GB2294266A - Reduction of the level of glucose in an organism - Google Patents

Abstract

Selective reduction of the level of glucose, with respect to other reducing sugars, in a cell, tissue, organ or organism, comprises altering the expression levels of alpha-amylase. The level may be reduced by inhibition of the activity of the enzyme, preferably using the anti-sense DNA gene thereto. The organism may be a plant, preferably a potato.

Description

A METHOD OF REDUCING THE LEVEL OF SUGAR IN AN ORGANISM The present invention relates to a method of reducing the level of sugar in an organism.

Niore in particular, the present invention relates to a plant or a part thereof (e.g. fruit, seed, tuber, shoot etc.) or a product thereof, having a reduced level of sugar, and the use of that plant or a part thereof or a product thereof as a foodstuff. Further in particular, the present invention relates to reducing the levels of the reducing sugar glucose in an organism, such as a plant - preferably a potato.

In some instances it is desirable to reduce the level of sugar in an organism, in particular a plant. For example, it may be desirable to have low levels of sugar, in particular glucose, because it is known that the presence of high levels of sugars in a foodstuff, such as a plant, can be undesirable in some instances for the food industry. For example, it may be desirable to have low levels of sugars in a foodstuff so as to reduce the calorific value of the foodstuff. One way to achieve this would be to extract the sugars before forming the foodstuff. However, this process is very laborious and time consumming.

It is also very expensive.

By way of further example, the presence of reducing sugars in a foodstuff can cause discolouration (such as blackening e.g. by the Maillard reaction (1)) of the foodstuff when it is subjected to high temperatures - such as through a baking or a frying process. In this regard, it is believed that the reducing sugar forms residues with protein material in the foodstuff giving materials that are often black in colour. Naturally, the higher the levels of reducing sugars so there is an increase in the formation of blackened residues.

In particular, this is a major problem in the potato industry wherein it is undesirable to have potatoes that produce, for example, blackened pomme frites, chips, crisps or the like by such a reaction.

It has been observed in natural potatoes that the total levels of reducing sugars are approx.

10 times greater in cold conditions (e.g. approx 3-4 C) than in warm conditions (e.g.

approx 10-16QC). Thus in the chip/crisp industry (as well as in the pomme frite industries) it is undesirable to store potatoes at low temperatures.

However it would be desirable to store potatoes at low temperatures because under those conditions there would be no need to apply chemicals to prevent Sprouting.

All the non- and phosphorylated reducing sugars (e.g. glucose and fructose) in a tuber contribute to the Maillard reaction (1, 2) while the non-reducing sugars sucrose do not.

Since a sucrose molecule comprises one glucose molecule and one fructose molecule the general view is that the build-up of reducing sugars during Storage of potato tubers, especially cold stored tubers (e.g. storage at 4cm), is due to a splitting of the sucrose molecule (3, 4, 5, 6). This indicates that sucrose is first accumulated and then split into glucose and fructose - more specificly the phosphorylated sugars - during cold storage as shown by Isherwood (1973) (4).

The accumulation of glucose and fructose during Storage of potato tubers is dependent on a whole range of factors. A major one is the actual varicty. Only a few varieties are known to accumulate low enough amounts of reducing Sugars, even at warm temperature storage (8-12"C), to be useful for the chipping and crisping industries As soon as the reducing sugars in e.g. a potato slice come into contact with the hot cooking oil (184"C warm) a non-enzymatic process, called the Maillard reaction (1, 2), starts in which the reducing sugars react with the free amino acids in the tuber slice to form long coloured polymeric molecules. The produced potato chips or crisps have a dark brown to black colouration and the chips taste bumed.

It is accepted that the actual pool of free amino acids in the potato tuber is not the limiting factor controlling the extent of the Maillard reaction (7). Instead, the general view is that the actual content of the reducing sugar in the tuber is the controlling factor.

Tubers with a reducing sugar level below 2.5-3 mg/g tuber will normally be acceptable for chip or crisp making although a content as low as 1 mg/g tuber is more ideal for the crisp industry (8, 9). It is possible to blanch the tuber slices so as to remove excess reducing sugars but still not good enough if the level is above 3 mg/g tuber (this is also an expensive method). For the French fry/chip industry a level as high as 5 mg/g tuber can be acceptable, but not if the tubers contain reducing sugars above that level (8, 9).

Two varieties which are typically used by the chip/crisp industry are Satuma and Record.

A variety like e.g. Bintje is a well-known table potato which can be used for making chips or crisps very early in the season, but soon its build-up of glucose and fructose is too high. A variety completely unsuited for chip/crisp making is Dianella, an old starch variety, its content of reducing sugar is much too high to be used in crisp/chip making.

Therefore, the chip or crisp industry is therefore very dependent on specific varieties which accumulate relatively low levels of reducing sugars compared with other varieties.

Another important factor in the determination of any variety's content of reducing sugars is the maturity of the harvested crop before it is stored. This is greatly influenced by the actual growing season, e.g. a cold, rainy season will produce a somewhat immature crop, which will accumulate higher and more fluctuating levels of reducing sugars than a fully mature crop (8, 2, 1, 10). A survey over 3 years of 100 crop sites in the United Kingdom involving monitoring at least 10 parameters (such as temperature, rainfall, soil cultivation etc.) was performed by Kirkman, Young et al (1991) (11). Their studies revealed that there is not a single factor which can be exclusively linked to the content (average) of reducing sugars of the harvested crops.However, the comprehensive analysis did come out with one result - namely the content of reducing sugars of the specific crop is site specific. A field producing a crop with a good crisp colour will on the average each year always perform better than a field that produces potatoes with a bad crisp colour.

Finally, the actual storage temperature where the potatoes are kept is essential. All known potato varieties used by the industry inevitably accumulate very high and unacceptable levels of reducing sugars if they are stored cold (below 7"C). This means that at present all potatoes used for processing are stored above 7"C (e.g. at 8-10"C) (8, 12). However, the storage season is often as long as 10 months and the potato tubers' rest is about 2-4 months and so this means that the tubers have to be chemically treated to avoid the sprouting (8). "Rest" here means tubers with no major visible sprouting.

Sprouting tubers create problems in the processing since the sprouts contain the toxic product solanin (as all the green part of a potato plant). Thus these sprouts have to be cut off before slicing of the tubers. In addition, the sprouts grow quickly at the warm storage temperature (above 7"C) and, in doing so, use the starch in the tuber leading to losses in production. If they are left unattended then the whole crop is unsuitable for chip or crisp production (12).

it is well known that potato tubers stored under cold conditions (e.g. 3-4"C) will not develop growing sprouts of any significance in more than 12 months since this is the storage temperature at which the seed potatoes are kept.

Thus from the industrial point of view it would be feasible to obtain potato varieties which could be stored in longer periods at cold temperatures.

It is generally accepted that the accumulating reducing sugars in stored potato tubers are produced by a breakdown of the potato starch grains.

Starch is an insoluble polysaccharide carbohydrate. It is one of the principal energy reserves of plants. It is often found in colourless plastids (amyloplasts), in storage tissue and in the stroma of chloroplasts in many plants. It comprises two main components: amylose and amylopectin. Both components consist of straight chains of a(1,4)-linked glycosyl residues but in addition the latter component includes a(1,6) branches (13).

The enzyme amylase is one of the many enzymes involved in the degradation of starch.

amylase is best known from cereals where it splits the a-1-4 glucosidic bindings in the amp lose and amylopectin molecules thereby liberating maltose, maltotriose and adextrins (molecules containing the a-1-6glucosidic bindings of the amylopectin branches (15, 16). After the initial attack on the starch grains other enzymes like starch phosphorylase, Amylase, a-glucosidase, debranching enzyme, disproportionating enzyme take over producing finally glucose and glucose-1-phosphate (16).Glucose is quickly phosphorylated by hexokinase to glucose-6-phosphate which is also the product of phosphoglucomutases action on glucose-1-phosphate. Glucose-6-phosphate is converted by phosphohexose isomerase to fructose-6- phosphate. Glucose- 1-phosphate reacts with UTP by the action of UDPG-pyrophosphorylase creating UDPG. This reacts with either fructose or fructose-6-phosphate by the action of either sucrose synthase or sucrose-P-synthase thereby creating sucrose-6-phosphate.

Finally, a phosphatase cleaves the sucrose-6-phosphate liberating sucrose, then invertase can split sucrose into glucose and fructose.

The overall breakdown of starch is thus a highly complex reaction including and involving the action of many enzymes, the formation of a number of intermediates and the formation of a number of equilibria reactions between some of the intermediate products.

But current understanding as to which enzyme(s) and exactly how they function in potato tubers during storage is still rather limited (1). In this regard, there have been few reports that the enzyme a-amylase has higher activity in cold stored than in warm stored tubers (23, 25. 63, 64). This has been reported for other enzymes as well, e.g. amylase, agiucosidase (23), and invertase (25).

Several workers have tried to sort out which specific enzymes should be responsible for the accumulation of reducing sugars at low storage temperatures. For example, studies have included investigations into: starch phosphorylase - which degrades starch primarily by a phosphorylytic reaction (as opposed to a hydrolytic reaction by amylases) (17, 18); phosphofructophosphorylase (PFP) - which is reported to be cold labile thereby blocking the entry of the hexoses into the glycolytic cycle and leading to an accumulation of hexoses (8); phosphofructokinase (PFK) - which is also reported to be cold labile thereby blocking the entry of the glycolytic cycle (8, 19, 20); and invertase - where the levels are higher in cold stored tubers compared with tubers kept at warmer temperatures (21).Also it has been reported that where a- and amylase as well as a-glucosidase have been measured in both warm and cold stored tubers the results indicate that these enzymes have higher levels in cold stored tubers compared with warmer stored tubers (22, 23, 24) and therefore might contribute to the build-up of reducing sugars.

On the other hand, other workers have concluded that there are negligible amounts of aamylase activity in stored potato tubers and so the breakdown must be phosphorolytic (3, 25, 26). Whether or not which enzymes are present in higher or negligible amounts in either warm or cold stored tubers, it is still not known which enzyme(s) are responsible for the actual accumulation of the reducing sugars glucose and fructose in stored potato tubers.

In the past, workers have even looked at ways of trying to reduce the levels of reducing sugars in potatoes. In particular, at the 4th International Congress of Plant Molecular Biology in Amsterdam (19-24 June 1994) three approaches for trying to achieve this aim were discussed.

The first approach was that of Rita Zrenner et al (27), and related to using a 35S promoter to express antisenSe vacuolar invertase in potatoes. The workers found that in potatoes containing the antisense invertase the amounts of starch, glucose, fructose and sucrose in tubers stored at room temperature showed no difference compared to the control (i.e. nontransformed potato). Tubers which were cold stored showed a strong inhibition of the invertase enzyme (up to 75%) contrary to nontransformed control stored tubers.

Apparently, the expression of the vacuolar invertase (mRNA level) was increased after 44 hours of cold storage and maintained this high level for up to 6 weeks, whereupon it fell again in nontransformed control tubers.

By measuring the levels of various sugars (glucose, fructose, sucrose) in the cold stored transgene potatoes the workers found that the total quantity was the same as with the control, but the ratio sucrose to hexoses (e.g. glucose and fructose) had changed. In this regard, there was approximately 50% less glucose and fructose, but in return there was an equivalent increase of sucrose.

It is important to note that even though some removal of the hexoses was observed for the first six weeks, despite the increase in the level of sucrose, this approach is not satisfactory as potatoes are stored on an industrial scale for periods much greater than six weeks, and often up to 10 months.

Thus on an industrial level, this approach does not solve the problem of reducing the levels of reducing sugars in potatoes over a long period of time, let alone the problem of reducing the levels of reducing sugars under cold conditions only decreasing with 50% compared to the control.

The second approach was that of Kishore et al, from Monsanto, and related to the use of the E. coli gene ADP-Glucose pyrophosphorylase (ADPGPP) to increase the starch content with the hope that the levels of reducing sugars would decrease. However, Kishore et al. did not find any correlation between the quantity of ADPGPP protein produced in the transgene tubers and an increase in the levels of starch, let alone a decrease in the levels of reducing Sugars. Further work with tubers with increased starch content which had been stored at 4"C for 4 months apparently produce lighter coloured pomme frites. However, the workers did not report on the sugar levels or other things which might explain the underlying mechanism.It is to be noted that the production of pommes frites is not quite so sensitive to increased levels of the hexoses as potato crisps.

Moreover, the workers did not report on crisp production from their transgenic potatoes with increased starch content. The workers did not show any other data concerning low temperature Storage. Therefore this approach does not solve the problem of reducing the levels of reducing sugars in potatoes, let alone the problem of reducing the levels of reducing sugars under cold conditions.

The third approach was that of Duvening et al (28), Virgin et al (29), Lorberth et al (30), Abel et al (31), Blundy et al (32), Donath and Druger (33), Geuveia (34, 35), Krause et al (36), Rouwendal et al (38), Burrell and Mooney (37) which related to the transformation of potatoes with gene sequences encoding various carbon metabolism enzymes from plants as well as E. co/i enzymes. In each of these instances there was either no effect on the sugar and starch quantities, or the levels of starch were lower whereas the level of sugar had considerably increased. Therefore, likewise, this approach does not solve the problem of reducing the levels of reducing sugars in potatoes, let alone the problem of reducing the levels of reducing sugars under cold conditions.However, this approach does clearly highlight the difficulties faced by the food industry to reduce the levels of reducing sugars - such as glucose and fructose - in the potatoes.

There is therefore a need to be able to reduce the levels of reducing sugars in organisms, particularly in potatoes. More in particular there is a need to reduce the levels of glucose.

There is also a particular need to reduce the levels of reducing sugars under cold conditions.

According to a first aspect of the present invention there is provided a method of selectively reducing the level of glucose, relative to the level of other reducing sugar(s), in an organism comprising at least partially inhibiting the activity of a-amylase in the organism.

According to a second aspect of the present invention there is provided a method of selectively reducing the level of glucose, relative to the level of other reducing sugar(s), in an organism comprising at least partially inhibiting the activity of a-amylase in the organism, wherein the organism is one that is capable of enzymatically degrading amylopectin or amylose.

According to a third aspect of the present invention there is provided a cell, tissue, organ or transgenic organism capable of enzymatically degrading amylopectin or amylose comprising an exogenous nucleotide sequence wherein a transcript from the expression thereof at least partially inhibits the activity of a-amylase in the cell, tissue, organ or transgenic organism.

According to a fourth aspect of the present invention there is provided a construct, a transformation vector or an expression vector comprising a promoter and an exogenous nucleotide sequence according to the present invention.

According to a fifth aspect of the present invention there is provided a foodstuff prepared from an organism according to the present invention.

According to a Sixth aspect of the present invention there is provided baked or fried potato prepared from an organism according to the present invention.

According to a seventh aspect of the present invention there is provided the use of antisense amylase to selectively reduce the level of glucose, relative to the level of other reducing sugar(s), in an organism capable of enzymatically degrading amylopectin or amylose.

Preferably the activity of the a-amylase is inhibited by expression in the organism of an exogenous nucleotide sequence coding for a first transcript capable of binding to a second transcript of a nucleotide sequence coding for a-amylase thereby preventing the translation of the second transcript.

Preferably the exogenous nucleotide sequence has a sequence that is at least partially antisense to the sequence shown as SEQ. ID. NO:1.

Preferably the exogenous nucleotide sequence has a sequence shown as SEQ. ID. NO:2, or is a variant, homologue or fragment thereof wherein the variant, homologue or fragment thereof can inhibit the activity of a-amylase (e,g. prevent translation of the second transcript).

Preferably the exogenous nucleotide sequence is expressed under the control of a 35S promoter.

Preferably the organism is a plant.

Preferably the organism is a potato.

Preferably the foodstuff is a crisp, chip or pomme frite.

In experiments relating to the present invention it was found that a-amylase activity increases in cold conditions. As explained earlier, a-amylase is one of the enzymes involved in the degradation of starch. A potato a-amylase is the subject of EP-B0470145.

Based on that observation, an anti-sense a-amylase was placed under the control of a 35S promoter and transformed into a potato plant. However, surprisingly, the present invention shows that expression of anti-sense a-amylase led to a significant reduction in the levels of just the reducing Sugar glucose - particularly under warm conditions - in the potato over a long period of time.

In particular, there was an overall decrease of the level of glucose, as distinct from the level of fructose which remained constant, of about 70% under warm conditions and an overall decrease of about 10% under cold conditions.

Thus, the present invention shows that at Icast partial inhibition of the endogenous aamylase enzyme in stored potato tubers both at warm and cold storage temperatures specifically affects glucose levels in tubers.

These results are very surprising as, in accordance with current thinking, one would have initially expected a reduction of each of sucrose, glucose and fructose due to starch breakdown in stored potato tubers (see before).

In addition at least one would have expected that a similar and proportional reduction in glucose and fructose would have occurred as a response to a reduction in the build up of sucrose.

Also, and following a survey of potato extracts (associated with the present invention) which revealed that the fraction having a-amylase activity and no amylase activity gives the products glucose and maltose when incubated with the substrate amylose, it is very surprising that at least partially inhibiting the a-amylase enzyme in a transgenic potato line transformed with the antisense sequence specifically reduces the level of glucose in any potato variety.

Therefore, even though potato a-amylase contributes directly to the glucose pool, it is also associated with the production of maltose and dextrins.

Thus it is surprising that a partial inhibition of the a-amylase activity leads to a specific reduction in the levels of glucose, without affecting the fructose pool.

In accordance with the present invention, a decrease as high as 70% in the glucose levels in warm stored tubers was found, but the fructose levels remained constant. Since other starch degrading enzymes like starch phosphorylase and Amylase are present in the tuber, one would have expected that they could have compensated for the removal of the a-amylase activity, especially starch phosphorylase. However, this expected compensation effect did not occur.

Other embodiments of the present invention include the expression of the exogenous nucleotide sequence according to the present invention under the control of a tissue specific promoter, a temperature inducible promoter or a native a-amylase promoter including combinations thereof. The present invention also includes constructs comprising the same.

An example of a preferred temperature inducible promoter is the promoter as shown in Figure 15. This promoter, which comprises the sequence shown as SEQ. ID. NO:3, is a promoter comprising a nucleotide sequence corrcsponding to the 5.5 Kb EcoR1 fragment isolated from Sotanum tuberosum, or a variant, homologue or fragment thereof.

An example of a highly preferred tissue specific promoter is the promoter sequence shown as SEQ. I.D. No. 3. This promoter is a tuber specific, cold-inducible promoter.

Further embodiments of the present invention include the presence of at least one inhibitor of at least one other of the enzymes involved in the degradation of starch. For example, the expression of the exogenous nucleotide sequence according to the present invention may be in conjunction with the expression of an exogeneous nucleotide sequence that at least partially inhibits the activity of vacuolar invertase - such as anti-Sense invertase.

The term "selectively reducing relative to other reducing sugar(s)" means that the levels of glucose are specifically reduced more so than the levels of other reducing sugars, in particular fructose. In a preferred embodiment the term means that the levels of glucose are lowered substantially more than the levels of fructose.

The term "reducing sugar" is used in its normal sense in the art - i.e. a sugar capable of acting as a reducing agent in solution as indicated by a positive Benedict's test and ability to decolourise potassium permanganate solution. Most monosaccharides are reducing sugars, as are most disaccharides exccpt sucrose. Typically reducing sugars include glucose and fructose.

The term "at least partially inhibiting the activity of a-amylase" means that preferably there is at least about 15% inhibition. More preferably there is at least about 50% inhibition. Even more preferably there is at least about 70% inhibition - or even higher (e.g. 95%).

The term "inhibiting the activity of a-amylase" includes the organism's a-amylase per se being inhibited. It also includes reducing the level of replication of the a-amylase gene. It also includes reducing the level of transcription of the a-amylase gene. It also includes reducing the level of translation of the transcript of the a-amylase gene.

Preferably, it means the latter.

The term "at least partially anti-SenSe to the sequence shown as SEQ. I.D. No. 1" means that the nucleotide sequence must express a transcript that is at least anti-SenSe to some of the nucleotide sequence of SEQ. I.D. No. 1 but wherein the nucleotide sequence must be able to inhibit at least partially the activity of a-amylase.

In a prefered embodiment the nucleotide sequence has a sequence that is at least substantially the same as that shown as SEQ. I.D. No. 2 - i.e. is the same as SEQ. I.D.

No: 2 or is a variant, homologue or fragment thereof. In a more prefered embodiment the nucleotide sequence is that shown as SEQ. I.D. No. 2.

The term "construct" - which is synonymous with terms such as "conjugate", "cassette" and "hybrid" - includes the exogenous nucleotide sequence directly or indirectly attached to a promoter. An example of an indirect attachment is the provision of a suitable spacer group such as an intron sequence, such as the Shl-intron or the ADH intron, intermediate the promoter and the GOI.

The construct may even contain or express a marker which allows for the selection of the genetic construct in, for example, a plant cell into which it has been transferred. Various markers exist which may be used in, for example, plants - such as mannose. Other examples of markers include those that provide for antibiotic resistance - such as resistance to G418, hygromycin, bleomycin, kanamycin and gentamycin.

The term "expression vector' means a construct capable of in vivo or in vitro expression.

The term "transformation vector" means a construct capable of being transferred from one species to another - such as from an E.Coli plasmid to a plant cell.

The term 'nucleotide' includes genomic DNA, cDNA and synthetic DNA.

The term "exogenous" means that the nucleotide sequence is not natural to the organism of the present invention. By way of example, in a preferred embodiment of the present invention the nucleotide sequence coding for anti-sense a-amylase is not natural to potato.

The term 'organism' in relation to the present invention includes any organism capable of enzymatically degrading amylopectin or amylose. Typical examples of such organisms include plants, algae, fungi and bacteria, as well as cell lines thereof. Preferably the term means a plant or cell thereof, preferably a dicot, more preferably a potato.

The term 'transgenic organism' in relation to the present invention means an organism comprising either an expressable construct according to the present invention or a product of such a construct.

For example the transgenic organism can comprise an exogenous nucleotide sequence (as herein described) under the control of a suitable exogenous promoter, such as the 35S promoter or the cold-inducible promoter (as described herein); or an exogenous nucleotide sequence under the control of a native promoter, such as the native amylase promoter.

The terms "variant", "homologue" or "fragmcnt" include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acid from or to the sequence providing the resultant nucleotide sequence has the abilitv to express a transcript that can prevent the translation of the afore-mentioned second transcript, or to act as a promoter in an expression system (such as the transformed cell or transgenic organism according to the present invention), respectively.In particular, the term "homologue" covers homology with rcspect to structure andlor function providing the resultant nucleotide sequence has the ability to express a transcript that can prevent the translation of the afore-mentioned second transcript, or to act as a promoter, respectively.

With respect to sequence homology, preferably there is at least 75%, more preferably at least 85%, more preferably at least 90% homology, more preferably at least 95%, more preferably at least 98% homology.

The basic principle in the construction of genetically modified plants is to insert genetic information in the plant genome so as to obtain a stable maintenance of the inserted genetic material. Several techniques exist for inserting the genetic information, the two main principles being direct introduction of the genetic information and introduction of the genetic information by use of a vector system.

A review of the general techniques may be found in articles by Potrykus (Annu Rev Plant Physiol Plant Mol Biol [1991] 42:205-225) and Christou (Agro-Food-lndustry Hi-Tech March, April 1994 17-97).

Thus, in one aspect, the present invention relates to a vector system which carries a nucleotide sequence or construct according to the present invention and which is capable of introducing the nucleotide sequence or construct into the genome of a plant such as a plant of the family Solanaceae, in particular of the genus Solanum, especially Solanum tuberosum The vector system may comprise one vector, but comprises preferably two vectors; in the case of two vectors, the vector system is normally referred to as a binary vector system.

Binary vector systems are described in further detail in Gynheung An et al. (1980), Binary Vectors, Plant Molecular Biology Manual A3, 1-19.

One extensively employed system for transformation of plant cells with a given promoter or construct is based on the use of a Ti plasmid from Agrobacterium tumefaciens or a Ri plasmid from.-lgrobacterium rhizogenes An et al. (1986), Plant Physiol. 81, 301-305 and Butcher D.N. et al. (1980), Tissue Culture Methods for Plant Pathologists, eds.: D.S.

Ingrams and J.P. Helgeson, 203-208.

Several different Ti and Ri plasmids have been constructed which are suitable for the construction of the plant or plant cell constructs described above. A non-limiting example of such a Ti plasmid is pGV3850.

The nucleotide sequence or construct of the present invention should preferably be inserted into the Ti-plasmid between the terminal sequences of the T-DNA or adjacent a T-DNA sequence so as to avoid disruption of the sequences immediately surrounding the T-DNA borders, as at least one of these regions appear to be essential for insertion of modified T-DNA into the plant genome.

As will be understood from the above explanation, the vector system of the present invention is preferably one which contains the sequences necessary to infect a plant (e.g.

the vir region) and at least one border part of a T-DNA sequence, the border part being located on the same vector as the genetic conStruct. Furthermore, the vector system is preferably an Agrobacterium tumefa ciens Ti-plasmid or an Agrobacterium rhizogenes Riplasmid or a derivative thereof, as these plasmids are well-known and widely employed in the construction of transgenic plants, many vector systems exist which are based on these plasmids or derivatives thereof.

In the construction of a transgenic plant the nucleotide sequence or construct may be first constructed in a microorganism in which the vector can replicate and which is easy to manipulate before insertion into the plant.

An example of a useful microorganism is E. coli, but other microorganisms having the above properties may be used. When a vector system as defined above has been constructed in E. coli, it is transferred, if necessary, into a suitable Agrobacterium strain, eg. Agrobacterium tumefaciens.

The Ti-plasmid harbouring the nucleotide sequence or construct of the invention is thus preferably transferred into a suitable Agrobacterium strain, e.g. A. tumefaciens, so as to obtain an Agrobacterium cell harbouring the nucleotide sequence or construct of the invention, which DNA is subsequently transferred into the plant cell to be modified.

Direct infection of plant tissues by Agrobacterium is a simple technique which has been widely employed and which is described in Butcher D.N. et al. (1980), Tissue Culture Me thods for Plant Pathologists, eds.: D.S. Ingrams and J.P. Helgeson, 203-208. See also Potrykus (Annu Rev Plant Physiol Plant Mol Biol [1991] 42:205-225) and Christou (Agro-Food-Industry Hi-Tech March/April 1994 17-27).

As reported in CA-A-2006454, a large amount of cloning vectors are available which contain a replication system in E. coli and a marker which allows a selection of the transformed cells. The vectors contain for example pBR 332, pUC series, M13 mp series, pACYC 184 etc. In such a way, the construct or nucleotide sequence can be introduced into a suitable restriction position in the vector. The contained plasmid is used for the transformation in E. coli. The E. coli cells are cultivated in a suitable nutrient medium and then harvested and lysed. The plasmid is then recovered. As a method of analysis there is generally used a sequence analysis, a restriction analysis, electrophoresis and further biochemical-molecular biological methods.After each manipulation, the used DNA sequence can be restricted and connected with the next DNA sequence. Each sequence can be cloned in the same or different plasmid. After each introduction method of the desired nucleotide sequence or construct in the plants further DNA sequences may be necessary. If for example for the transformatinn, the Ti- or Ri-plasmid of the plant cells is used, at least the right boundary and often however the right and the left boundary of the Ti- and Ri-plasmid T-DNA, as flanking areas of the introduced genes, can be connected. The use of T-DNA for the transformation of plant cells is being intensively studied and is well described in EP 120 516; Hockema, in: The Binary Plant Vector System Offset-drukkerij Kanters B.B., Alblasserdam, 1985, Chapter V; Fraley, et al., Crit.

Rev. Plant Sci., 4:1-46 and An et al., EMBO I. (1985) 4:277-384.

Direct infection of plant tissues by Agrobacterium is another simple technique which may be employed. Typically, a plant to be infected is wounded, e.g. by cutting the plant with a razor or puncturing the plant with a needle or rubbing the plant with an abrasive. The wound is then inoculated with the Agrobacterium, e.g. in a solution.

Alternatively, the infection of a plant may be done on a certain part or tissue of the plant, i.e. on a part of a leaf, a root, a stem or another part of the plant. The inoculated plant or plant part is then grown on a suitable culture medium and allowed to develop into mature plants.

When plant cells are constructed, these cells may be grown and maintained in accordance with well-known tissue culturing methods such as by culturing the cells in a suitable culture medium supplied with the necessary growth factors such as amino acids, plant hormones, vitamins, etc. Regeneration of the transformed cells into genetically modified plants may be accomplished using known methods for the regeneration of plants from cell or tissue cultures, for example by selecting transformed shoots using an antibiotic and by subculturing the shoots on a medium containing the appropriate nutrients, plant hormones, etc.

In summation therefore a highly preferred embodiment of the present invention therefore relates to a method of lowering the level of a specific reducing sugar (i.e. glucose) in an organism capable of enzymatically degrading amylopectin or amylose by at least partially inhibiting the activity of a-amylase in the organism.

The following sample has been deposited in accordance with the Budapest Treaty at the recognised depositary The National Collections of Industrial and Marine Bacteria Limited (NCIMB) at 23 St Machar Drive, Aberdeen, Scotland, AB2 1RY, United Kingdom, on 20 October 1994: DH5a-pJK4 Deposit No. NCIMB 40691 This sample is an E. Coli bacterial (DHSa-) stock containing plasmid pJK4 (described later).

The following sample has been deposited in accordance with the Budapest Treaty at the recognised depositary The National Collections of Industrial and Marine Bacteria Limited (NCIMB) at 23 St Machar Drive, Aberdeen, Scotland, AB2 1RY, United Kingdom, on 26 August 1994: DHSa-gPAmy 351 (Deposit No. NCIMB 40682).

This sample is an E. Coli bacterial stock containing the plasmid pBluescript containing the EcoR1 5.5 genomic DNA fragment isolated from potato (Solanum ruberosum). The EcoR1 5.5 fragment contains the promoter region and part of the 5' untranslated sequence of the structural gene of a potato a-amylase gene. The plasmid was formed by inserting the EcoR1 5.5 kb potato fragment into the polylinker of the vector pBS (Short et al [1988] Nuc. Acid. Res. 16:7583-7600). The promoter may be isolated from the plasmid by enzyme digestion with EcoR1 and then extracted by typical separation techniques (e.g.

gels).

The present invention will now be described only by way of examples, in which reference shall be made to the following Figures.

Figure 1 is a map of each of pIV21 and pEPL; Figure 2 is the nucleotide sequence of the AmyZ4 clone; Figure 3 is a restriction map of the pBKS- plasmid; Figure 4 is a restriction map of gPAmy 351 clone.

Figure 5 is the anti-sense nucleotide sequence of the AmyZ4 clone; Figure 6 is a restriction map of pJK-; Figure 7 is a restriction map of pJK4; Figure 8 presents graphs of glucose and fructose levels of a transgenic potato according to the present invention and a control potato at 4oC (cold conditions) and 120C (warm conditions); Figure 9 presents graphs of glucose and fructose levels of a transgenic potato according to the present invention and a control potato at 4oC (cold conditions) and 12oC (warm conditions); Figure 10 presents graphs of a-amylase activity of a transgenic potato according to the present invention and a control potato at 40C (cold conditions) and 120C (warm conditions);; Figure 11 presents graphs of beta-amylase activity of a transgenic potato according to the present invention and a control potato at 4oC (cold conditions) and 120 C (warm conditions); Figure 12 presents graphs of glucose and fructose levels of a transgenic potato according to the present invention and a control potato at 40C (cold conditions) and 120 C (warm conditions); Figure 13 presents graphs of a-amylase activity of a transgenic potato according to the present invention and a control potato at 40C (cold conditions) and 120C (warm conditions); Figure 14 presents graphs of beta-amylase activity of a transgenic potato according to the present invention and a control potato at 40C (cold conditions) and 120C (warm conditions); and Figure 15 is the sequence of the promoter in the gPAmy 351 clone (see above).

In more detail, Figure 4 is a pictorial rcpresentation of plasmid gPAmy351. The highlighted portion is a EcoR1 - SalI fragment isolated from potato (Solanum tuberosum).

The EcoR1 - SalI fragment contains the EcoRI 5.5 kb fragment (called subclone Eco 5.5) - which is indicated by the line shown at the bottom of the drawing. The EcoRI 5.5 kb fragment contains the promoter region and part of the 5' untranslated sequence of the structural gene of a potato a-amylase. The following restriction enzyme sites are shown in Figure 4: E: EcoRI, Ha: HaelII, Sp: Sspl, H: HindIII, P: Pvul, S: Sall. In addition putative CAAT and TATA boxes and the ATG initiation site are shown. Introns are shown as open bars and exons as closed bars. The Eco 5.5 may be cloned into a pBluescript M13-plasmid or a pBSK-plasmid.

Materials and Methods Tuber of Solanum tuberosum L. ssp. tuberosum cvs Dianella, Saturna, Bintje and Record were harvested from a field located near Holeby in the southern part of Denmark. All four varieties are well established. Dianella is used for starch production, Bintje is predominantly a table potato, and Satuma and Record are used by the chip/crisp industry.

The 'Ceral?ha" kit and the "Betamvl" kit were obtained from Nfegazvme (Aust) Pty.Ltd., 6 Altona Place, North Rocks, NSW 2151, Australia.

The sugar determination kit (Sucrose/D-Glucose/D-Fructose) was obtained from Boehringer Mannheim. All other chemicals were obtained from Sigma, Merck, Ferak, Difco Lab. or BDH Chemicals Ltd.

Bacterial strains and plasmids JM109 (40): rec Al, end Al, gvr A96, thi, hsd R17, supE44, relA1, ## (lac-proAB), [F', tra D36, DRAB, LacIqZ#M15] DH5 αTM(BRL): F-, end A1, bsd R17 (rk-, Mk+) sup E44, third, V, rec Al, gyr A96, rel Al, (argF-lacZYa) U169, 80 cl lac ZAM15 LBA4404 (41): contains the disarmed pTI AchS plasmid p AL4404 in the streptomycin resistant derivative of the Agrobacterium tumefaciens strain Ach5.

pBI121: see references (42,43) pCaMVCN see references 44,45 pBR322: see reference 46 pUC19: see reference 40 pHC79: see reference 47 Media and plates L-Broth (LB) medium: Per litre: 5g of yeast extract, 5g of NZ-amide, Sg of NaCI, 5g of bacto-peptone. Autoclave.

LB-plates: LB medium plus 15g Bacto agar per litre. Autoclave. Pour into plastic petri dishes (25 ml/dish).

ANI-plates As LB-plates plus 35 mg ampicillin, 120 mg IPTG (isopropylthiogalactoside), 40mg Xgal (dissolved in dimethylformamide) per litre after autoclaving. [Xgal: 5-bromo-4chloro-3indolyle-8- D-galactoside.] Kan-plate: As LB-plates plus 50 mg kanamycin per litre after autoclaving.

YMB medium: Per litre:0.66 g K2HPO4 - 3H2O. 0.2 g MgSO4. 0.1 g NaCI. 10.0 g Mannitol. 0.4 g Yeast extract. Adjust pH to 7.0. Autoclave.

Liquid MBa medium: Per litre:4.4 g MS salts (Murashige and Skoog basal salt (48),Sigma). 20 g sucrose. pH is adjusted to 5.7 with NaOH.

Solid MBa medium: As liquid MBa medium plus 0.8 % Difco agar.

MBa co: As solid MBa medium plus 0.5 mg t-Zeatin (trans-isomer, Sigma) and 2.0 mg 2,4 D (2,4-dichlorophenoxacetic acid, Sigma) per litre.

Solid MBb medium: As solid MBa medium but instead of 20 g sucrose 30 g sucrose is added per litre.

Water: The water used in Materials and Methods was always distilled and autoclaved before use.

Handling of Plants Transgenic as well as control lines of cv Dianella, cv Satuma, cv Bintje and cv Record were micropropagated in vitro under sterile conditions. Independent transgenic lines transformed with either pJK4 or pJKS and non-transformed control lines were maintained sterile in agar in a growth chamber.

Single node (containing a leaf) cuttings from the steril plants were planted in 5.5 cm pots and placed under cover in a greenhouse. The cover was gradually removed (over 20 days) to harden the plants. The in vitro micropropagated potato plants were planted in a field by hand when they were approx. 10-12 cm tall. The field was managed using ordinary agronomic handling for potato ficlds. Each line (whether it was transgenic or a nontransgenic control line) was placed in two or four repeats. At harvest all tubers from every line in the two or four repeats were collected, mixed and randomly divided into two portions. One was placed at 4"C (cold storage conditions) and the other at 120C (warm storage conditions) in the dark.

Extraction Procedure Each tuber was washed in running water, blotted dry and a slice was cut out midway from the tuber apex to its point of attachment (longitudinal direction). The slice was weighed (approx. 20 g) and immediately cut into smaller pieces before it was homogenized with the extraction buffer using a 37 ml cup to a Waring blendor. The extract buffer used was essentially as described by Fan, 1975 (49) and consisted of 4 ml 0.05M acetate buffer pH 5.0, with 0.01M Calcium acetate and 1 ml H2O with 0.02 g sodium sulfite per sample.

The homogenate was filtered through miracloth and the filtrate centrifuged at 10 krpm for 10 min at 4"C in a Sigma 202MK centrifuge to give a clear supernatant.

The extracts were divided into two tubes. One for enzyme analyses, which was stored at 200C immediately and the other for sugar analysis was boiled for 10 min. and then stored at -20C.

Determination of Enzvme .Activity The "Ceralpha" or "Betamyl" method for determination of a-amylase or amylase activity, respectively, was used. The enclosed substrates in the kits (Ceralpha or Betamyl) were prepared according to suppliers' instructions. The substrate contains blocked p-nitro phenyl maltoheptaoside (BPNPG7), glucoamylase and a-glucosidase in the Ceralpha kit and p-nitrophenyl maltopentaoside (PNPGS) and a-glucosidase in the Betamyl kit. 0.1 ml substrate was added to 0.35 ml 50 mM Sodium-citrate pH 6.5 and 0.05 ml potato extract and incubated at 30 C for 30 min. At time 0, 10, 20 and 30 min., 0.1 ml was removed and mixed with 0.15 ml stop solution to terminate the reaction. Stop solution was 0.5 M glycin, 2 mM EDTA adjustcd to pH 10.0 with NaOH.

Each sample was transferred to an Elisa-plate and the absorbance measured at 405 nm using an Elisa-reader. The solution containing 50 mM Sodium-citrate pH 6.5 was used as blank. If necessary the potato extract was diluted 5, 10 or 20 times in the Sodiumcitrate buffer.

The activity was expressed as OD,JNmg extracted protein. Each sample was determined in duplicate. In the a-amylase assay, the amount of p-nitrophenol released was directly proportional to the incubation time. This was also the case with the amylase assay after the first 10 min. incubation.

Amylose hydrolysis was followed by 12/KI staining method. 250 l 1,2 % potato amylose in 0.1 M Na-acetat pH 7 was incubated with 50,ul 4o mg/ml BSA and 100,ul extract.

The enzymatic reaction was performed at 40"C. The reaction was stopped by adding 20 ,ul 3 M HCl to 50 ,ul enzym reaction sample. 1 ml t2 (0.026 g/ml 12 + 0.26 g/ml ç diluted 1: 1000) was added and the amount of amylose was measured spectrophotomerically at 620 nm.

Protein concentration was determined according to Bradford (50) using the Bio-Rad protein assay kit (Ca, USA) with y-globulin as standard.

Separation of enzymes bv chromatography In order to more thoughly investigate starch hydrolysing enzymes in potatoes, peeled potatoes were extracted and proteins were separated by affinity chromatography followed by gelfiltration of the fraction binding to the affinity gel material. The eluted proteins were used for hydrolysing starch related substrates and their products were anaiysed on Dionex HPLC.

100 g peeled potatoes were homogenised with 100 ml of buffer (50 mM acetate pH 5.5, 20 mM DTT) and 10 g Dowex in a Warring Blender. After homogenisation the extract was filtered through miracloth and the filtrate was centrifuged at 14.000 rpm for 10 min.

at 4"C.

The supernatant was precipitated with 3C)"/o (NH4)-,SO4 by slowly stirring for 30 min. The precipitated proteins were collccted by centrifugation and discarded. The supernatant was then applied on a Blue Sepharose column.

The affinity column material Blue Sepharose CL-6B (Pharmacia) is the dye Cibacron Blue F3G-A covalently attached to the cross-linked agarose gel Sepharose CL-6B. A 32 ml column was used, equillibrated with 50 mM Tris pH 6.4. The flow was 30 ml/h. 40 ml supernatant sample was applied to the column. Unbound proteins were eluted with 64 ml 50 Tris pH 6.4. The proteins that bound to the affinity material were then eluted with a increasing gradient from 0 - 0,5 M NaCI in total 400 ml of the buffer. Fractions of 8 ml were collected. All operations were performed at 4"C. The fractions were analysed for a-amylase, amylase and protein.

The fractions containing B-amylase activity were pooled and concentrated appr. 5 times by pressure dialysis using Amicon (membrane YM-10) to 6 ml. This fraction was desalted on a PD-10 gelfiltration column (Pharmacia) equillibrated with 50 mM Tris, 0.1 M NaCi pH 7.

Fractions of 200 ul sample was chromatographed on a gelfiltration column FPLC Superose 12 (Pharmacia). The column was equillibrated with 5O mM Tris, 0.1 M NaCl pH 7. The run time was 80 min. Samples were collected in eppendorf tubes manually. Protein was recorded by UV 280 nm. In total 6 gelfiltration runs were performed. Peaks from the runs were pooled. The samples were defined as peak 1 to peak 9.

Product analysis For analysis of products from hydrolysis of carbohydrates with the samples peak 1 to peak 9 the following substrates were used: Maltopentaose (8 mg/ml), soluble starch (2 mg/ml) and amylose (5 mg/ml). The enzymatic reaction mixture contained 200 ,ul substrate, 200 ,ul 50 mM Na-acetate pH 6.5 and 100 ul enzyme preparation. The mixture was incubated at 37 C for 20 hr. The reaction was stopped by boiling for 5 min. 20 ul sample was then analysed on Dionex HPLC.

The analysis was performed on a Dionex 4500i with a pulsed electrochemical detector used in pulse amperometric detection mode. A CarboPac PA1 column (4 x 250 mm) was used. The samples were eluted with the following gradient: Buffer: Gradient: Time t Buffer min 1 2 3 1) 0.2 M NaOH 0 5 95 2) 1.5 M NaOAC 5 5 95 3) H2O 10 22 78 15 50 10 40 20 50 20 30 25 5 95 Determination of Sugar The sugar analysis was done with the boiled potato extracts (see previously) and a SucroselD-Glucose/D-Fructose test kit from Boehringer Mannheim was used according to supplier's instructions. The reactions were performed in triple in Elisa-plates by dividing the volume of all ingredients with 12 compared to the recommended method in the kit.An ELISA-reader was used for measuring at 340 nm.

Isolation of DNA Plasmid DNA transformed into E.coli cells of JM109 or DH5a was isolated as described in EP-B-0470145. Plasmid DNA transformed into LBA4404 was isolated as follows: The plasmid preparation was as described in EP-B-0470145. In particular, small scale preparation of plasmid DNA was performed as follows.

Bacterial strains harbouring the plasmids were grown overnight in 2 ml L-Broth (LB) medium with ampicillin added (35,ug/ml). The operations were performed in 1.5 ml Eppendorf tubes and centrifugation was carried out in an Eppendorf centrifuge at 4"C.

The cells from the overnight culture were harvested by centrifugation for 2 min., washed with 1 ml 10 mM Tris-HCI (pH 8.5), 50 mM EDTA and centrifuged for 2 min. The pellet was suspended in 150 ul of 15% sucrose, 50 mM Tris-HCI (pH 8.5), 50 mM EDTA by vortexing. 50 ,ul of 4 mg/ml lysozyme was added and the mixture was incubated for 30 min. at room temperature and 30 min. on ice. 400 ,ul ice cold H20 was added and the mixture was kept on ice for 5 min, incubated at 70-720C for 15 min. and centrifuged for 15 min. To the supernatant was added 75,ul 5.0 M Na-perchlorate and 200 us isopropanol (the isopropanol was stored at room temperature), and the mixture was centrifuged for 15 min. at 40C.

The pellet was suspended in 300 ,us 0.3 M Na-acetate and 2-3 vol. cold ethanol was added. Precipitation was accomplished by storing at either 5 min. at -80 C or O/N at -'0 C, centrifuging for 5 min., drying by vacuum for 2 min. and redissolving the pellet in 20 ul H.O. The yield was 5-10 ug plasmid DNA.

Large scale preparation of plasmid DNA was accomplished by simply scaling up the small scale preparation ten times. Working in 15 ml corex tubes, all the ingredients were scaled up ten times. The centrifugation was carried out in a Sorvall cooling centrifuge at 4"C.

Only changes from the above will be mentioned in the following. After incubation at 70-79 C, the centrifugation was for 30 min. at 17,000 rpm. After adding isopropanol and after adding cold ethanol, the centrifugation was for 15 min. at 17,000 rpm. The final plasmid DNA pellet was suspended in H.O and transferred to an Eppendorf tube and then given a short spin to remove debris. The supernatant was adjusted to 0.3 M Na-acetate and 2-3 vol. cold ethanol were added. The pellet was resuspended in 40,ul H,O. The yield was usually 20-28 ,ug plasmid DNA.

To obtain very pure plasmid DNA, 200-300 cg of isolated plasmid DNA from the upscaled method were banded on a CsCl gradient. Solid CsCl was mixed with H20 (1:1 wiv) and 0.2 mg/ml ethidium bromide was added. The solution was poured into a quick-seal polyallomer tube and the plasmid DNA, mixed with solid CsCl (1:1 w/v). The tube was filled, sealed and centrifuged in a Bcckman VTI 65 rotor at 15"C, 48,000 rpm for 16-18 hours. The centrifuge was stopped by without using the brake. The banded plasmid DNA was withdrawn from the tubes using a syringe and the ethidium bromide was extracted with CsCl-saturated isopropanol 7-8 times.The CsCl was removed by dialysis in 10 mM Tris-HCI (pH 8.0), 1 mM EDTA for 48 hours with three changes of buffer. The DNA was precipitated by adjusting to 0.3 M Na-acetate and adding 2-3 vol.cold ethanol.

The small scale plasmid preparation from E. coli was usually followed by a LiCI precipitation to remove RNA from the DNA solution. The small scale prepared plasmid DNA was dissolved in 100 ,ul destillcd water. 1 vol of SM LiCl was added and the mixture incubated at -200C for 30 min followed by centrifugation at 12,000 rpm. for 15 min, 4"C. The supernatant was transferred to a new eppendorf tube and 2 vol TE buffer or water was added. Precipitation with 2.5 vol of 96% ethanol was accomplished by storing either 10 min. at -800C, or O/N at -'0 C. The DNA was precipitated by centrifuging for 15 min. 12,000 rpm ,at 4"C, drying by vacuum for 2 min and redissolving in 18 l of TE or water.

Genomic potato DNA was isolated according to Dellaporta et al (1983) and also the work reported in ref. 51.

Construction of pEPL and pIV2l(DW2t) Restriction enzyme digestion, gel electrophoresis, fragment isolation, subcloning and sequencing were done as outlined in EP-B-0470145 et al. (1989), (52).

pEPL is constructed from pCaMVCN (44, 45) in which the CAT gene is removed by a PstI digestion. A small linker (linker: PstI-BamHI-BaII-PstI) is inserted into this plasmid PstI site, giving the plasmid called pLise(pL). pL is digested with HincII and BglII and the resultant fragment containing the 35S promoter and the NOS terminator is cloned into another pL plasmid digested with EcoRV and BgIII. Both EcoRV and HinclI are blunt ended sites. The resulting construct is called pEnhanced-Lise (pEL). pEL differs essentially from pCaMVCN in that it contains a variant 35S promoter with a tandem duplication of the 250 bp of the upstream sequence of the promoter.The variant 35S promoter has a transcriptional activity approximately ten times higher than the natural 35S promoter (53).

pEL is digested with PstI and BgIII, thereby removing the NOS terminator, and a CaMV terminator (DW2t) is inserted instead. Finally, a linker (PstI-BamHI-SmaI-SacI-SaII- SphI) is inserted into the PstI site situated between the enhanced E35S promoter and the CaMV terminator. This plasmid is called pEPL (see Figure 1).

pit91 is constructed from pHC79 (47) in which a PvuII and BglII digest removed the major part of sequences which are not pBR39 (46) sequences. A PvuII-BglII fragment containing streptomycin resistant (SpRSmR, R70') was ligated in and the resulting plasmid is called pit1. A HindlII plus EcoRI digest of plV1 removed the rest of the non-pBR322 sequences.A pUC19 (40) EcoRI/HindIII polylinker was inserted between the HincIUEcoRI sites in pIV1, and this plasmid is called pIV1O. The 800bp 35S promoter from pBI121 (digested with BamHI and HindIlI) was cloned into the xbal-HindlII sites in pIV10 thereby creating the plasmid pIV21. Finally an BamHI/EcoRI fragment of approx. 230 bp from pEPL, containing the DW2 termination was inserted into plV21 thereby creating the pIV21 (DW2t) plasmid which is shown in figure 1.

Hvbridization of DNA Genomic potato DNA or plasmid DNA was digested with appropriate restriction enzymes and used for Southern transfer (Southern) using Hybond N or N+ membranes (Amersham International) and hybridized according to supplier's instructions (55).

Transformation of Agrnbactenum tumefaciens The LBA 4404 strain was kept at YMB plates (pH 7.0) containing 100 mg/ml of rifampicin (Sigma) and 500 mg/ml of streptomycin (Sigma). 2.5 ml of LB medium (pH 7.4) was inoculated with the bacteria. The suspension was left growing for 24 hours at 28"C in an incubation shaker at 300-340 rpm. The suspension was then diluted 1:9 with LB and incubated for another 2-3 hours at 280C and 300-340 rpm.When OD was 0.5-1, 25 ml aliquots of the cells were harvested in 50 ml tubes in a cooling centrifuge at 10.000 rpm, 5 min, 4"C. The tubes were placed on ice and the pellet resuspended in 0.5 ml of 20 mM CaC12. 0.1 ml aliquots of the resuspended cells were quickly frozen in 1 ml cryotubes in liquid nitrogen and stored at -80 C.

Transformation was accomplished using the freeze-thaw method (56) as follows: A 0.1 ml aliquot of Cacti2 competent LBA 4404 cells was thawed on ice and added 1 ,ug of plasmid DNA. The mixture was incubated at 370C for 5 min. and added 1 ml LB (pH 7.4). Incubation at room temperature with shaking (100 rpm.) for 4 hours was followed by a quick spin at 10.000 rpm, 4"C for 30 sec. The pellet was resuspended in 100 iil LB and plated on a YMB plates containing 50 mg/l of kanamycin (Sigma).

The plates were incubated for 48 hours at 280C or until the colonies had a suitable size.

This was the first round of selection. Only bacteria transformed with a plasmid containing the NPT II gene conferring kanamycin resistance is able to survive on the kanamycin plate.

For the second round of selection six of the obtained colonies were transferred to a YMB plate containing 100 mg/l of rifampicin, 500 mg/l of streptomycin and 50 mg/l of kanamycin. LBA 4404 is resistant to rifampicin and streptomycin and the plasmid confers resistance to kanamycin. The plates were incubated at 280C until the colonies reached a suitable size (approx. 4-5 days).

The colonies were tested for their plasmid content. Plasmid preparations of the colonies were generated and the DNA was digested with appropriate restriction enzymes and run on a 1 % agarose gel to ensure that the plasmid and the insertcd fragment had the right size. The digested DNA was blotted onto a Hybond N+ membrane and hybridised with an appropriate radioactively labelled probe (a fragment of the plasmid DNA or insert).

Storage of the transformed LBA 4404 was at -80 C. 2 ml LB medium containing 100 mg/l of rifampicin, 500 mg/l of streptomycin and 50 mg/l of kanamycin was inoculated with bacteria and incubated at 28"C for 48 hours with shaking (300-340 rpm). The suspension was diluted 1:1 with sterile 35 % glycerol and aliquoted into cryotubes, 800 ,ul per tube and stored at -80"C.

Plant transformation A culture of the transformed LBA 4404 bacteria were made by inoculating 2 ml of YMB (pH 7.0) with the bacteria and incubating at 280C for 24 hours. The suspension was diluted 1:10 and incubated for another 18 hours. The bacteria was centrifuged at 10.000 rph, 4"C for 10 min. and the pellet rinsed twice with 2.5 ml of 2 mM magnesium sulfate, before resuspension in liquid MBa to an OD660 nm of 0.5.

The potato plant material used for transformation was maintained in vitro at MBa medium added 2 uM STS (57, 58). By multiplication top shoots as well as nodes were applied, if the leaves were big they were removed. 5 shoots per container with 80 ml medium was left growing at 75"C and 30-35 days after subcultivation the nodes could be used for transformation.

The stems of micropropagated plants were cut just above and beneath the node so that only the internodes are used, these may possibly be divided so that the explants are approx. 4 mm long. The explants were floatcd in the bacterial suspension for 30 min. and blotted dry on a filter paper and transferred to co-cultivation plates (MBa co). The explants were covered with filter paper moistened in liquid MBa, and the plates were covered with cloth for 3 days and left at 250 C. The explants were then washed in liquid MBa containing 800 mg/l. 2 explants per ml were shaken for 18 hours, then blotted dry and transferred to selection medium.

The selection medium was solid MBb added 50 mg kanamycin, 800 mg carbenicillin (Duchefa), 0.1 mg GA3 (Gibberellic Acid,Sigma) and 1 mg t-Zeatin per litre. The carbenicillin was added to kill any remaining Agrobacteria.

The selection medium was subcultivated every 3 weeks.

Regeneration of whole potato plants Shoots from the explants which by subcultivation was more than 1 cm were harvested and transferred to a solid MBa medium containing 400 mg/l of carbenicillin, 2,uM STS and 0.5 mg/l t-Zeatin. After approx.2 weeks the shoots were transferred to root-formation medium, that is solid MBa with 2 ,uM STS added.

AS uM stock of STS was made from 0.19 g of Na2S2O3-5H20 and 10.19 mg of ARNO: dissolved in 7 ml of water and sterilised by filtration.

After approx. 2 weeks the shoots had rooted and were ready for planting in soil.

The plantlets were rinsed in lukewarm water to remove residues of media and planted in small pots with TKS 2 instant sphagnum (Flora Gard, Germany). The plantlets were kept moist during the planting and watered after. The pots were placed in a "tent" of plastic with 100 % humidity and 21-230C, until the plantlets were rooted in the soil. Then the tent was removed and the plants watered rcgularly.

After 4 weeks of growth the plants were potted into large pots (diameter 27 cm) and transferred to a growth chamber with 16 hours day 22"C and 8 hour night 15"C. When the plants had wiltered down, the tubers were harvested.

Histochemical localisation of beta-elucuronidase (GUS) activity The tissue was cut in small sections with a razor blade and placed in X-gluc (X-gluc: 5-bromo-4-chloro-3-indolyl- 13-glucoronide) is a solution of 50 mg X-gluc dissolved in a buffer with: 0.1 M NaPO, (pH 7.0), 1 mM K3(Fe(CN)6), 0.1 mM K4( Fe( CN ) 6 ) -3H2O, 10 mM Na2EDTA and 3 % sucrose (59) solution to cover the section.

The sections were incubated in X-gluc for 2-12 hours at 37"C. Care was taken to prevent evaporation. The X-gluc was removed and 96 % ethanol was added to the tissue sections to extract chlorophyll and other pigments. Incubation in ethanol was overnight at 5"C and the following day the tissue was transferred to a 2 % sucrose solution and after approx.

1 hour examined in a dissection scope.

Statistical analysis One-way analysis of variance was performed to examine the significant differences between the individual sugar content at both storage temperatures and the measured enzyme activities during the storage period.

The statistical analysis was performed using the program JMPR version 2 from SAS Institute. JMP is a statistical visualization software for the AppleR MacintoshR.

Constructions of the binary plasmids pTKA and p.JK5 The potato a-amylase encoding sequences originate from plasmid pAmyZ4 (see detailed description in EP-B-0470145). Briefly pAmyZ4; encodes a 407 amino acid long potato a-amylase precursor and in addition contains 149 bp 5' and 201 bp 3' untranslated sequences (see figure 9 for the potato AmyZ4 sense sequences) positioned in the EcoRI site of the plasmid pBSK-'s polylinker. See map of pBKS- in figure 3. This cDNA clone is used to prepare two anti-sense clones as follows.

An a-amylase antisense construction was made by digesting the pAMYZ4 cDNA clone with SaCI and EcoRV. This 1640 bp fragment was then subcloned into the SmaI and SacI digested pIV21 (DW2t) plasmid creating the plVZ4 Sac-Eco plasm id. This places the aamylase encoding sequences in the antisense direction downstream of the 35S promoter and upstream of the DW2t terminator. (Sce figure 5 for the antisense sequence). A partial HindIII digest of pIVZ4Sac-Eco liberates a fragment of approx. 2580bp containing the 35S promoter followed by the antisense a-amylase and the DW2t terminator.This fragment was subcloned into a HindIII digested pBI191 vector and the resulting binary plasmid is called pJK5 (see figure 6).

Another antisense a-amylase construction was made by again using the SacI and EcoRV fragment from pAMYZ4 but this time subclone it into a Smal and Saci digested pEPL plasmid (see Materials and Methods for construction of the pEPL plasmid). This places the antiSense sequence downstream of an enhanced 35S promoter (E35S) and upstream of the DW2t terminator. This plasmid is called pEPLZ4Sac-Eco and a partial HindIII fragment containing the E35S promoter, the antisense potato sequence and the DW2t terminator was further subeloned into a HindIII digested pB1121, thereby creating the binary plasmid pJK4, see figure 7.

DNA of the constructs pJK4 and pJK5 was purified from E.coli JM109 cells (plasmid isolation, see Materials and Methods) and the specific direction of the insert in pJK4 was verified by sequencing using a primer which primes 51 bp before position -90 in the 35S sequence.

Transformation and production of potato plants After appropriate control tests of the pJK4 and pJK5 constructions, they were transformed into the.4grobacterium strain LBA4404 as explained in Materials and Methods. The transformed Agrobacteria were used to transform potato stem segments of the varieties Dianella, Saturna, Record and Bintje as described in Materials and Methods.

A leaf from each produced plantlets was GUS-tested (see Materials and Methods) and if it was positive then the plant was transferred into a pot and grown in a growth chamber at 220C 15 h day and 15"C 8 h night. Tubers from pot-grown plants were likewise GUStested to verify the transgenic status, and then genomic DNA was isolated as described in Materials and Methods.

Table 1 shows the distribution of integrated copies of 40 independent transgenic potato plants. Genomic DNA was isolated from each plants and digested with HindIII. This cleaves at one end of the GUS gene in all the constructions leaving the GUS gene intact and the left border of the T-DNA at the other end (see e.g. figure 6) of the GUS gene.

Table 1. Estimated copy number in the transgenic potato plants No of transgenic No of copies potato plants inserted 18 1 11 2 3 3 i 1 1-2 2 2-3 3-4 1 3-s Hybridizing with the SstI-BamHt digested fragment from pBI 121 containing the GUS gene and then counting the number of positive bands in each lane gives a good estimate of the number of integrated copies. Transformed potato plants with integrated antisense a-amylase sequences, growing on kanamycin and showing GUS-positive tubers were chosen for micropropagation to a field test. For handling the plants for field testing, see Materials and Methods.

Tables 2 and 3 show the number of independent transgenic lines chosen for the field test 1993 and 1994, respectively.

Table 2. Overview of the lines in the 1993 field trial.

Construct E35S- 35S- Non anti sense anti sense transformed Variety JK4,1 JK5,1 control Saturna 10 10 1 Record 5 5 1 Bintje 5 1 Dianella 5 5 1 Table 3. Overview of the lines in the 1994 field trial E35S- 35S- non Construct anti- anti- trans sense sense Control formed pJK4 oJK5 pBI121 control Variety Saturna 15 15 5 1 Bintje 5 5 - 1 Record 7 - - 1 Dianella 5 5 5 1 A leaf of all the in vitro micropropagatcd transformcd potato plants was stained for GUS activity. This result was later compared with the GUS-staining activity of a leaf from the field grown plants. Comparing these results showed that the individual potato lines are true to type and stably express the GUS genes in their leaves.In addition, careful observations on both growth chamber transgenic plants as well as field grown plants reveal that the stable integrated constructions apparently do not exert an effect on the transgenic plants phenotype. There were no clear differences seen between transgenic or non-transgenic control plants in their appearance in the field or in the growth chamber.

The genes encoding either the in vivo Amy 1 type or the Amy 3/4 type genes are not expressed in the potato leaves so even though the constructions (pJK4 and pJKS) contain the predominantly constitutive 35S promoter (53) in front of the potato a-amylase sequences this does not cause a problem in the leaf metabolism due to the apparent lack of a-amylase type 1 and 3i4 sense messenger RNA in the leaf.

In summary, the produced constructs pJK4 and plK5 do not have any undesirable effects on already well-known existing potato varieties. This is important for an exploitation of the present invention. The transgenic potato plants behave and resemble the specific variety that was originally transformed (cvs Saturna, Record, Dianella or Bintje, see Materials and Methods), and thus were phenotypically true to type.

Storage experiments with tubers harvested in 1993 and 1994 Field grown tubers from 28 independent transgenic lines (in four repeats in the field 1993) and 40 also independent transgenic lines (in two repeats in the field 1994) were stored in the dark at both 4"C and 120C for 4-6 months. Every 2 weeks, 3 or 5 tubers (in few cases 2 tubers) from each line and storage temperature were sampled of the 1993 field stored material. The sampled tubers were then processed as explained in "Extraction Procedure" in Materials and Methods. Selected lines from the 1994 field material were sampled (3-5 tubers) per storage temperature and these were processed as explained above.

Sugar analysis of stored tubers from the 1993 field The sugar analysis was performed as described in Materials and Methods. Each single tuber was analysed for its content of the disaccharide sucrose (composed of one glucose linked to one fructose molecule), glucose and fructose. The kit used for measuring these sugars (and especially the reducing phosphorylated sugars glucose and fructose) measures the total amount in the tuber extract. Two lines (a transgenic Record and a transgenic Bintje) showed significantly lower levels of reducing sugars compared with the corresponding in vitro micropropagatcd, non-transformed control lines. Then a possible effect of antisensing the tuber c-amylase can be seen in 2 out of 28 independent transgenic potato lines.This observed frequency corresponds very well to the frequency observed by (60) where 4 out of 80 independent potato lines transformed with a construct antisensing the potato uridinediphosphonate-glucose pyrophosphorylase enzyme showed clear reduction in the specific messenger RNA level. Only the results obtained with the transgenic Bintje line (called K125-2.1) will be described as outlined in the following paragraphs.

Reducing sugar levels in tuber of the transgenic K125-2.1 and control line after 4"C and 120C storage The individual reducing sugar levels (glucose and fructose) of respectively the transgenic Bintje line K125-t'.1, transformed with pJK5 (see Materials and Methods) and the nontransformed Bintje control line, after respectively 4"C and 120C storage are shown in figures 8A and SB. It is clear from the figures that the sampled tubers from both K125 2.1 and Bintje control accumulate the reducing sugars after storage at 4"C compared with tubers sampled after 12 C storage. Five tubers from each line and storage temperature were sampled on the indicated dates and individually analysed as explained in Materials and Methods. The average reducing sugar content of the sampled five tubers is shown in figure 8. Variation in the sugar content was much larger from tuber to tuber than e.g.

between individual plants, as also observed by others with field grown material (22, 23).

All results were therefore verified by a statistical analysis. Then, the level of reducing sugars in tubers stored at 4"C is, as expected, significantly higher in both lines compared with the 120C stored tubers. There is however a difference in the exact levels when Bintje control and K125-?.1 are compared. The dotted line in figures 8A and 8B indicates the upper limit of reducing sugar content a potato tuber may contain if it should be used for making French fries (chips) (5 mg/g tuber fresh weight, 8). Then, neither the tubers from the control Bintje line nor from the K1'5-2.1 line could have been used for chip/crisps after 4"C storage.

The tubers from both lines stored at 19 C are usable for chips/crisps.

The upper limit content of reducing sugars in potato tubers used for crisping (chip/crisp making) is shown in figures SA and SB by a dashed line (?.5 mg/g tuber fresh weight, 8). The potato variety Bintje is often used as a table potato and in some cases for French fries production. Only very early in the new potato season can Bintje be used for chip/crisp production, this is normally long before the month of October, at that time chips/crisp produced from cv Bintje will be dark brown and taste badly. The reason for this is clearly seen in figure 8B where the tubers from the control Bintje line contain much too high levels of glucose and fructose.From October to January (sampling time) even in tubers stored at 12 C (the curve lay above the dashed line, but below the dotted line which is the French fries limit). In contrast to this, the transformed Bintje line K1242.1 has produced tubers with a reducing sugar content after 12"C storage, clearly low enough for this line to be used for not only French fries but also for chip/crisp production (see figure 8A).

Comparing the reducing sugar levels of K125-2.1 and control Bintje tubers after respectively 4"C and 12"C storage reveals that not only after 120C storage, but also after 4"C srorage K125-'.1 has significantly lower reducing sugar levels than control Bintje tubers.

This comparison is shown in figures 9A (comparing the reducing sugar content of tubers sampled from 4"C storage) and 9B (tubers sampled from 12"C storage. The results of a statistical analysis (see Materials and Methods) are shown in Table 4.

Table 4. Statistical analyses of the sugar data obtained from Bintje control and K125-2,1 PART ONE 4 C storage Variableb LINEC numberd Meanc tstd Error K125-2,1 34 5,23 t 0, 28 Glucose Bintje 29 8 54 + 0,30" K125-2, 1 34 3,65 t 0,14n.s.

Fructose Bintje 29 3,74 + 0,15n.s.

K125-2,1 34 5,28 + 0,27' Sucrose Bintje 20 4,14 + 0,36 Reducing K125-2,1 34 8,85 + 0,36*** Sugar Bintje 29 12,28 + 0,39"' Total K125,2,1 34 14,13 + 0,45"' Sugar Bintje 20 16,26 + 0,58" Table 4 contd. Statistical analyses of the sugar data obtained from Bintje control and K125-2,1 PART TWO 12 C storage Variableb LINE" numberd Mean" #std Error K125-2,1 35 0,83 + 0,28 Glucose Bintje 29 2,76 + 0 ao K125-2,1 35 0,40 + 0,14 n.s.

Fructose Bintje 28 0,65 + 0,15n.s.

K125-2,1 35 2,57 + 0,27n' Sucrose Bintje 25 1,74 + 0,32n.s.

Reducing K125-2,1 35 1,24 # 0,35*** Sugar Bintje 28 3,44 + 0,40*** Total K125,2,1 35 3,81 t 0,44" Sugar Bintje 24 5,12 + 0,53** Notes: a) Indicates whether the data obtained were from tubers stored at 4 or 12 C; b) Indicates which sugar data sets were used in the comparison.

Reducing sugar: Glucose + Fructose; Total sugar: Glucose + Fructose + Sucrose.

c) Indicates which lines were compared under each variable heading.

d) Number of individual tubers analysed for the indicated sugar.

e) The mean and standard error generated and used is a one way anova varians analysis (the actual values used are sugar in mg/g tuber fresh weight).

n.s.: not significant, the two sets of data are not significantly different.

*, **, ***,: The two sets of data are significantly different with P,0,05, P < O,O1 and P < 0,001 respectively.

Significant tests were performed using a Students t test, comparing each pair of lines under the individual variable heading.

In the line called "Reducing Sugar" are the results of analysing 34 individual tubers from K125-2.1 and 29 tubers from control Bintje for their content of reducing sugars shown.

The mean standard error is indicated and one, two or three stars indicate the level of significance. The amount of reducing sugars in tubers from K125-2.1 is significantly (P > 0.001) lower than both 4 C and 120C storage compared with tubers from control Bintje at the same storage temperature.

a-amvlase activities in tubers of K125-2.1 and control Bin tie after storage at 40C and 12 C a-amvlase activity was measured in the tuber extracts as explained in Materials and Methods. The enzyme activity was measured in the extracts produced from tubers from which also the sugar content (see above) was measured. The sampling period and dates therefore correspond with the sugar dates (figures 8 and 9). In figure 10 is shown the aamylase activity expressed as OD405n",'h/mg protein and each point represents the average of in most cases five individually analysed tubers.

a-amylase activity (using the megazyme kit) has previously been measured in stored potato tubers by Cochrane et al. (22, 23), but these results cannot be directly compared with the results presented here, since the extraction procedures used are different. The data obtained by Cochrane are expressed as activity/g tuber dry weight while those presented here are expressed as activity/mg extracted protein, and finally the exact assay conditions (amount of extract, incubation buffcr, pH etc.) are also different.

Figure 10 and Table 5 show that tubers (on the average) from K125-2.1 have significanyly lower a-amyiase activity compared with the control line Bintje after both 4 C storage (P > 0.001) and 19 C storage (P > 0.01). Figure 10A and table 5 line a-amylase "4 C storage" show the results with the 4 C stored tubers and Figure 10B and table 5 with the 4 C stored tubers and Figure 10B and Table 5 line a-amylase "12"C storage".

Table 5. Statistical analyses of the enzyme data obtained from Bintje control and Kl25-2,1 a-amylase Variablea LINEb numberC Mean" tstd Error 4 C K125-2,1 35 6,08 + 0,50*** storage Bintje 29 8,63 + 0,54*** 12 K125-2,1 35 4,83 + 0,58 storage Bintje 29 7,62 + 0,63" 12 C Bintje 24 6,20 + 0,64 4"C Bintje 24 8,45 + 0,64' 12 C K125-2,1 35 4,83 + 0,38 4 C K125-2,1 35 6,08 + 0,39 Table 5 continued.Statistical analyses of the enzyme data obtained from Bintje control and K125-2,1 amylase Variable LINEC numberd Meand +std Error 4 C K125-2,1 35 12,05 t O,85- storage Bintje 29 13,87 + 0,82n.s.

12 C K125-2, 1 35 8,93 + 0,68n.s.

storage Bintje 29 9,30 + 12 C Bintje 29 9,30 + 0,62' 4 C Bintje 29 13,87 + 0,82' 12 C K125-2,1 35 8.93 + 0,74' 4 C K125-2,1 as 12,05 t O, 74 a) Indicates whether the data compared were obtained from tubers stored at 40C or 12"C.

b) Indicates which lines were compared under each variable heading.

c) Number of individual tubes analysed for either a or ss amylase activity.

d) The mean and standard error generated and used in a one way anova varians analyses. (The actual values used are enzyme activity expressed as OD units/mg extracted protein).

Significans test was performed using a Students t test, comparing each pair of lines under the individual variable heading.

not not significant, the two sets of data are not significantly different.

, , ) "') the two sets of data are significantly diffrent with P < 0,05, P < 0,01 and P < O,OO1 respectively.

In conclusion, K125-2.1's tubers not only have a significantly lower reducing sugar level than the control Bintje line, but also significantly lower a-amylase activity compared with the control line, whether the tubers are stored at 4"C or 120C.

13-amylase activities in tubers of K125-2.1 and control Bintje after storage at 4"C and 120C amylase was measured in the same tuber extracts as a-amylase. The exact method for determination of amylase is explained in Materials and Methods. As with the sugar measurement there are larger variations seen between the tubers so the average of five (in most cases) individually analysed tubers is shown per point in Figure 11A and B. In Figure 11A are shown the amylase activity results of the sampled 4"C stored tubers, obtained from K125-2.1 and control Bintje line.A statistical analysis obtained from the results shows that there is no significant differcnce in the amylase activity from K125 2.1 or control Bintje tubers (see table 5 amylase line "4 C storage". Likewise as shown in Figure 11B and table 5 amylase line "1' C storage", there is no significant difference in the tubers sampled from 1' C storage, 13-amylase activity between K125-2.1 and control Bintje.Then, the reduction in a-amylase activity seen in the transgenic Bintje line K125-2.1 compared with the non-transformed control Bintje is the result of specific inhibition by the a-amylase antisenSe construct (pJKS) and not the result of any coarse control mechanism since only the activity of a-amylase is affected while amylase stays the same as the control line.

Measuring the individual reducing sugars, glucose and fructose as well as sucrose, revealed a completely new and unexpected result. These findings are now discussed.

Onlv the olucose level is affected in antisensed inhibited r-amvlase tubers. not fructose Comparing individual glucose and fructose levels bctween K125-2.1 and control Bintje tubers at both 4"C and 120C storage showed that only the specific glucose levels are reduced in the transgenic tubers.

Figure 13B shows the level of fructose in both K125-2.1 and control Bintje tubers (sampled at the indicated dates, in most cases the average of 5 individually analysed tubers are shown). It is evident from the figure that there is no significant difference seen between K125.2,1 and control Bintje tubers whether these are sampled from 4"C or 120C storage (see also Table 3 line "Fructose"). On the contrary, there is a large difference seen in the specific glucose levels of K135-2.1 and control Bintje tubers as shown in Figure 12A.

In tubers stored at 1' C, K125-2.1 has a significantly lower glucose level (P > 0.001) compared with control Bintje amounting to a reduction (on the average) of 70% compared with the control.

Again the reduction seen previously in the reducing sugar level in K125-2.1's tubers stored at 40C compared with control Bintje is exclusively in the glucose level (see Figure 1'A).

The lower glucose level after 4"C storage in K125-2.1 is significantly (P > 0.001) lower than the levels of control Bintje tubers, and here the reduction (on the average) is 40%, while the fructose level stays the same (see table 3, 4"C storage line "Glucose" and "Fructose").

The results with the constructs of the present invention are therefore very surprising because one would have expected fructose to be converted into glucose to supply enough substrates to build up sucrose, which then would be split into equal amounts of glucose and fructose. The fact that the fructose pool seems to be unaffected in the transgenic Bintje line especially at 4"C storage, while the glucose is reduced by 40% leaving fructose unaffected, clearly points to the direct participation of the a-amylase enzyme in the specific glucose levels of potato tubers stored at either 4"C or 12"C.

Further Characterisation Of Enzyme Activities In order more thoroughly to characterize starch degrading enzyme activities from potatoes, potatoes were extracted and the enzymes were partially separated by affinity chromatography (Blue Sepharose CL-6B). The proteins binding to the affinity column was further separated by gelfiltration chromatography, resulting in 9 peaks; peak 1 to peak 9. The purification steps are described in detail in Materials and Methods.

The hydrolysis of starch related polysaccharides were analysed spectrophotometrically partly with amylose as substrate measured with I2JKI staining and partly for a- and ss- amylase using p-nitrophenyloligosaccharides as substrates (Megazyme kit) (see Materials and Methods). The results are shown in Table 6.

Table 6: Amyolytic activities in preparations from potato.

1 2 3 4 5 6 7 8 9 &alpha;-amylase' 0 0 0,019 0 0 0 0,071 0 0 ss-amylasel 0 0,006 0 0 0 0 0 0 O Amylose 0 0,025 0,060 0,027 0 0 0,093 0 0 . hydrolysis~ The numbers 1 to 9 refer to peak 1 to peak 9 from gelfiltration chromatography.

The activities are calculated as 1) OD/2h and ') OD/h.

The enzymatic activities in table 6 show that peak 3 and peak 7 contain a-amylase activity whereas peak 2 mainly contains amylase activity. These three peaks also degrade amylose, where the highest amylytic activity is found in peak 3 and peak 7.

Analysis of the end products on Dionex HPLC showed that peak 7 produces glucose and maltose from hydrolysis of amylose. Similar results are also seen for peak 3, however, the increase of glucose and maltose was much lower than for peak 7. These endproducts were not detected for the remaining peak fractions. The amylolytic activity isolated in peak 7 only accumulates the endproducts, glucose and maltose, with amylose as substrate but not with soluble starch or maltopentaose used as substrates. Peak 3 on the contrary degrades maltopentaose into maltose, maltotriose and maltohexaose. No glucose was detected.

Conclusively, the enzyme activity separated in peak 7, a-amylase, produces glucose and maltose from amylose. The enzyme activity from peak 3, however, also contains aamylase but in addition other impurities like e.g. disproportionating enzyme.

These results clearly indicate that a-amylase from potatoes hydrolyses amylose into glucose and maltose. Moreover, when these results are viewed in combination with the results obtained with the transgenic K125-2.1 Bintje line they show that potato tuber aamylase directly affects the glucose content of the tubers, and by partially inhibiting the production of the enzyme a substantial reduction of glucose can be achieved. This leads to a surprising and significant reduction in the total amount of glucose. Thus the present invention provides new potato lines useful for the chip and pomme frite and saute potato and crisp industries.

The reducing sugar level in potato tubers stored at cold temperatures (e.g. 4"C) is up to 10 times higher than the sugar level seen in tubers stored at warm temperatures (e.g.

120C, see e.g. Figures 8A and B). Therefore, by reducing the glucose level as much as 70CTa in warm stored tubers (and possibly even more with a stronger antisense effect), one can convert many already known varieties into either a chip and/or a crisp variety if they are kept at warm storage. This is beneficial to the industry since many other characteristics are important in selecting a good variety besides its sugar content (e.g.

resistance against diseases, yield, eye depth, skin colour form of tuber etc.), and these do not always go together with low reducing sugar content.

However, as will now be described, a further surprising advantage is that tubers according to the present invention can be stored cold (below 7"C) and still not accumulate reducing sugars compared with warm stored tubers. This is very beneficial.

a- and amylase activities are higher in cold stored tubers Comparing the specific a-amylase activity seen in K125-2.1 cold and warm stored tubers showed that there is more activity in the cold stored as shown in Figure 13A. This is likewise the case with the cold store control Bintje tubers compared with warm stored tubers as shown in Figure 13B. Each sampling point is the average level obtained from 5 (in most cases) individually analysed tubers. The higher a-amylase activity seen in tubers stored at 4"C compared with tubers stored at 120C is significant (P > 0.05) higher in both K125-2.1 and control Bintje (sce table 5, a-amylase the two "120C 40 C" lines).

This is likewise the case with the amylase activity as shown in Figures 14A and B and table 5 amylase line "12"C 4"C" (P > 0.05).

This rise, especially in the a-amylase activity, explains why the specific reduction in glucose, in cold stored K125-2.1 tubers, on the average is 40% and not the 70% seen in the warm stored tubers, compared with the control. The promoter in front of the antisense a-amylase sequence (see Figure map JK5) in K125-2.1 is the predominantly constitutive 35S promoter and therefore it is not believed to show a stronger expression at cold temperatures (4 C) compared with warm temperatures (12"C).

It is therefore believed that with a more appropriate promoter greater amounts of antisense a-amylase will be expressed thus lowering the levels of glucose by more than 40%, such as by 70%.

Producing a Potato variety which can be stored in the cold A potato variety which produces tubers that do not accumulate reducing sugars after cold (below 7"C) storage or at least do not have reducing sugar content above the limit for chipping or crisping can be achieved as follows: A construction containing a cold inductive promoter as e.g. gPAmy351 (see Figure 4) or (go), in front of the antisense a-amylase sequence (e.g. as used in pJK4, pJK5), possibly with an enhancer in front, is transformed into potato varieties like e.g. Saturna or Record (low sugar varieties) or other varieties, as explained in Materials and Methods. According to the present invention it is possible to store field grown tubers (either in vitro micropropagated or from planted tubers) at cold temperatures.These tubers are then suitable for frying.

Another embodiment of the present invention is the combination of the antisense aamylase sequence (e.g. pJK4, pJKS) with the antisense vacuolar potato invertase sequence.

The two antisense sequences can either be placed one in direct connection with the other under the control of one promoter (or thcy could have their own promoter and terminator).

This could be for example the E35S promoter (as e.g. seen in pJK4) or the 35S promoter (as seen in plK5) or alternatively a cold inductive promoter as explained above, e.g.

gPAmy 351 eventually with enhancer - since antisensing the vacuolar invertase is only effective in cold stored tubers as reported (27).

This dual construct would (partly or nearly completely) keep glucose from being released from the degradation of the starch grain by the action of a-amylase. It would also keep at least partially the glucose and fructose in the form of sucrose molecules thereby removing at least some fructose and perhaps nearly the rest of the glucose. In this way a substantial amount of the glucose and fructose otherwise present in non-transformed control tubers will be absent from these transgenic tubers. Since the amount of glucose and fructose is up till 10 times higher in cold stored tubers at least 90-95% has to be removed by the dual construction in order for the tubers to be stored at cold temperatures.

Yet another embodiment of the present invention is a combination of the sequence encoding for either E.coli ADP-glucose pyrophosphorylase (WO91/19806) or the barley ADP-glucose pyrophosphorylase or potatoes own ADP glucose pyrophosphorylase (EP An0455316) in a sense orientation in combination with the antiSense a-amylase sequence.

Either in a dual construct with each sequence having its own promoter like e.g. the ones specified above or by transforming the same potato plant twice - first with e.g. pJK4, selected by kanamycin resistance and then later transformed with e.g. a vector containing the mannose-6-phosphate isomerase gcne from E.coli and then selecting on mannose. The transformed potato varieties could, as described before, be fried after cold and warm storage.

It would also be feasible to combine the antisense a-amylase sequence with any of the other degradative starch enzymes. This is because the levels of other enzymes such as amylase goes up as well in cold stored tubers (23). Thus, combining antiSense sequences for a-amylase and amylase (obtained by low stringency hybridization with e.g. the soybean clone (66) or the sweet potato clone (67) as teached in (EP-B-0470145) would lower the amount of glucose even further.

For other combinations, the antisense a-amylase could be beneficially to combine with the PFK gene (sense or antisense), or the PFP(sense or antisense) gene or any other genes participating in either the starch synthesis or starch degradation. Further examples of combinations of the present invention include anti-sense a-amylase and at least any one of: branching enzyme (sense or antisense), or sucrose synthase (sense or antisense), or sucrose-P-synthase (sense or antisense), or UDP-glucosepyrophosphorylase (sense or antisense), or ADP-glucose phyrophosphorylase (sense or antisense), or starch phosphorylase (sense or antisense), or beta-amylase (sense or antisense), or a-glycosidase (sense or antisense), or B-glycosidase (sense or antisense), or disproportionating enzyme (sense or antisense), or debranching enzyme (sense or antisense), or phosphoglucomutase (sense or antisense), or phosphohexose isomerase (sense or antisense), or hexokinase (sense or antisense), or fructose-6 phosphate-2 kinase (sense or antisense), or fructose-26-bisphosphatase (sense or antisense), or other invertases (sense or antisense).

Other types of combinations could be the antisenSe potato a-amylase with either another plant gene (monocot as well as dicot e.g. ADP-glucose phyrophosphorylase(e.g. from barley) or a bacteria! or fungi gene like e.g. the pullanase from bacteria, or other suitable bacterial enzymes. A preferred combination is anti-sense potato a-amylase with ADP-glucose phyrophosphorylase.

It is also possible to have a triple construction, such as that containing sense ADP-glucose pyrophosphorylase, antisense a-amylase and antiscnse vacuolar invertase. This could lower the reducing sugar level as much as 955 in cold stored tubers.

Other modifications of the present invention will be apparcnt to those skilled in the art without departing from the scope of the invention.

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40. Yanisch-Perron, C. et al. (1985) Gcne 33: 103-119 41. Hoekema et al. (1983) Nature 303: 179-180 42. Bevan, M.W. (1984) Nucleic Acid Res 12: 8711-8721 43. Jefferson R.A. et al. (1987) EMBO I 6: 3901-3907 44. Fromm, M. et al. (1985) Proc. Natl. Acad. Sci. U.S.A. 82: 582Y 45. Fromm, M. et al. (1986) Nature 319: 791 46. Bolivar, F. et al. (1977) Gene 2: 95-113 47. pHC79 48. Murashige, T. et al. (1962) Physiol Plantarum 15: 473-497 49. Fan, M.L. (1975) Taiwania 20(1): 71-76 50. Bradford, M.M. (1976) Anal Biochem 72: 248-254 51. Dellaporta, S.L. et al. (1983) Plant Mol Biol Rep 1(4): 19-21 52. Maniatis, T. et al. (1989) Molecular Cloning A Laboratory Manual. Second Edition. Cold Spring Harbour Laboratory Press 53.Kay et al. (1987) Science 236 pp. 1299-1302 54. Southern, E.M. (1975) J. Mol. Biol 98: 503-517 55. Amersham Life Science Protocol: HybondrM - N (N+: positively charged nylon membrane) Version 2.0 56. Holters et al. (1978) Mol Gen Genet 163:181-187 57. Linsmaier, E.U. et al. (1965) Physiol Plant 18: 100-127 58. Chang, H.H. et al. (1991) Bot Bull Academia Sinica 32: 63-70 59. Jefferson, R.A. (1987) Plant Mol Biol Rep 5:387 60. Zrenner, R. et al. (1993) Planta 190: 247-252 61. Beers, E.P. et al. (1990) Plant Physiol 92: 1154-1163 62. Handbook of Amylases and related enzymes. Their Sources Isolation Methods, Properties and Applications. Edited by The Amylase Research Society of Japan, Pergamon Press.

63. Samotus, B. et al. (1973) Ziemniak, 21-29 64. Bielinska-Czarnecka, M. et al. (1977): 95-101 65. Saez-Vasquez, J. et al. (1993) Plant Mol Biol 23:1211-1221 66. Yoshida, N. et al. (1991) J. Biochem 110:196-201 67. Mikami, B. et al. (1988) Seikaguku (in Japanese) 60: 211-216 SEOUENCE LISTING (1) GENERAL INFORMATION NAME OF APPLICANTS: DANISCO A/S BUSINESS ADDRESS: Langebrogade 1 DK-1001 Copenhagen K Denmark TITLE OF INVENTION: A Method Of Reducing The Level Of Sugar In An Organism SEQ. ID.NO. 1 SEQUENCE TYPE: Nucleotide MOLECULE TYPE: DNA ORIGINAL SOURCE: Solanum Tuberosum SEQUENCE LENGTH: 1570 SEQUENCE: 10 20 30 40 TGTGGTGATC GAATTTTCAA TTTTTTTACT GAGTATCTAG 50 60 70 80 GTTGAGGAAC GTAATTTCAA GCTGCGATCG GCTTTTTCCC 90 100 110 120 CTGAACGAGC AAACACAGGT TGTGGGTTCG AGTTAGCAAG 130 140 150 160 GGACGTATAA TCTCAACTAC AATCCATTAT GGCGCTTGAT 170 180 190 200 GAAAGTCAGC AGTCTGATCC ATTGGTTGTG ATACGCAATG 210 220 230 240 GAAAGGAGAT CATATTGCAG GCATTCGACT GGGAATCTCA 250 260 270 280 TAAACATGAT TGGTGGCTAA ATTTAGATAC GAAAGTTCCT 290 300 310 320 GATATTGCAA AGTCTGGTTT CACAACTGCT TGGCTGCCTC 330 340 350 360 CGGTGTGTCA GTCATTGGCT CCTGAAGGTT ACCTTCCACA 370 380 390 400 GAACCTTTAT TCTCTCAATT CTAAATATGG TTCTGAGGAT 410 420 430 440 CTCTTAAAAG CTTTACTTAA TAAGATGAAG CAGTACAAAG 450 460 470 480 TTAGAGCGAT GGCGGACATA GTCATTAACC ACCGTGTTGG 490 500 510 520 GACTACTCAA GGGCATGGTG GAATGTACAA CCGCTATGAT 530 540 550 560 GGAATTCCTA TGTCTTGGGA TGAACATGCT ATTACATCTT 570 580 590 600 GCACTGGTGG AAGGGGTAAC AAAAGCACTG GAGACAACTT 610 620 630 640 TAATGGAGTT CCAAATATAG ATCATACACA ATCCTTTGTT 650 660 670 680 CGGAAAGATC TCATTGACTG GATGCGGTGG CTAAGATCCT 690 700 710 720 CTGTTGGCTT CCAAGATTTT CGTTTTGATT TTGCCAAAGG 730 740 750 760 TTATGCTTCA AAGTATGTAA AGGAATATAT CGAGGGAGCT 770 780 790 800 GAGCCAATAT TTGCAGTTGG AGAATACTGG GACACTTGCA 810 820 830 840 ATTACAAGGG CAGCAATTTG GATTACAACC AAGATAGTCA 850 860 870 880 CAGGCAAAGA ATCATCAATT GGATTGATGG CGCGGGACAA 890 900 910 920 CTTTCAACTG CATTCGATTT TACAACAAAA GCAGTCCTTC 930 940 950 960 AGGAAGCAGT CAAAGGAGAA TTCTGGCGTT TGCGTGACTC 970 980 990 1000 TAAGGGGAAG CCCCCAGGAG TTTTAGGATT GTGGCCTTCA 1010 1020 1030 1040 AGGGCTGTCA CTTTTATTGA TAATCACGAC ACTGGATCAA 1050 1060 1070 1080 CTCAGGCGCA TTGGCCTTTC CCTTCACGTC ATGTTATGGA 1090 1100 1110 1120 GGGCTATGCA TACATTCTTA CACACCCAGG GATACCATCA 1130 1140 1150 1160 GTTTTCTTTG ACCATTTCTA CGAATGGGAT AATTCCATGC 1170 1180 1190 1200 ATGACCAAAT TGTAAAGCTG ATTGCTATTC GGAGGAATCA 1210 1220 1230 1240 AGGCATACAC AGCCGTTCAT CTATAAGAAT TCTTGAGGCA 1250 1260 1270 1280 CAGCCAAACT TATACGCTGC AACCATTGAT GAAAAGGTTA 1290 1300 1310 1320 GCGTGAAGAT TGGGGACGGA TCATGGAGCC CTGCTGGGAA 1330 1340 1350 1360 AGAGTGGACT CTCGCGACCA GTGGCCATCG CTATGCAGTC 1370 1380 1390 1400 TGGCAGAAGT AATCTTACAG CTATTCCGTT ACTTAATATA 1410 1420 1430 1440 TTAGTAGAAA TATATATGTT TTAAACCCGA GCACCTACTT 1450 1460 1470 1480 CTAACACTAG ATCCGCCTCT ACAGGCTTGG ATGGAGTGAT 1490 1500 1510 1520 GAGTTTTTTT TTCCTGTTCA TTAGACATTG CAACATGGGA 1530 1540 1550 1560 TGTATGTTTT GTTAATAAAA GTGTTCTTGA TCAATGCAAT 1570 GTAATAAGGG SEQ. ID. NO. 2 SEQUENCE TYPE: Nucleotide MOLECULE TYPE: DNA ORIGINAL SOURCE: Solanum Tuberosum SEQUENCE LENGTH: SEQUENCE: 1570 10 20 30 40 ACACCACTAG CTTAAAAGTT AAAAAAATGA CTCATAGATC 50 60 70 80 CAACTCCTTG CATTAAAGTT CGACGCTAGC CGAAAAAGGG 90 100 110 120 GACTTGCTCG TTTGTGTCCA ACACCCAAGC TCAATCGTTC 130 140 150 160 CCTGCATATT AGAGTTGATG TTAGGTAATA CCGCGAACTA 170 180 190 200 CTTTCAGTCG TCAGACTAGG TAACCAACAC TATGCGTTAC 210 220 230 240 CTTTCCTCTA GTATAACGTC CGTAAGCTGA CCCTTAGAGT 250 260 270 280 ATTTGTACTA ACCACCGATT TAAATCTATG CTTTCAAGGA 290 300 310 320 CTATAACGTT TCAGACCAAA GTGTTGACGA ACCGACGGAG 330 340 350 360 GCCACACAGT CAGTAACCGA GGACTTCCAA TGGAAGGTGT 370 380 390 400 CTTGGAAATA AGAGAGTTAA GATTTATACC AAGACTCCTA 410 420 430 440 GAGAATTTTC GAAATGAATT ATTCTACTTC GTCATGTTTC 450 460 470 480 AATCTCGCTA CCGCCTGTAT CAGTAATTGG TGGCACAACC 490 500 510 520 CTGATGAGTT CCCGTACCAC CTTACATGTT GGCGATACTA 530 540 550 560 CCTTAAGGAT ACAGAACCCT ACTTGTACGA TAATGTAGAA 570 580 590 600 CGTGACCACC TTCCCCATTG TTTTCGTGAC CTCTGTTGAA 610 620 630 640 ATTACCTCAA GGTTTATATC TAGTATGTGT TAGGAAACAA 650 660 670 680 GCCTTTCTAG AGTAACTGAC CTACGCCACC GATTCTAGGA 690 700 710 720 GACAACCGAA GGTTCTAAAA GCAAAACTAA AACGGTTTCC 730 740 750 760 AATACGAAGT TTCATACATT TCCTTATATA GCTCCCTCGA 770 780 790 800 CTCGGTTATA AACGTCAACC TCTTATGACC CTGTGAACGT 810 820 830 840 TAATGTTCCC GTCGTTAAAC CTAATGTTGG TTCTATCAGT 850 860 870 880 GTCCGTTTCT TAGTAGTTAA CCTAACTACC GCGCCCTGTT 890 900 910 920 GAAAGTTGAC GTAAGCTAAA ATGTTGTTTT CGTCAGGAAG 930 940 950 960 TCCTTCGTCA GTTTCCTCTT AAGACCGCAA ACGCACTGAG 970 980 990 1000 ATTCCCCTTC GGGGGTCCTC AAAATCCTAA CACCGGAAGT 1010 1020 1030 1040 TCCCGACAGT GAAAATAACT ATTAGTGCTG TGACCTAGTT 1050 1060 1070 1080 GAGTCCGCGT AACCGGAAAG GGAAGTGCAG TACAATACCT 1090 1100 1110 1120 CCCGATACGT ATGTAAGAAT GTGTGGGTCC CTATGGTAGT 1130 1140 1150 1160 CAAAAGAAAC TGGTAAAGAT GCTTACCCTA TTAAGGTACG 1170 1180 1190 1200 TACTGGTTTA ACATTTCGAC TAACGATAAG CCTCCTTAGT 1210 1220 1230 1240 TCCGTATGTG TCGGCAAGTA GATATTCTTA AGAACTCCGT 1250 1260 1270 1280 GTCGGTTTGA ATATGCGACG TTGGTAACTA CTTTTCCAAT 1290 1300 1310 1320 CGCACTTCTA ACCCCTGCCT AGTACCTCGG GACGACCCTT 1330 1340 1350 1360 TCTCACCTGA GAGCGCTGGT CACCGGTAGC GATACGTCAG 1370 1380 1390 1400 ACCGTCTTCA TTAGAATGTC GATAAGGCAA TGAATTATAT 1410 1420 1430 1440 AATCATCTTT ATATATACAA AATTTGGGCT CGTGGATGAA 1450 1460 1470 1480 GATTGTGATC TAGGCGGAGA TGTCCGAACC TACCTCACTA 1490 1500 1510 1520 CTCAAAAAAA AAGGACAAGT AATCTGTAAC GTTGTACCCT 1530 1540 1550 1560 ACATACAAAA CAATTATTTT CACAAGAACT AGTTACGTTA 1570 CATTATTCCC SEQ. ID. NO. 3 SEQUENCE TYPE: Nucleotide MOLECULE TYPE: DNA ORIGINAL SOURCE: Solanum Tuberosum SEQUENCE LENGTH: 1734 SEQUENCE: 10 20 30 40 TCTTTAAGTT GTTTGCTTGA TTTTTCTTCT TCAATCTTCT 50 60 70 80 ATATTTAATT CGTTTTAGCT TCAAACTTCT TCAATTTTAT 90 100 110 120 TTCAATTTAA TTCTACAAAA AAAATCTCTA TTTAGCACCA 130 140 150 160 TTCATAAAAT TCATGCTCAA AATGGGCAAA CATAAATAAT 170 180 190 200 AAATGTGAAG TAAATAATGG ATTAAAATAT ATATTTTTGG 210 220 230 240 GCCTCACATC AACCTTCATA ATTCTTGAAT GAATGAATGA 250 260 270 280 TAGACTTCAT AATTTTTTAA CCTATACATA TAAGAAAATT 290 300 310 320 GAGAGTAACT CAAATAACAA GTTGTAGTAT CACATCTTTA 330 340 350 360 CTATTTGATA ACATTATGAA GGTGATTATA CATTACGTAA 370 380 390 400 CATTTCTTTT AAAAATATGT AAGCAAATTT ACTTTTTAAC 410 420 430 440 TTATCATTGA TCTTCATGGT TTTGTCATAA ATCTCAAAGT 450 460 470 480 TATCATATTT TATATAGCTA TTTGAAAGTA ATTTTATTTT 490 500 510 520 TACTCATCAT TGAGTGATGC TTTTATTATA ATACTAGTAA 530 540 550 560 GTTTTATTTA TTATTTTCTT TTAGGGGTGA ATTGTATAAT 570 580 590 600 ATAATAAAAA ATATATTTTT AGAAATAATG ATTCTTTTAT 610 620 630 640 TATTAAAAAG TTAAGATATT AGATTATTTA TGCTTGTATA 650 660 670 680 ATAATGAACG AAGTTTTATT TTCTATGAGT TTCATTAATC 690 700 710 720 ATGTTTGTAA TTATTTCAAA TTTTGATGTA TTTTTATAAT 730 740 750 760 TTTGTATTAT TATATTATTA TACTATATTT AAAAATTTAA 770 780 790 800 AGATCCATAG GGCTTACGCC CCACGTCAAG AGGCTTGCGC 810 820 830 840 CTTTCCCTAA ATTAAGTAAA ACTCTTCGCC TCATGCCTTA 850 860 870 880 CGCCTCCGCC TTTTAAAACA CTGATTCCTT TCCTCATATA 890 900 910 920 GCTTGAGGCG AAAATATTTA ATAAAAACAC TTCTTAATTT 930 940 950 960 GTTTATATGT TCAATTGAAC ATGTCCGTGA TTAGAAAATT 970 980 990 1000 AAATTAAATT CAATGACAAA TTTAATAATT TGACACAAAA 1010 1020 1030 1040 TTTATGAAAA AAATATCAAA ATATAAAGAA ATATTTTTTT 1050 1060 1070 1080 TGAAATGGAT TAAAAAGAAA AAAAAAACAA ATAAATTGAA 1090 1100 1110 1120 CCGGGATAAG TTGGTTGTTT AATTGATTAT TGATTATGAT 1130 1140 1150 1160 CTCAATTTGA CATTTTGCGC GATCTTTCGA CCTCAATTCG 1170 1180 1190 1200 TATGAACTGA CACTACGCCA ATGGACAGTC GCCGTCGTCA 1210 1220 1230 1240 CCGCCACCGC ACTATTCTCG ACGCGTCGTC TATCTCCTCC 1250 1260 1270 1280 ACCCCACAGC CGTCAATTCC AAGCTTCCAA TGAACCGTTG 1290 1300 1310 1320 CCATGTGTCA CTGCCTATTC ACCGCGAAAC ATGAATATCA 1330 1340 1350 1360 CTGACGAACG ATTTCGGAGC GGAACGAATC CAGAAAATGG 1370 1380 1390 1400 ATTACTTTCT ATAAATTCCT CGAATCTCAA CTCCATTTCG 1410 1420 1430 1440 TAAAAATAAA ATTAAAAATA TTGTTTCTTT TTGTATTTCT 1450 1460 1470 1480 TTTTGTATTT CTGGTTTATG TGGTGATCGA ATTTTCAATT 1490 1500 1510 1520 TTTTTACTGG TAGTGATTCC TACTTTTCTT CAATTGCATT 1530 1540 1550 1560 TCTCCTTTTT CCATTTCACG GTTGAGAATT CATGATTCCT 1570 1580 1590 1600 TATCAGAGGA ATCGATCCGA TTTGACTAAT TTCACTTTTC 1610 1620 1630 1640 GTCTGTATAA ATACCAGAGT ATCTAGGTTG AGGAACGTAA 1650 1660 1670 1680 TTTCAAGCTG CGATCGGCTT TTTCCCCTGA ACGAGCAAAC 1690 1700 1710 1720 ACAGGTTGTG GGTTCGAGTT AGCAAGGGAC GTATAATCTC 1730 AACTACAATC CATT

Claims (1)

1. CLAIMS l. A method of selectively reducing the level of glucose, relative to the level of other reducing sugar(s), in a cell, tissue, organ or organism comprising altering the expression levels a-amylase in the cell, tissue, organ or organism.

   2. A method of selectively reducing the level of glucose, relative to the level of other reducing sugar(s), in a cell, tissue, organ or organism capable of enzymatically degrading amylopectin or amylose comprising at least partially inhibiting the activity of amylase in the cell, tissue, organ or organism.

   3. A method according to claim 1 or claim 2 wherein the activity of the - amylase is inhibited by expression in the cell, tissue, organ or organism of an exogenous nucleotide sequence coding for a first transcript capable of binding to a second transcript of a nucleotide sequence coding for amylase thereby preventing the translation of the second transcript.

   4. A method according to claim 3 wherein the exogenous nucleotide sequence has a sequence that is at least partially anti-sense to the sequence shown as SEQ. ID.

   NO:1.

   A A method according to claim 4 wherein the exogenous nucleotide sequence has a sequence shown as SEQ. ID. NO:2, or is a variant, homologue or fragment thereof wherein the variant, homologue or fragment thereof can prevent translation of the second transcript.

   6. A method according to any one of claims 3 to 5 wherein the exogenous nucleotide sequence is expressed under the control of a 35S promoter.

   7. A method according to any one of the preceding claims wherein the cell, tissue, organ or organism is a component of or is a plant.

   S. A method according to claim 7 wherein the plant is a potato.

   9. A cell. tissue, organ or transgenic organism capable of enzymatically degrading amylopectin or amylose comprising an exogenous nucleotide sequence wherein a transcript from the expression thereof at least partially inhibits the activity of - amylase in the cell, tissue, organ or organism.

   1 0. A cell, tissue, organ or transgenic organism according to claim 9 wherein the exogenous nucleotide sequence is that defined in claim 4 or claim 5.

   11. A cell, tissue, organ or transgenic organism according to claim 9 or claim 10 wherein the organism is that defined in either claim 7 or claim 8.

   12. A construct, a transformation vector or an expression vector comprising a promoter and an exogenous nucleotide sequence as defined in either claim 4 or claim 5.

   13. A foodstuff prepared from the cell, tissue organ or organism according to any one of claims 9 to II.

   14. A foodstuff according to claim 13 wherein the foodstuff is a fried or baked potato.

   15. A foodstuff according to claim 14 wherein the foodstuff is a crisp, chip or pomme frite.

   16. Use of anti-sense amylase to selectively reduce the level of glucose, relative to the level of other reducing sugar(s), in a cell, tissue, organ or organism capable of enzymatically degrading amylopectin or amylose.

   17. A method substantially as described herein.

   13. A foodstuff substantially as described herein.

   19. A cell. tissue, organ or transgenic organism substantially as described herein.

   20. A construct, a transformation vector or an expression vector substantially as described herein.