A. tumefaciens strain LBA4404 cells (GibcoBRL) were electroporated using an E coli Pulser (BioRad, Bio-Rad Laboratories Ltd., Hemel Hempstead) connected to a Gene Pulser (BioRad) with 2 μl of plasmid (an excess volume) at 12.5 kv cm^-1. L broth was added instantly after electric shock treatment and cells left to recover for 2 h at room temperature in the dark. Cells were pelleted in a bench centrifuge, the supernatant removed, the cells resuspended in the remaining media and spread on an L broth^* plate supplemented with 5 mg 1^_1 tetracycline. The plates were incubated in the dark at 28 °C. A single colony was selected and incubated for 48 h in 10 ml L broth and 5 mg l^"1 tetracycline at 28 °C in the dark on a shaker and streaked out on L broth and tetracycline agar plates that were incubated at 28 °C in the dark.

Glycerol stocks of the Agrobacterium were prepared by taking 750 μl of the liquid culture and adding 250 μl 60

% (w/v) glycerol to give a final glycerol stock of 15 % to store at -80 °C. The streaked plate was sealed and stored at 4 °C.

7 Regeneration of lettuce plants in vitro

Seeds were germinated and cotyledons removed and plants regenerated by the method of Curtis et al . (1994).

Cotyledons were scored on their abaxial surface using a scalpel blade and floated for 10 min on liquid UM medium

(Uchimiya and Murashinge, 1974) . Sterile 7 cm diameter

Whatmann filter papers were soaked in UM medium and placed on 9 cm Petri dishes containing 20 ml UM agar medium. The cotyledons were blotted dry and placed scored surface down on the filter paper, 8 per dish. A dry filter paper was placed on top to keep contact between media and cut surface. The cotyledons were incubated in the conditions described (4, above) for 48h.

Explants were transferred on to Petri dishes containing 20 ml solid shoot initiation media (SI medium) with the petiole end of the cotyledon entered into the media and the abaxial surface facing the medium, and incubated as before. The explants were subcultured every 14 d on 3 occasions. Explants exhibiting callus or shoots were moved, 1 explant per jar, into 175 ml "baby-food" sterile glass jars containing 40 ml SI media, supplemented with 0.11 % (w/v) 2- [N-morpholino] ethanesulphonic acid (MES) to permit further shoot growth.

Shoots of 1 cm were excised from the callus and transferred to Magenta GA-7 boxes (Sigma) with 90 ml of rooting medium. Three shoots were rooted in each box. Rooted shoots were carefully removed from the media, the media washed off using water, and planted in 9 cm pots containing sterile compost (medium grade Vermiperl Vermiculite: Levingtons F2, 2:1; v/v). The pots were enclosed in polythene bags and incubated in a growth room (23 ±2 °C with a 12 h photoperiod and white fluorescent light at 30 μmol m^~2 s^-1) . At 7 days the first corner of the bag was removed, after 14 d the second corner was removed and after 21 d the bag was removed and the plant allowed to set seed in a contained glasshouse.

Transformation techniques

8.1 Transformation method 1

Liquid cultures of Agrobacterium were prepared by selecting a single colony into 10 ml of L broth supplemented with 5 mg l^"1 tetracycline, in a sterile 20 ml universal tube. The culture was grown in the dark for 48 h, at 28 °C, on a horizontal rotary shaker set to 180 rpm. The OD₆₀o was measured on a spectrometer and the culture diluted with liquid UM medium, to be equivalent to a reading of 1.1-1.6 ODeoo prior to a 1:10 (v/v) dilution (Curtis et al . , 1994).

Scored explants were exposed to the Agrobacterium diluted with UM medium, and plants regenerated using protocol 2.3.7. SI media was supplemented for selection of transformants with 50 mg l^-1 kanamycin monosulphate, 500 mg l^"1 carbenicillin and 100 mg I^"1 cefotaxime (all Melford Labs., Ipswich). Carbenicillin was not used in the baby- > food jars and 100 mg l^"1 kanamycin was used in the rooting medium. Controls using 0 mg 1^_1 kanamycin and no Agrobacterium were incorporated.

8.2 Transformation method 2

Modifications were made to the method described in transformation 1 (8.1). An A. tumefaciens single colony was liquid cultured in 5 ml minimal A media supplemented with 5 mg 1^_1 tetracycline. After a 48 h culture period the media and tetracycline were removed by pelleting the cells at maximum speed for 5 min in an Eppendorf centrifuge 5415D, using an F45-24-11 rotor (Fisher) . The cells were resuspended in 15 ml liquid UM medium and transformation carried out as described above. Additional trials were made using cotyledons that were not scored but had the tip and base removed to produce a squared piece of tissue.

8.3 Transformation method 3

A final modification was made to the transformation method. Agrobacterium potency was maintained by re- streaking on minimal A media plates (made solid with 0.8 % agar (w/v) ) , supplemented with 5 mg l^-1 tetracycline, prior to selection into liquid culture. Liquid cultures were grown for 48 h, as previously described, in minimal A and resuspended in 5 ml liquid UM medium. Cotyledons were exposed to the transformation vector by dipping the scored cotyledon in the liquid culture for 3 sec before blotting dry. The regeneration protocol was then followed as described using kanamycin selection. Rooted plants were transferred to soil and directly into the glasshouse in 9 cm pots, enclosing the plants in a mini propagator to reduce plant bolting at early stages. Vents were opened after 7 and 14 d, the propagator lid was removed after 21 d and plants were re-potted into 15 cm pots to set seed.

9 GUS activity histochemical stain

Leaves from regenerated plants exposed to Agrobacterium were tested for the activity of the GUS gene. A section of leaf (10x10 mm) was vacuum infiltrated with 1 ml X- Gluc stain for 5 min. The leaf was left in the stain, in the dark, overnight at 37 °C. The stain was replaced with 70 % ethanol and the extent of the stain revealed. As a control GUS positive Arabidopsis were assayed.

10 On-line sequence analysis

Amino acid sequence alignment was carried out on a personal computer using Clustal W, a multiple alignment software program (Thompson et al . 1994) via the Pasteur Institute's website. (http: //bioweb.pasteur . fr/seqanal/interfaces/clustalw- simple.html on 17/06/02.)

Multiple sequences were loaded to a single file through "loadseq" (http: //bioweb.pasteur. fr/seqanal/interfaces/loadseq- simple.html on 17/06/02) . The Clustal W alignment on the loadseq.out file was performed using the default settings and the alignment output (loadseq. loadseq.aln) entered into the "boxshade" program

(http: //bioweb.pasteur. fr/cgi-bin/seqanal/lib/connect .pl on 17/06/02) with output set to rich text format and 30 characters per line.

The resulting file (boxshade. result) was saved to disc and inserted into a Microsoft Word document (Microsoft Corp., Seattle, USA) for viewing, analysis and manipulation.

11 RNA extraction

Total RNA was extracted from lettuce leaf material using Qiagen RNeasy kit following the manufacturer' s instructions. 50-100 mg of fresh weight tissue was used and ground to a powder using liquid nitrogen in a microfuge tube. Buffer RLT was used and RNA was eluted in 30 μl RNase-free H₂0. 1 μl of the RNA was determined on an agarose gel and approximately quantified using known weight markers (taking into account that RNA stains with half the intensity of DNA) . Samples were stored at -80 °C.

12 Reverse transcriptase (RT) reaction

cDNA from the total RNA was prepared by an RT reaction. 5 μg of extracted RNA in 9.5 μl H₂0 was denatured at 70 °C and quenched on ice. To this 4 μl 5x 1^st strand buffer and 2 μl 0.1 M DTT (a stabiliser) were added from a

Superscript-RT kit (GibcoBRL) plus 2 μl 5 mM dNTPs; 1 μl

RNase inhibitor (5 U μl^-1) ; 1 μl Oligo-dT primer (500 ng μl^"1) , from a Reverse-iT kit (AB Gene, Epsom) . The reaction was heated at 37 °C for 2 min, 0.5 μl

Rtranscriptase added (Superscript-RT kit) , and the reaction returned to 37 °C for 60 min. The cDNA was cleaned up with microCLEAN (Microzone Ltd., Lewes) using the manufacturer's instructions, and stored at -20 °C.

13. DNA Extraction

10x10 mm sections of lettuce leaf material were harvested from whole plants into microfuge tubes, frozen in liquid nitrogen and stored at -80 °C until extraction. The tissue was ground to a powder in liquid nitrogen using a pestle and 500 μl of nuclear extraction buffer was immediately added. Subsequently 100 μl of 5 % (w/v) sarkosyl was added, the tube inverted 5 times to mix the sample and incubated at 65 °C for a minimum of 20 min, inverting occasionally.

A phenol/chloroform extraction was performed on the sample. 500 μl phenol/chloroform was added, the tube inverted 20 times and the phases separated with a maximum speed spin in the microfuge described in 8.2, above. The upper phase was transferred to a clean tube containing 300 μl isopropanol, inverted 5 times, and centrifuged at maximum speed for 1 min to recover the genomic DNA. The supernatant was removed, the pellet of DNA washed in 300 μl 70 % ethanol and centrifuged again for 1 min at maximum speed. The supernatant was carefully removed and the pellet dried at room temperature. The dry pellet of gDNA was resuspended in lx TE and determined on an agarose gel with known weight standards to approximately quantify. gDNA was stored at -20 °C.

14 Gradient PCR

PCR was performed using a Peltier thermal cycler, the PTC-225 DNA Engine Tetrad Cycler (Genetic Research Instrumentation, Braintree) . Minimum and maximum annealing temperatures were estimated for the degenerate XET primers by adding 4 °C for each G or C and 2 °C for each A or T. Annealing temperatures were subsequently examined experimentally using gradient PCR. Conditions for the reactions were: hot start at 94 °C for 2 min, 35 cycles of [94 °C (20 s) ; 42-56 °C (20 s) ; 72 °C (90 s) ] and an extended elongation of 72 °C for 5 min. Each reaction used 1 unit AGSGold Taq polymerase, lx polymerase enzyme buffer, 10 % (final volume) enhancer (Hybaid, Ashford) ; 200 μM dNTPs; 2 μM XETf2 and XETr2; approximately 70 ng gDNA; AnalR water (BDH Lab. Supplies, Poole) to a final reaction volume of 20 μl. Products were visualised by agarose gel electrophoresis to determine the optimum annealing temperature.

15 XET PCR

XET PCR with degenerate primers (Figure 2) as carried out with a hot start of 94 °C for 2 min, 35 cycles of [94 °C (20 s); 48°C (20 s) ; 72 °C (90 s) ] and an extended elongation of 72 °C for 5 min. Each reaction used 2 units Pwo DNA polymerase (Roche) , and lx buffer (including MgS0₄); 2 μM XETf2 and XETr2; 200 μM dNTPs; 1-5 μl cDNA; AnalR water (BDH Lab.), to give a final reaction volume of 40 μl. The PCR was performed in Gene-Amp PCR System 9700 (PE Applied Biosystems Ltd.). Products were visualised by agarose gel electrophoresis.

16 Cloning vector preparation

A high copy number cloning vector pGEM 3Zf(+) (Promega) was used to clone and sequence XET fragments. The vector was restricted with Sac I for an hour at 37 °C in multi- core buffer and BamH I for the second hour. The restricted vector was visualised by agarose gel electrophoresis, the band excised and electro-eluted.

17 Electro-elution of gel separated fragments

Bands were excised from agarose gels on a UV transilluminator with a clean scalpel blade, talking care to keep the agarose to a minimum. A clean dialysis tube, containing the gel fragment and 500 μl lx TE, was placed between 2 dialysis clips. Care was taken to remove any air bubbles. The gel fragment was moved to one side of the elution tube, toward the negative electrode, and placed in a gel tank under lx TAE running buffer. Current was passed through the tank at 120 V for 1 h. The charge was reversed for 20 sec to take the product away from the side of the dialysis tube and the buffer containing the nucleic acid removed to a new microfuge tube.

The gel eluted product was cleaned sequentially with phenol, phenol/chloroform and an ethanol precipitation. An equal volume of phenol was added, mixed and the tube centrifuged to separate the phases at top speed for 1 min. The top layer was transferred into a microfuge tube containing an equal volume of phenol/chloroform, the contents mixed by inverting the tube 20 times and centrifuged as before. The top layer was transferred to a third tube containing 10 % volume 3M sodium acetate (pH

5.2) and 1 μl glycogen, and mixed with the pipette. 2.5x volume 100 % ethanol were added and tubes held overnight at -20 °C to precipitate.

The precipitated product was collected by centrifugation at maximum speed on a bench centrifuge for 20 min. The supernatant was removed and the pellet washed in 70 % ethanol (as in 13, above) and re-suspended in 20 μl lx TE. The concentration was determined by spotting the 0.5 μl of the product on an ethidium bromide plate (agarose gel containing ethidium bromide in a Petri dish) with known standards or by running 1 μl on an electrophoresis agarose gel with standards to estimate the quantity.

18 Ligation of vector and PCR product

Eluted and cleaned PCR products were cut with BamR I and Sac I for 2 h at 37 °C, phenol/chloroform extracted and ethanol precipitated. Ligation reactions were carried out overnight at approximately 14 °C. Reactions were performed using T4 DNA ligase (GibcoBRL) , with a molar excess of vector to insert ratio (>2:1), in a 7 μl reaction. 19 Blue/white screen of transformed E. coli

To select for positive ligation and heat shock transformation of E. coli cells the transformation mix was streaked out on L broth plates supplemented with 50 mg l^-1 ampicillin and with a layer of 1.2 mg X-Gal (5- Bromo-4-Chloro-3-Indolyl-Beta-D-Galactopyranoside) and 0.8 mg IPTG (isopropyl β-D-thiogalactoside) . Plates were incubated in the dark overnight at 37 °C and screened for blue or white colonies. White colonies indicate an insert into the lacZ α-peptide coding region of the vector, preventing the production of β-galactosidase by α-complementation.

20 Universal primer PCR screen of transformed E. coli

Selected white colonies from the blue/white screen were grown in 400 μl liquid cultures of L broth plus 50 mg l^"1 ampicillin, on a shaker at 37 °C for 2 h. PCR was performed directly on 1 μl of the culture mix.

Conditions for the reactions were: 35 cycles of [94 °C

(20 s); 53 °C (20 s) ; 72 °C ( 40 s) ] and an extended elongation of 72 °C for 5 min. Each reaction used 1 unit

AGSGold Taq polymerase, lx polymerase enzyme buffer, 10 % (final volume) enhancer; 200 μM dNTPs; 0.5 μM M13 universal forward and reverse primers (Applied Biosystems) ; 1 μl culture; AnalR water, to a final reaction volume of 16 μl. Products were determined on a gel to visually determine if there was an insert.

21 Sequencing preparation

Liquid cultures that displayed bands of interest following universal primer screen were added to 4 ml of 50 mg l^"1 ampicillin supplemented L broth, in a 20 ml sterile universal tube and cultured overnight at 37 °C on a shaker. The culture was pelleted by centrifugation for 2 min at 2600 g, the supernatant removed and the bacterial pellet resuspended in 200 μl Sol .1 buffer, pipetting up and down. 300 μl of fresh 0.2 M sodium hydroxide / 1% (w/v) SDS was added and mixed by inversion with the bacterial suspension until clear. The cells were left to lyse on ice for 5 min, the solution neutralised with 300 μl 3 M potassium acetate (pH 4.8), mixed by inversion and incubated on ice for a further 5 min. Cellular debris was removed by centrifugation for 10 min at maximum speed on a bench-top centrifuge and 700 μl of the supernatant recovered and RNase A treated at 37 °C for 20 min. (RNase A final concentration of 20 μg ml^"1.) DNA was precipitated with an equal volume of 100 % isopropanol and centrifuged as previously. The pellet was washed with 70 % ethanol, resuspended in 50 μl TE and cleaned up with a NucleoSpin column (AB gene) following the manufacturer's instructions.

22 BigDye Cycle sequencing

Sequencing PCR reactions were carried out using the ABI Prism BigDye Terminator method (Perkin Elmer, USA) . In brief, reactions were set up with forward and reverse primers for each DNA preparation using 4 μl of 1/10 stock sequencing mix, 1 μl primer, 3 μl DNA and water to 10 μl. The conditions for the reactions were: [96 °C (10 s) ; 50 °C (5 s); 60 °C (4 min)] for 25 cycles.

Post PCR the reaction volumes were doubled to 20 μl with water, l/10^th volume of low pH sodium acetate was added and 2.5x volume 100 % ethanol to precipitate the products in the dark for >30 min at room temperature. The pellet was recovered by centrifugation for 20 min, washed in 70 % ethanol, the ethanol removed and the pellet dried.

23 Sequencing and analysis

Sequencing reactions were run on an automated ABI Prism 377 DNA sequencer (Applied Biosystems, California, USA) . The sequence data were visualised using ABI Sequencing Analysis 3.3 and consensus sequences assembled using ABI Auto Assembler 2.1 software.

24 Production of SLJ732-3B1

Plasmid SLJ732 and the vector pGEM containing 3B1 were restriction digested with Sac I and then BamH I, cleaned by electro-elution and a nucleopsin column respectively and ligated as described before (18, above) using a molar excess of insert, (5:1 insert : ector) , resuspending in 3 μl of TE for use in electro-transformation. The plasmids were electro-transformed into E. coli (25, below) and screened by XET PCR for the insert (15, above) . Positive screened cultures were set up for plasmid purification (3, above) and following test digests to confirm the plasmid and insert were present A. tumefaciens cells were electro-transformed (6, above) .

25 Electro-transformation of E. coli

Electro-competent E. coli cells (Electromax DH10B cells, GibcoBRL) were thawed and placed on ice for transformation. The protocol used was similar to that used in the electro-transformation of Agrobacterium cells (6, above) . 25 μl of cells were mixed with 2 μl plasmid and rested on the ice for 1 min. The cells and DNA were transferred to a chilled electroporation cuvette and pulsed once at 12.5 kv cm^"1. One ml of SOC medium was instantly added and the cells resuspended by gentle inversion. The culture was then incubated at 37 °C for an hour on a shaker, recovered, spread on a series of L broth tetracycline (5 mg I^"1) plates and incubated in the dark at 37 °C overnight.

26 nptll fragment PCR

Primers to a fragment of the nptll gene (1: 5'GTCGCTTGGTCGGTCATTTCG3' (SEQ ID NO 6); 2:

3'GTCATCTCACCTTGCTCCTGCC5' (SEQ ID NO 7) Curtis et al . ,

1994) were used to detect the presence or absence of the gene in gDNA from transformed lines. Reactions contained lx BioRed 2.0 mM MgCl₂ (Bioline, London), 0.5 μM primer 1, 0.5 μM primer 2, 1 μl gDNA and distilled water to 20 μl .

Conditions were: 95 °C (10 min) and 40 cycles of [95 °C

(45 s); 58 °C (40 s) ; 72 °C (2 min)] and 72 °C (3 min).

Products were visualised on an agarose gel.

27 Quantification of RNA by absorption

Amounts of RNA extracted were quantified accurately using a spectrometer. Wavelength of 1 μl sample in 250 μl across a 0.5 cm path was recorded from 300-200 nm. A₂βo was used to calculate the quantity of RNA from the equation below (derived from the Lambert-Beer law) .

Cone (μg/μl) of RNA = A₂₆o x 20 28 Denaturing RNA agarose gel

A denaturing agarose gel was prepared by dissolving 1.5 % (w/v) agarose in water, before the addition of lx MOPS and cooling to hand warm. 40 % (v/v) formaldehyde was then swiftly added, swirling in the formaldehyde and rapidly pouring the gel in a fume hood. The gel was allowed to set.

Samples of 10 μg total RNA in 10 μl water were added to 16.25 μl RNA gel loading buffer and held at 65 °C for 10 min, and transferred on to ice prior to gel loading. The gel was loaded and using lx MOPS as the running buffer was run at 40 V h^"1 until the dye band had travelled a suitable distance.

29 Northern transfer of RNA

The capillary transfer of RNA from the denaturing gel to an Amersham Hybond-N membrane (Amersham Biosciences U.K. Ltd., Bucks) was set up as shown in Figure 2.4. In brief, the gel was trimmed to the size used and a corner removed for orientation purposes. Whatmann 3M paper strips were cut to size, 2x for wicks and 5x gel piece size. The transfer set up was built with care to leave no air bubbles and left overnight to transfer. The transfer was dismantled and the membrane was baked at 80 °C for >2 h, RNA side up and stored in a dry box prior to hybridisation. (Figure 3) .

30 Blot hybridisation

The membrane was wetted in 6x SSC in a lunchbox container and given a 4 h pre-hybridisation treatment at 42 °C on a shaker using just enough pre-hybridisation buffer to cover the membrane. A probe was prepared with Rediprime II (Amersham Biosciences) following the manufacturer's instructions using a PCR product cDNA probe and radioactively labelled with ³²P dCTP (Amersham Biosciences) . The pre-hybridisation was replaced with hybridisation buffer and the probe, and hybridised overnight at 42 °C on a shaker.

A series of stringency washes were performed on the membrane to first reduce the background radiation by removing the non-specifically bound probe (high salt, low temp) and secondly remove unwanted low homology hybrids

(lower salt, higher temp) . Wash 1 was at room temperature with 5x SSC, 0.1 % SDS. Wash 2 was at 42 °C, for 15 min on a shaker with 5x SSC, 0.1 % SDS. Wash 3 was as wash 2 with 2x SSC, 0.1 % SDS. Wash 4 was for 10 min at 42 °C with lx SSC, 0.1 %SDS, and the radioactive counts were checked before the final fifth wash for 10 min at 42 °C with 0. lx SSC, 0.1 % SDS. All excess fluid was removed from the membrane, the membrane wrapped in cling film and exposed to film for a suitable time period.

31 Stripping and re-hybridising membrane with 18S rRNA probe

To re-probe a membrane with a control probe the membrane was stripped by washing twice with water at 80 °C for 20 min. The blot was then hybridised as before using an 18S rRNA probe to normalise for loading differences. The probe was an oligo designed to anneal to the 18S rRNA of flax and was obtained by EcoR I and Kpn I digest of plasmid pBG35S to release a 1.5 kb fragment (Goldsbrough and Cullis, 1981) . Levels were normalised to 18S levels using spot density analysis in AlphaEase (Flowgen) .

Biochemical analysis of 2SE

1 SζET e zyme activity assay

The activity of the enzyme XET was determined using the method of Fry et al . (1992) Biochemical Journal 282:821-

828, a widely used and sensitive assay for XET activity

(Fry, 1997) The Plant Journal 11:1141-1150. The assay uses XET to join a radiolabelled oligosaccharide

(acceptor substrate) to a xyloglucan polysaccharide (donor substrate) . After incubation the reaction is stopped and spotted onto filter paper, washed and scintillation counted for the radiolabel. Unincorporated labelled acceptor does not hydrogen bond to the filter paper because of its low molecular mass and is washed off, and therefore only the labelled donor-acceptor product is detected by the counter, producing a quantifiable measure of XET activity in terms of label counts .

XET was extracted from fresh leaf tissue discs of a known weight (approx 30 mg) in ice cold XET extraction buffer at a ratio of 4 parts buffer to 1 part tissue by homogenisation and incubation for 1 h on ice. The extraction mix was centrifuged and the supernatant used as the XET preparation for all assays. For each assay 30 μl of XET preparation was mixed with 20 μl XET assay mix and incubated at room temperature for 45 min. The reaction was stopped with an equal volume of formic acid and spotted on to 4x4 cm squares of Whatmann 3M paper and dried at room temperature. Filter paper squares were washed overnight, dried at 105 °C and scintillation counted for [³H] on a Beckman LS6500. XET enzyme activity was presented as [³H] counts per min unit fresh weight.

2 Total protein quantification

Protein levels were assayed using Bradford reagent (Bradford, 1976) . 10 μl of each sample was assayed in a well of 96-well plate with known standards of BSA 0.5-10 μg in the same buffer to produce a standard curve. 100 μl of reagent was added and absorbance read at 570 nm before a precipitate formed.

3 Co-localisation of endogenous XET enzyme activity and donor substrate

XET action was localised using a donor substrate of a sulforhodamine conjugate of xyloglucan oligosaccharide (XGO-SR) vacuum infiltrated into leaf tissue sections. The method used followed that described in Vissenberg et al . (2000) The Plant Cell 12:1229-1237 and the XGO-SR used (Fry, 1997) was provided as a suspension of 90 μM substrate in 1 ml 25 mM MES buffer (pH 5.5) from ICMB, University of Edinburgh, Edinburgh. A 4x4 mm² section of leaf tissue was vacuum infiltrated with 100 μl of fluorescent substrate in a 1 ml syringe using 4 times an up and down action, and incubated in the substrate for 2 h at room temperature. The tissue was transferred to 70 % ethanol overnight to remove the unincorporated, ethanol soluble, XGO-SR and washed 3 times in 70 % ethanol before storing in 70 % ethanol, in the dark, prior to microscopy. Fluorescence and transmission pictures were made from tissue sections using a Bio-Rad 1024 ES confocal laser scanning microscope. A krypton/argon laser at 568 nm was used, at low intensity, for excitation of sulforhodamine and fluorescence was collected with at 585 nm long pass filter. A range of Z-series images were collected and observed in Confocal Assistant version 4.02 (Todd Clarke Brelje) . MetaMorph imaging system (Universal Imaging Corp., PA, USA) was used to compare fluorescence between control tissue and sample sections when the gain and iris settings on the confocal microscope were identical.

Results

Establishment of a transformation system

The lollo rosso cultivar Valeria had a germination efficiency of 99%. Lollo rosso was chosen as the target for transformation partly because the variety has good germination in vitro and poor shelf-life.

The success of any tissue culture system is dependent on the suitability of the selection system used. The antibiotic kanamycin was chosen for selection in the system and its applicability was determined by testing a range of kanamycin concentrations in the regeneration media and determining at what concentration untransformed cotyledons will not produce callus (data not shown) . Kanamycin at 50 mg I^"1 was used for selection of kanamycin resistant transformants.

The vector SLJ732 (Figure 4) containing the selectable marker gene nptll for kanamycin resistance, was obtained from the Sainsbury Lab, Norwich for use in transformation. The vectors were successfully transformed, by heat shock, into competent cells of Escherichia coli to bulk up the plasmid prior to cleanup, verification by restriction digests (data not shown) and transfer by electroporation into the transformation vector Agrobacterium tumefaciens . To test the transformation system two independent transformations were performed with Agrobacterium containing SLJ732 selected because of the right border orientation of the GUS gene. This was important because when the T-DNA is incorporated into the genome the right border is inserted first with higher precision than the left border. Thus shoots selected on kanamycin media are likely to contain the complete XET fragment. In the first transformation, cotyledons of Valeria were exposed to the Agrobacterium containing the SLJ732 construct using the method of Curtis et al . (1994) J. Exp. Botany 45:1441-1449.

Table 1 Shoot, callus and transgenic seed setting plants regeneration from L. sativa cultivars exposed to A. tumefaciens containing the vector SLJ732 or SLJ732-3B1. Cotyledons all treated as Curtis et al . (1994) .

Transformation event Cotyledons % Callus % Shoots Positive with exposed to (on 50 mg (on 50 mg transgen

A. tumefaciens vector r¹ r¹ ic kanamycin) kanamycin) plants

Valeria + SLJ732- 80 53 44 19

3B1 (i)

Valeria + SLJ732- 104

3B1 (ii)

Valeria + SLJ732- 64

3B1 (iii)

Valeria + SLJ732 (2) 50 38 Agrobacterium was cultured in minimal A media supplemented with magnesium to produce a less "clumpy" bacterial suspension and permit an improved exposure between cotyledon and transformation vector for a second transformation (2 in Table 1) . Using the scoring method of Curtis et al . (1994) Valeria developed callus on 4 explants, 3 producing shoots from 8 cotyledons.

Two plants of Valeria were successfully regenerated after 4 months in tissue culture and leaf sections were stained for activity of the GUS gene (data not shown) . One of the plants stained positive, from a total of 24 cotyledons exposed to the vector.

XET analysis and sequencing from L. sativa, "lollo rosso'

A native XTH gene fragment was cloned from young leaves of lollo rosso to facilitate future transformation. A selection of amino acid sequences representing the 3 main groups of Arabidopsis XETs (groups 1, 2 and 3; Campbell and Braam (1999) Trends in Plant Science 4:361-366), were aligned to locate conserved domains. Degenerate oligonucleotide primers were designed to two regions for subsequent PCR (Figure 2). The DEIDFEFLG (SEQ ID NO 3) domain is highly conserved and was selected for our forward primer. A MIYNYCT (SEQ ID NO 8) region is also conserved, following a region of sequence divergence, and was selected for our reverse primer. These primers (XETf2 and XETr2, Figure 2) flank a region of divergence toward the carboxyl terminus of the enzyme that allows for classification into groups and are thought to allude to specific function. The primer pair was designed to conserved regions of the sequences and as a result antisensing may lead to the down regulation of several specific XETs or an entire group of XETs. The inclusion of the divergent region between the primers should permit a more targeted down regulation of specific XETs that have modifications in the less conserved, function specific, protein domain, towards the C terminus.

To facilitate cloning restriction enzyme sites were incorporated into the 5' ends of the PCR primers. BamKl was incorporated into the forward (f) primer and Sstl into the reverse (r) primer, so that any XET inserts could be cloned into the vector SLJ732, replacing the GUS open reading frame (Figure 4) . Specific selection of this restriction site and primer combination ensured that any cloned XET fragment would be transcribed in an antisense orientation. Three extra bases (ATG) were added in front of the restriction sites to optimise restriction, as many restriction enzymes have reduced cleavage activity at the end of DNA fragments. The degenerate PCR primers were tested over a range of annealing temperatures considering the base pair ratios of the primers (42°C-56°C) and 48 °C was selected as the annealing temperature for future PCR with the XET degenerate primer set (data not shown) .

Multiple efforts were made to clone XET fragments from lollo rosso. RT-PCR using the designed primers was performed on total RNA extracted from young, yet established, expanding leaves of Ravita that had been grown with care to avoid mechanical stimulation and activation of the touch related wall strengthening genes. All the PCR products were separated on agarose gels (data not shown) and fragments of approximately 500-600 bp were isolated from the gel, cloned using a pGEM vector in E. coli and sequenced using BigDye terminators. Many of the sequenced clones contained no insert or produced poor sequence data as a result of being from either a mixed E. coli colony or an escape from the blue-white selection. Of the readable sequence a range of products were sequenced. Many of the cloned fragments showed homology to a maize zein protein, a class of storage protein, and two showed homology to XET sequences. The sequencing results are shown in Table 2, with homology to published sequences from BLAST search results summarised. A consensus sequence of the 0.5 Kb XET-like 3B1 fragment was produced and it contained the XET primer regions and both restriction sites to facilitate cloning and insertion into the SLJ732 vector.

Of all the RT-PCR products cloned and sequenced only 2 fragments, R31 (sequence not shown) and 3B1 showed sequence homology to known XETs, with most of the fragments sequenced not showing any homology to any XET sequence. The 2 XET-like fragments were different and were aligned to the region between the PCR primers to known XET sequences (Figure 5) and homology between the cloned fragments and named XETs was observed. Interestingly both of the isolated fragments lacked a second pair of cysteine residues, in contrast to the majority of XETs that have 2 pairs of cysteines.

Table 2 L. sativa cv Ravita XET PCR sequencing results and homology to database sequences. Sequencing was with BigDye termination and homology identified through BLAST searches of sequence data at the National Centre for Biotechnology Information (NCBI, Bethesda, USA. http://www.ncbi.nlm.nih.gov/BLAST/ on 15/09/2001.) Details Homology

RavXetl No insert (BamHI site lost) RavXet3 No insert (BamHI site lost) RavXet4 Consensus sequence built (no 99% to Zea mays

XET primer sequence) zein protein

Ra Se δ No consensus sequence

(primer sequence absent)

600-1 Poor sequence 600-2 Poor sequence 600-7 Consensus sequence built (no 100% to Z. mays primer sequence) zein protein 600-9 312 bp consensus sequence to Z. mays zein between cut sites (no XET primer) 600-12 Poor sequence

RavXetZ Poor sequence containing forward and reverse XET primers. Mixed seq.

Z12 Poor sequence (no primer sites)

Z14 Poor sequence (no primer sites)

R31 Consensus sequence XETs and EXGTs R33 Good reverse sequence (XET Tobacco TJ primer) . Forward sequence sequence. Rice has no primer gDNA.

3B1 Consensus sequence XET sequences 3B3 No consensus (primers present)

Transformations with antisense XET

To reduce XET activity in lollo rosso plants, Valeria was transformed with SLJ732 containing the reversed sequences isolated from Ravita showing homology to XETs. Valeria was targeted for transformation ahead of Ravita because it displayed superior in vitro plant regeneration, in respect of time taken to produce shoots (data not shown) and success rate. The XET like fragments 3B1 and R31 replaced the GUS open reading frame in the SLJ732 plasmid by restriction with BamHI and Sad , ( Sacl is an isoschizomer of Sstl) and re-ligation with the XET insert, to create SLJ732-3B1 (Figure 7) and SLJ732-R31. The SLJ732-3B1 construct was successfully incorporated into Agro actei'ium in preparation for transformation, but SLJ732-R31 could not be cloned in E. coli .

Valeria cotyledons were transformed on 3 separate occasions with the SLJ732-3B1 construct using transformation method 3 (see Table 1, (i)-(iii)). The first transformation alone was successful for the regeneration of plants through tissue culture technique using selective media, the second transformation did not produce callus and the third transformation generated shoots that after 5 months had not rooted in a kanamycin rich media and were not considered for further study as a result of this delay.

The 19 regenerated plants from transformation (i) were labelled 1-19, genomic DNA and total RNA were extracted for analysis from the primary transformants as they set seed. PCR with primers to a fragment of the nptll gene showed that the primary transformants contained the kanamycin resistance gene in their genome indicating the successful transformation of the SLJ732-3B1 T-DNA, in conjunction with the antibiotic resistance shown by regeneration on kanamycin rich media. To confirm expression of the antisense 3B1 fragment gene expression analysis was performed on the transformants where RNA was successfully extracted (all Ti lines with the exception of plants #13 and #18) probing total RNA with radiolabelled 3BlFrom the probed blots endogenous 3B1 expression was shown in all the plants including the wild type. A second smaller band indicating the additional expression of the antisense 3B1 fragment was found only in Ti plants #2, #3, #6, #7, #8, #9, #10 and #19. _x plant #5 showed a larger sized band in addition to the endogenous sequence, the nature of this fragment is unknown. The RNA sample for plant #19 was loaded incorrectly on to the denaturing gel and did not have a reduced level of XET RNA as the photograph of the probed blot would suggest. From the northern analysis on the primary transformants it was not possible to detect an antisense phenotype for XET levels and the leaf physiology could not be examined due to possible somaclonal variation as a result of the tissue culture process and the need to nurture the plants to a produce seed so that traits could be studied in the T₂ progenies.

The Ti plants flowered in the glasshouse and seed was collected over a 2 month period from the self pollinated lines, with the exception of Ti plants #6, #10, #13, #14 and #16 where the plants failed to flower and produce seed. Lines #2, #3 and #8 all produced only 5 seeds, line #15, #4 and #11 produced 17 seeds, 48 seeds and 53 seeds respectively; all the remaining lines produced more than 100 seeds. Up to 96 seeds from each parental Tiline were germinated prior to selection with a kanamycin spray and the germination rates are shown in Table 5.4 along with the number of green plants, those resistant to the antibiotic and therefore positive for the nptll gene and the non-resistant white plants that lack the nptll gene. Green plants were shown to carry the nptll gene in their genome and the white plants lack the gene by PCR with primers to a region of the gene as used on the DNA of i plants. The genomic DNA from both the white and green plants were visualised on an agarose electrophoresis gel and looked identical. The method of screening for plants inheriting the kanamycin resistance gene was successful and provided a far quicker set of T₂ plants than germination on kanamycin plates and subsequent transfer to growth room and glasshouse would have permitted, with the additional advantage of removing the undesirable traits of bolting and somaclonal variation in

Table 3 Germination success of T₂ seed produced from T_α plants of L. sativa cv. Valeria transformed with SLJ732- 3B1 and the percentage of plants germinated resistant to kanamycin applied as a spray during growth.

Parental Number of Germination Resistant line seeds ratio plants number sown

1 96 39% 89%

2 5 80% 50%

3 5 100% 60%

4 48 54% 77%

5 96 39% 65%

7 96 46% 68%

8 5 100% 40%

9 96 63% 33%

11 48 67% 69%

12 96 78% 88%

15 17 71% 75%

17 96 52% 68%

18 96 72% 80%

19 96 47% 13%

in vitro grown plants. Germination success varied between 39% and 100% with the ratio between resistant and non-resistant plants dependent on the Ti parental line.

From the kanamycin screened population of T₂ plants 16 resistant plants were selected (Table 4) for further characterisation of XET enzyme activity, XET action co- localisation with a donor substrate, leaf processability traits and northern analysis. These plants were selected following the 3B1 expression analysis and selected from the parents showing high levels of expression. The T₂ plants were labelled with the parental line first and then assigned a number, for example the progeny from line #7 will be 7-1, 7-2 etc., for the analysis. The T₂ plants were allowed to self fertilise and seeds for a T₃ generation were collected from the plants that survived. The seed was cleaned up from the flower heads, counted, entered into a database and stored for future application.

Table 4 T₂ plants selected for phenotype analysis.

Plant Ti Plant fate T₃ seed name parent quantity

5-1 5 Selfed 200

5-2 5 Plant died -

5-21 5 Plant died -

7-4 7 Selfed 60

7-5 7 Aborted ~~ seeds

7-7 7 Selfed 250

7-8 7 Selfed 100

7-10 7 Plant died -

7-11 7 Plant died -

7-25 7 Selfed 80

7-28 7 Selfed 30

9-11 9 Selfed 50

19-3 19 Selfed 100

19-4 19 Plant died -

19-5 19 Selfed 200

19-6 19 Plant died -

Material Testing

The biophysical properties of leaves and the leaf cell walls were determined using an instron apparatus (named after Instron the equipment manufacturer) used by Ferris and Taylor (1994£>) New Phytologist 127: 297-307, Gardner et al . (1995) New Phytologist 131:81-90 and Ferris et al . (2001) Plant Cell and Environment 24: 305-315 from the technique described by Cleland RE (1967) Extensibility of isolated cell walls: measurement and changes during cell elongation. Planta 74:197-209 and Van Volkenburgh et al . (1983) A simple instrument for measuring cell-wall extensibility. Annals of Botany 51:669-672.

Sections of leaf tissue were stretched twice between two small brass clamps to a known weight load. The gradient of the first slope is equivalent to the total extensibility of the leaf section, plasticity and elasticity (P+E) , the same section stretched a second time outputs a curve with a steeper gradient and this is the elastic (E) extensibility only. Plastic (P) extensibility of the leaf tissue sample is calculated as the difference between the two curves, P = (P+E) - (E) , (Figure 8) .

Leaves were harvested and stored individually in 20 ml of methanol in a universal tube. The tubes were stored at <4 °C in the dark and tested after at least 24 h in methanol. Leaves of lollo rosso and salad rocket have been stored and successfully tested up to 6 months later. Leaves were re-hydrated in 150 ml of distilled water in an 250 cm³ vessel on an orbital shaker (LH Engineering Co. Ltd., Stoke Poges) set at a low to medium speed for 10 min (optimum re-hydration time was determined experimentally for lollo rosso and baby salad leaves, data not shown) . Immediately a strip of leaf tissue was excised parallel to the mid-vein, 10 mm in and 10 mm down, cut 5 mm thick and 10 mm long, using scalpel blades fitted to a Perspex block to ensure a standard width over which the force would be exerted. The sample was blotted dry and fixed between a pair of small brass clamps, set 5 mm apart. The leaf section was held in the clamps with a small section of masking tape attached to the inside of the clamps to prevent slippage as the load was applied. The section was stretched vertically to 20 g

(experimentally determined as a suitable load) reversed to a 0 g load and stretched again to 20 g. Leaf section reversible (elastic) and irreversible (plastic) extension were determined from the pen chart output, where distance stretched and load (force applied) were plotted on the x and y axes respectively.

The specific leaf area (SLA, cm² g^"1) was recorded for the leaf strip tested by cutting a 5x5 mm section and drying at 80 °C for 48 h to give a dry weight. The specific leaf area was determined using the formula (leaf area) / (leaf dry weight) . SLA were calculated to determine if there were significant differences between leaf sample thicknesses that may have influenced their extensibility, because material stiffness depends on cross-section thickness (Cleland 1967, supra) and a correction was not needed if there was no significant change between sample tissue weight (Van Volkenburgh et al . , 1983, supra).

Physical Observations on Leaves of Til9 plants

The data for the traits of processability is presented for the progeny of the primary (Ti) transformant 19 as the plant group of highest interest due to the reduced XET activity and reduced XET gene expression phenotype of plants 19-5 and 19-3. The youngest emerged leaf was labelled A and the leaf that emerged immediately prior to A was labelled B and so on down the plant. Leaves B, D and F were sampled and consequently B was the youngest leaf sampled and F the most mature leaf sampled 10 weeks after seed sowing.

Leaf processability traits in the T₂ progeny of the primary transformant #19 expressing an XET fragment (3B1) in antisense orientation where B, D and F represent the youngest leaves in the head wherein the youngest leaf on the plant was labelled A 10 weeks after the seed was sown.

1) Epidermal cell area, values represent the mean of 20 cells for each sampled leaf.

2) The visible leaf area from a flatbed scanned image.

3) Leaf epidermal cell number determined from the cell area and visible leaf area.

4) Leaf plasticity sampled with an Instron like device. The leaf was stretched twice to a 20 g load and the irreversible extensibility determined from the chart output. 5) Leaf disc fresh and dry weight were used to calculate the % dry weight. 6) Specific leaf areas were obtained from the dry weight data to give an indication of leaf thickness.

(For wild type plants n=7, ±SE)

Leaf epidermal cell area was reduced in comparison to the wild type mean, 2382 μm², for plants 19-3 and 19-5 in the most mature leaf, leaf F, 1900 μm² and 1837 μm² respectively. The epidermal cell area of 19-3 was lower than the wild type across all three leaves, indicating small cells in the leaves of this transformant. The leaf B cell areas of plants 19-4 and 19-6 (1338 and 1188 μm² respectively) are equivalent to the wild type mean cell area, 1208 μm², for leaf B and cell area was higher than plant 19-3 (875 μm²) . Visible leaf area was determined for the plants and was higher than the wild type mean across the range of leaves sampled for 19-5. The visible leaf area data was combined with the epidermal cell area data to calculate the leaf epidermal cell number to correct for changes in cell size due to the size of the leaf. The plants 19-3 and 19-5 at leaf F contain significantly more cells (4,382,502 and 6,987,131 respectively) than the wild type plant mean (3,188,968).

A key processability trait is the irreversible extensibility of the leaf (% P) and it was altered in the T₂ progeny of 19. % Plasticity was reduced in the plants 19-3 and 19-5 across all three ages of leaf sampled in comparison to the wild type mean % plasticity. Taking a mean value of the 3 assayed leaves for each plant, the irreversible extensibility (% plasticity) of the whole plant was reduced in comparison to the wild type (1.36%) in 19-3 (1.05%), 19-5 (0.69%) and 19-6 (1.26%) and is increased in 19-4 (1.50%).

Leaf dry weight as a percentage of the fresh weight was reduced in the younger leaves of plants 19-3 and 19-5 at leaf B and D, however at leaf F the ratio of dry to fresh weight is higher in all the transgenic T₂ plants studied from the Ti plant 19. Specific leaf area is higher in the T₂ 19 transgenics across all the leaves, with the exception of 19-5 at leaf B, therefore all the transgenic plant leaves are thinner than those of the wild type. Solutions and buf ers

lOx Agarose gel loading dye. 0.25 % (w/v) Orange G, 15 % (w/v) ficoll in water.

Bradford reagent. 10 mg Coomassie Brilliant Blue G-250 (dissolved in 5 ml 95 % ethanol), 10 ml 85 % (w/v) phosphoric acid, brought to 100 ml with water. Filtered through Whatmann #1 and store at 4 °C in the dark.

CHO extraction ethanol. 50 mM HEPES-KOH, 5 mM MgCl₂.6H₂0, in 80 % ethanol. pH to 7.4 with 10 % (w/v) KOH.

100X Denhardt's solution. 2 % w/v each of Ficoll-400, bovine serum albumin, polyvinylpyrrolidone. Sterilised by filtration and stored at -20 °C.

HEPES buffer. 100 mM HEPES-KOH, 5 mM MgCl₂.6H₂0. pH to 7.5.

Hybridisation buffer. 5x SSPE, 50 % (v/v) formamide, lOx Denhardt's solution, 0.5 % (w/v) SDS, denatured salmon sperm.

Luria Broth, (L Broth) . 10 g l^-1 bacto tryptone, 5 g l^"1 yeast extract, 5 g l^"1 sodium chloride, 1 g l^"1 D-glucose. (Made solid with 1 % (w/v) agar.) Media is autoclaved.

MES buffer. 500 mM MES. pH to 4.5 with 10 % (w/v) KOH.

Minimal Α media. 10.5 g l^"1 K₂HP0 , 4.5 g l^"1 KH₂P0₄, 1 g l^"1 (NH₄)₂S0₄, 0.5 g I^"1 sodium citrate, 2 g l^"1 glucose. Media is autoclaved. 1 ml l^"1 IM MgS0 (H₂0) was added immediately prior to use. lOx MOPS. 0.2 M MOPS (3- (iV-Morpholino) propane-sulphonic acid), 0.05 M Na acetate (pH 7.0), 0.01 M Na₂EDTA (pH 8.0). Buffer is made up with RNase free water, kept dark, in refrigerator and disposed off if yellow.

MSO medium. strength MS salts and vitamins (Duchefa) ,

1100 gg l1^"11 ssuuccrroossee.. ppHH aatt 55..88.. (Made solid with 0 (w/v) agar.) Media is autoclaved.

Nuclear extraction buffer. 120 mM tris, 30 mM EDTA, 1.2 M NaCl, 1.2 % (w/v) CTAB. pH at 7.5 (using HC1) . Prior to use add 0.38 % (w/v) sodium bisulphite.

Pre-hybridisation buffer. 5x SSPE, 50 % (v/v) formamide, 5x Denhardt's solution, 0.5 % (w/v) SDS, denatured salmon sperm.

RNA gel loading buffer. 1000 μl formamide, 300 μl formaldehyde, 250 μl lOx MOPS buffer, a few grains of Orange G.

Rooting medium. strength MS salts and vitamins (Duchefa), 30 g l^"1 sucrose. pH at 5.8. (Made solid with 0.25 % (w/v) Phytagel.) Media is autoclaved.

SI medium. 4.71 g l^"1 MS salts and vitamins, 30 g l^"1 sucrose, 0.04 mg l^"1 α-naphthaleneacetic acid (NAA) , 0.5 mg I^"1 6-benzylaminopurine (BAP). pH at 5.8. (Made solid with 0.8 % (w/v) agar.) Media is autoclaved.

SOC medium. 2 % Bacto tryptone, 0.5 % Bacto yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgS0, 20 mM glucose. Add 10 mM MgCl₂ prior to use. Sol.l. 50 mM Tris/HCl, 10 mM EDTA, 100 μg RNase Ami¹.

20x SSC. 3 M NaCl, 0.3 M sodium citrate. pH to 7.0 with 1 M NaCl. Autoclave.

10 x SSPE. 3.6 M NaCl, 0.2 M sodium phosphate (pH 7.7), 0.2 M Na₂EDTA (pH 8.0). Buffer is autoclaved for 20 min to remove RNase activity.

5x TAE. 24.2 g 1^_1 trisma base, 1.86 g I^"1 EDTA. pH at 8.0 (using acetic acid).

lOx TE. 100 mM trisma base, 10 mM EDTA. pH at 8.0 (using HC1) .

TFB, (transformation buffer). 10 mM MES (pH 6.3), 45 mM MnCl.4H₂0, 100 mM KC1, 3 mM hexamminecobalt chloride, 10 % (v/v) glycerol.

UM medium. 4.71 g l^"*1 MS salts and vitamins, 30 g 1^_1 sucrose, 2 g l^"1 casein hydrolysate, 2 mg I^"1 2,4- dichlorophenoxyacetic acid (2,4-D; Sigma), 0.25 mg l^"1 kinetin, 9.9 mg l^"1 thiamine-HCl, 9.5 mg l^"1 pyridoxine- HC1, 4.5 mg l^"1 nicotinic acid. pH at 5.8. (Made solid with 0.8 % (w/v) agar.) Media is autoclaved.

XET assay mix. 0.3 % (w/v) tamarind-XG (from Fry lab stock) , 10 mM calcium chloride, 300 mM succinic acid (pH 5.5 with 10 mM NaOH) . Made in 0.5 % (w/v) chlorobutanol .

[³H]XLLGol (from Fry lab stock) added at 400,000 [³H] scintillation cpm per 50 μl assay. XET extraction buffer. 300 mM succinic acid (pH to 5.5 with 10 M NaOH) , 10 mM calcium chloride, 10 mM ascorbic acid, 10 % (v/v) glycerol. Made in 0.5 % (w/v) chlorobutanol .

X-Gluc stain. 1 mM EDTA, 0.5 g 1 ^x X-gluc in DMF, 50 mM phosphate buffer pH 7.0, 0.05 % (v/v) Triton X-100.

Claims

1. A method of altering the xyloglucan polymer content of a plant cell wall in a plant cell of an edible, broad leaf plant that comprises introducing into said plant cell an isolated nucleic acid that comprises a XET nucleic acid sequence that is operably linked to an exogenous promoter that drives expression in the said plant cell.

2. A method according to claim 1 wherein the introduced isolated nucleic acid sequence comprises a XET nucleic acid sequence in the sense orientation that is operably linked to an exogenous promoter that drives expression in the said plant cell.

3. A method according to claim 1 wherein the introduced isolated nucleic acid comprises a XET nucleic acid sequence in the anti-sense orientation that is operably linked to an exogenous promoter that drives expression in the said plant cell.

4. A method according to any one of the preceding claims wherein XET activity is down-regulated in the plant cell wall upon expression of the introduced XET nucleic acid sequence.

5. A method according to any one of claims 1, 2 or 4 wherein the XET sequence is the sequence shown in Figure 1 in the sense orientation.

6. A method according to any one of claims 1 , 3 or 4 wherein the XET sequence is the sequence shown in Figure 1 in the anti-sense orientation.

7. A method according to any one of the preceding claims wherein the isolated nucleic acid is derived from a XET nucleic acid sequence selected from plant tissue from a broad leaf edible plant.

8. A method according to claim 7 wherein the isolated nucleic acid is derived from a XET nucleic acid sequence from a leaf from a broad leaf edible plant selected from the group lactuca sativa, Mizuna, lollo rosso, Frisee, rocket, wild rocket, lambs lettuce, little gem, cos, spinach, chard, ruby chard, watercress, red oak leaf (salad leaf), green oak leaf (salad leaf), and Apollo.

9. A method according to claim 7 or claim 8 wherein the isolated nucleic acid is derived from a XET nucleic acid sequence from a leaf from plant selected from lactuca sativa such as lollo rosso, cos, iceberg, little gem, Frisee, lambs lettuce, red oak leaf (salad leaf), green oak leaf (salad leaf), and Apollo.

10. A method according to claim 9 wherein the lactuca sativa plant is a lollo rosso plant.

11. A method according to any one of the preceding claims wherein the exogenous promoter is selected from inducible, chemical-regulated, constitutive, developmental and tissue specific promoters.

12. An isolated polynucleotide sequence from a broad leaf edible plant that encodes a XET nucleic acid sequence for use in a method according to any one of claims 1 to 11.

13. An isolated polynucleotide sequence according to claim 12 wherein the XET nucleic acid sequence is from a lactuca sativa plant.

1 . An isolated polynucleotide sequence according to claim 13 wherein the XET nucleic acid sequence is from lollo rosso.

15. An isolated polynucleotide sequence according to claim 13 or claim 14 wherein the XET nucleic acid sequence is that of Figure 1.

16. An isolated polynucleotide sequence according to any one of claims 12 to 15 comprising genomic DNA.

17. An isolated polynucleotide sequence according to any one of claims 12 to 15 comprising cDNA.

18. A nucleotide sequence from a broad leaf, edible plant comprising a DNA sequence encoding an antisense RNA molecule operably linked to a promoter and a terminator, said promoter and terminator functioning in a plant cell, wherein said antisense RNA molecule is complementary to a portion of the coding sequence for a protein having XET activity associated with the plant cell wall wherein said protein is a XET.

19. A nucleotide sequence according to claim 18 wherein the antisense RNA molecule is complementary to a sense mRNA molecule encoding for a XET or a fragment thereof of a lactuca sativa plant.

20. A nucleotide sequence according to claim 18 or claim 19 wherein the antisense RNA molecule is complementary to a sense mRNA molecule encoding for a XET sequence of Figure 1.

21. A nucleotide sequence according to any one of claims claim 18 to 20 wherein the antisense RNA molecule is complementary to a sense mRNA molecule encoding for a XET amino acid sequence comprising: DEIDFEFLG

22. The nucleotide sequence of any one of claims 12 to 21 wherein said nucleotide sequence further comprises a DNA sequence encoding a marker protein, said marker protein operably linked to a promoter and a terminator, said promoter and terminator functioning in a plant cell.

23. A nucleic acid vector suitable for transformation of an eucaryotic cell and including a polynucleotide according to any one of claims 12 to 22.

24. A nucleic acid vector according to claim 23 suitable for transformation of a plant or bacterial cell.

25. A nucleic acid vector suitable for transforming a procaryotic cell and including a polynucleotide according to any one of claims 12 to 22.

26. A nucleic acid vector according to claim 25 suitable for transforming an Agrobacterium cell.

27. A host cell containing a heterologous polynucleotide or nucleic acid vector according to any one of claims 12 to 26.

28. A host cell according to claim 27 which is a plant or a bacterial cell.

29. A host cell according to claim 28 which is a bacterial cell.

30. A host cell according to claim 28 which is a plant cell.

31. A host cell according to any one of claims 27, 28 and 30 which is comprised in a plant, a plant part or a plant propagule, or an extract or derivative of a plant or in a plant cell culture.

32. A method of producing a cell according to any one of claims 27 to 31, the method including incorporating said polynucleotide or nucleic acid vector into the cell by means of transformation.

33. A method according to claim 32 which includes regenerating a plant from a cell according to any of claims 27, 28 or 30 from one or more transformed cells.

34. A plant comprising a plant cell according to any one of claims 27, 28 or 30.

35. A plant comprising a plant cell according to claim 34 wherein the percentage plasticity of plant tissue comprised of said plant cell according to any one of claims 27, 28 or 30 is reduced when compared to the percentage plasticity of plant tissue of a wild type or control plant of the same type grown under similar growth conditions to that of the plant comprised of said plant cell.

36. A plant according to claim 35 wherein the percentage plasticity of plant tissue comprised of a plant cell according to any one of claims 27, 28 or 30 lies in the range of from about 0.50 to about 4.00 percent.

37. A plant according to claim 36 wherein the percentage plasticity of said plant tissue lies in the range of from about 0.60 to about 3.50 percent.

38. A plant according to any one of claims 34 to 37 wherein the plant is a lollo rosso plant and the percentage plasticity lies in the range of from 0.60 to about 1.50 percent.

39. A method of producing a plant, the method including incorporating a polynucleotide or nucleic acid vector according to any of claims 12 to 26 into a plant cell and regenerating a plant from said cell.

40. Use of a polynucleotide according to any one of claims 12 to 22 in the production of a transgenic plant.

41. An edible broad leaf plant obtainable by the method as claimed in any one of claims 1 to 11.

42. A lactuca sativa plant obtainable by the method as claimed in any one of claims 1 to 11.

43. A plant according to claim 42 which is a lollo rosso lettuce plant.