AU3702299A - Method for increasing vitamin c content of plants - Google Patents

Description

WO 99/53041 PCT/EP99/02046 Method for increasing vitamin C content of plants Field of the invention 5 The invention relates to the achievement of an increased content of Vitamin C in plants or products derived therefrom by methods of genetic modification. Background of the invention 10 Vitamin C (L-ascorbic acid) is essential to man, who is unable to synthesise or store any significant quantity within the body and it is necessary that the diet contains a regular, adequate supply of this vitamin. Plant-derived 15 vitamin C is the major source of vitamin C in the human diet. From "in vitro" experiments of the prior art wherein plant cells in the form of calli or dissected leaf tissues are 20 exposed to media with extremely high soluble sugar concentrations, in particular with D-glucose, it has been noted that such conditions have some influence on vitamin C content. However said conditions are deemed so far removed from those of the "in vivo" cellular environment that little 25 of significance can be deduced as to the true effects of cellular levels of soluble sugars on vitamin C in plants. The biosynthetic pathway of vitamin C in plants is still unknown. 30 WO 99/53041 PCT/EP99/02046 -2 There is a need to provide plants with increased vitamin C and accordingly, there is a need to modify plants so as to increase "in vivo" vitamin C production, thereby increasing their nutritional value and other beneficial properties. 5 In WO-A-98/01574, which is incorporated herein by reference, pea seeds are disclosed lacking plastidial phosphoglucomutase activity which provides higher sucrose levels at the end of the vining period and the freedom for 10 vining over an extended period compared to conventional varieties. A pathway of starch synthesis starting from sucrose is suggested in this application. Intermediates in this pathway are said to include UDP glucose, fructose, glucose-l-phosphate, glucose-6-phosphate, fructose-6 15 phosphate, ADP glucose. Mutants resulting in mutations at the loci r, rb, rug3, rug4, rug5, are also disclosed. Particular attention is paid to rug3 mutants which show an increased sucrose level 20 compared to wild-type pea seeds. The present invention is directed towards the need for plants with higher levels of vitamin C. In particular the present invention provides for the modification of plants 25 such that vitamin C production is increased. SUMMARY OF THE INVENTION The present invention is based on the surprising realisation 30 that an increased sucrose content of genetically modified plants results in a significant increase of vitamin C WO 99/53041 PCT/EP99/02046 -3 content in said plants. Without wishing to be bound to any theory, it is therefore assumed that there is a correlation between the biosynthetic pathway of sucrose, as referred to in figure 1, and that of vitamin C. 5 Accordingly, the invention is based on the finding that genetic modifications affecting the biosynthesis pathway from sucrose to starch, result in an increased vitamin C level in plant tissues. 10 In the first aspect the invention therefore comprises the use in a plant of a genetic modification capable of increasing the content of sucrose or any intermediate compound in, or precursor compound to the biosynthetic 15 pathway from sucrose to starch to produce an increase in vitamin C content in said plant. These and other aspects of the invention will be described in further detail in the description which will follow 20 hereinafter. Detailed description of the invention Description of the Figures 25( i.) Figure 1 provides a representation of the suggested pathway of starch synthesis starting from sucrose. ii.) Figure 2 shows a graphical representation wherein mg vitamin C per 100g of peas is plotted against 30 tenderometer reading values. At low tenderometer readings peas with increased sucrose have up to 50% more vitamin WO 99/53041 PCTIEP99/02046 -4 C. Values for tenderometer readings of the lines in table 2 have been plotted against vitamin C content. iii.) Figure 3 shows a graphical representation of the 5 correlation between vitamin C mg vit C/100g peas and % sucrose for standard peas and peas with increased sucrose when measured after blanching. where the axes: 10 (a) = mean vitamin C level for modified peas (b) = mean vitamin C level for standard peas (c) = mean % sucrose for standard peas (d) = mean % sucrose for modified peas 15 For the purpose of the invention the term plants is intended to embrace both plants and parts thereof such as for example leaves, stems, roots, propagating material such as seeds, flowers, fruits and tubers. 20 For the purpose of the present invention the term "genetic modification" is intended to encompass any alteration to the gene sequence that has the desired effect of increasing vitamin C, this may comprise one or more of a mutation, 25 antisense or co-suppression techniques, gene promoters, terminators or enhancers. The skilled person would also appreciate that some marker assisted breeding techniques may be used to achieve the desired effect. 30 The term "vining peas" is intended to refer to peas that are typically harvested at low maturity and low tenderometer WO 99/53041 PCT/EP99/02046 -5 readings (80-140 TR) for fresh consumption, for canning or for freezing. Genetic modifications in the form of mutations at the r, 5 rug3, rug4, rug5 and rb loci have previously been described (mentioned in WO 98/01574 and Bhattacharyya et al, Plant Molecular Biology 22, 525, 1993) in the context of analysing the relationship between sucrose and starch synthesis in peas. No previous consideration has been paid to the 10 influence on vitamin C synthesis. We have found that an increase in the level of any compound or precursor in the biosynthetic pathway of starch from sucrose, can be used to increase the vitamin C level of at 15 least part of a plant, particularly the seeds as compared with conventional commercial varieties. The loci have been identified, characterised and classified in peas and are known to encode the following enzymes, r: 20 starch branching enzyme, rug3: plastidial phosphoglucomutase, rb: ADP glucose pyrophosphorylase, rug4: sucrose synthase, rug5: starch synthase. The present invention is largely described by way of the enzyme and gene characteristics that have been classified in peas and 25 tomatoes, however the skilled person in the art would appreciate that directly analogous loci and enzyme activities could easily be found across the plant species to which the invention extends. 30 The biosynthesis of starch from sucrose is shown in figure 1.

WO 99/53041 PCT/EP99/02046 -6 Without wishing to be bound by any theory applicants believe the relationship between the biosynthesis of starch and vitamin C is that both pathways have one or more 5 intermediates in common. Moreover it is believed that the steps of the sucrose to starch synthesis pathway are closely related to the synthesis of vitamin C. It will be well within the capability of the skilled person 10 to identify the suitable genetic modifications that provide the desired changes in reaction equilibria within the biosynthetic pathway from sucrose to starch, that have the effect of increasing the desired intermediates in the pathway. 15 We believe that it is particularly useful to increase the content of the following compounds sucrose, glucose, UDP glucose, glucose-l-phosphate, glucose-6-phosphate, fructose, fructose-6-phosphate or ADP glucose. 20 Within this group it is preferred that the compounds sucrose, UDP glucose, glucose-l-phosphate or glucose-6 phosphate are increased. 25 Within plants of the invention with non-photosynthetic tissues the biosynthetic pathway from sucrose to starch is believed to occur in two phases. In the first phase sucrose is synthesised or imported into the cytosol and then transferred via a number of intermediates to the second 30 phase within sub-cellular organelles called plastids, where the final starch synthesis steps occur.

WO 99/53041 PCT/EP99/02046 -7 In the present invention the applicants have shown that genetic modifications that alter the equilibria of cytosolic reaction steps and effectively cause a build-up of 5 intermediates in the cytosolic phase of the pathway have the greatest impact on increasing vitamin C content. It is therefore further preferred that the intermediate compounds in the cytosolic phase of the pathway are 10 increased, the most preferred compounds being at least one of the group comprising sucrose, UDP glucose, or cytosolic glucose-l-phosphate or glucose-6-phosphate. More preferably the level of cytosolic glucose-l-phosphate 15 or glucose-6-phosphate are increased. The applicants have further shown by way of the present invention that a modification wherein the activity of plastidial phosphoglucomutase is substantially reduced, has 20 a particularly desirable effect of significantly increasing vitamin C content. The use of such a modification to increase the content of vitamin C provides a particularly preferred embodiment of the present invention. 25 The plastidial phosphoglucomutase is believed to catalyse the conversion of glucose-6-phosphate to glucose-l-phosphate in the first step in the plastidial synthesis of starch. A modification at this locus therefore has the desired effect of reducing the activity of this enzyme. 30 WO 99/53041 PCT/EP99/02046 -8 In line with previous comments, although the Applicants would not wish to be bound by any particular theory, it is believed that by inserting a genetic modification at the plastidial phosphoglucomutase locus and thereby reducing the 5 capacity for the plastidial conversion of glucose-6 phosphate to glucose-i-phosphate, a build-up of glucose-6 phosphate occurs in the plastid which reduces the capacity for translocation of further substrate into this organelle. In this way the reaction equilibria and concentration 10 gradients in the pathway as a whole are altered and this results in increased concentrations of the cytosolic intermediates that in turn are linked to an increase in vitamin C synthesis. 15 We have therefore found that a reduction in plastidial phosphoglucomutase mutant activity results in particularly enhanced vitamin C levels and accordingly the use of modification at this locus to increase vitamin C content in a plant provides a most preferred embodiment of the 20 invention. This modification resulting in an increased vitamin C level in peas is a mutation at the rug3 locus and has been described in WO-A-98/01574, in the context of increasing 25 sucrose content in peas. The Applicants have also demonstrated that a reduction in sucrose synthase activity, achieved in peas by way of example by a modification at the rug4 locus has a 30 detrimental effect i.e. a reduction in vitamin C content.

WO 99/53041 PCT/EP99/02046 -9 The enzyme sucrose synthase is believed to catalyse the conversion of sucrose to UDP glucose in the first step of the biosynthetic pathway from sucrose to starch. A reduction in the activity of this enzyme will accordingly reduce the 5 cellular content of soluble sugar intermediates further down the pathway, thereby reducing the extent of vitamin C production. This result achieved in peas using a rug 4 mutation is 10 consistent with the present invention and it is likely that such an effect is due to a reduction in the flow of intermediates through particularly the cytosolic phase of the biosynthetic pathway. 15 In a further aspect of the invention the increased vitamin C content may alternatively be obtained by a reduction in the activities of one or more of starch branching enzyme, ADP glucose pyrophosphorylase, starch synthase. These enzymes that catalyse further steps in the pastidial phase of the 20 biosynthetic pathway which are closer to final starch synthesis. It is strongly believed that modifications that can reduce the activities of the plastidial enzymes can be used to 25 increase the content of vitamin C in plants by incorporating relevant SIM lines into appropriate breeding programmes, as has been demonstrated by the rug3 example where the locus encoding plastidial phosphoglucomutase has undergone modification. 30 WO 99/53041 PCT/EP99/02046 - 10 Analysis of tomatoes with low levels of sucrose invertase have also provided support for the unexpected link between the levels of sucrose and intermediate compounds in the pathway to starch and vitamin C production by plants. 5 Sucrose invertase in standard plants catalyses the conversion of sucrose to glucose and fructose. The applicants have demonstrated by way of the present invention that where such enzyme activity is reduced a significantly 10 increased level of vitamin C can be achieved at all stages throughout the fruit maturation in the plant. Although again not wishing to be bound by any theory it is believed that a reduction in sucrose invertase activity 15 causes an increase in vitamin C production by way of increasing the cellular content of sucrose, glucose-1 phosphate and glucose-6-phosphate and the other important intermediates in the biosynthetic pathway to starch. 20 Therefore, for the purpose of the present invention particularly preferred genetic modifications cause a reduction in the activity of one or more of the enzymes starch branding enzyme, ADP glucose pyrophosphoylase, starch synthase, plastidial phosphoglucomutase encoded accordingly 25 in the pea at the r, rb, rug5, or rug 3 loci, or where a reduction in the activity of sucrose invertase is achieved. More preferably the genetic modifications cause a reduction in the activity of one or more of the enzymes starch 30 branching enzyme, ADP glucose pyrophosphorylase, plastidial phosphoglucomutuase accordingly encoded in the pea at the r, WO 99/53041 PCT/EP99/02046 - 11 rb and rug3 loci. Most preferably the modification causes a reduction in the activity of plastidial phosphoglucomutase. The mutations as indicated above can be applied to a variety 5 of plant species. Preferably the plants are suitable for human consumption. Suitable plants are for example vegetables, fruits, nuts, herbs, spices, infusion materials. Suitable vegetables are for example from the Pisum family such as peas, the family of Brassicae, such as green 10 cabbage, Brussel sprouts, cauliflower, the family of Phaseolus such as barlotti beans, green beans, kidney beans, the family of Spinacea such as spinach, the family of Solanaceae such as potato and tomato, the family of Daucus, such as carrots, family of Capsicum such as green and red 15 pepper, and berries for example from the family of Ribesiaceae, Pomaceae, Rosaceae, for example strawberries, black berries, raspberries, black current and edible grasses from the family of Gramineae such as maize, and citrus fruit for example from the family of Rutaceae such as lemon, 20 orange, tangerine. Also preferred are plants which can form the basis of an infusion such as black tea leaves, green tea leaves, jasmin tea leaves. The invention further provides genetically modified plants 25 having an increased vitamin C content. In view of the nutritional value and their consumption on large scale peas, spinach and tomatoes are the preferred plants. 30 WO 99/53041 PCT/EP99/02046 - 12 A most preferred embodiment of the invention comprises the use in a pea plant of a genetic modification capable of increasing the content of sucrose or any intermediate compound in, or precursor compound to the biosynthetic 5 pathway from sucrose to starch to produce an increase in vitamin C, wherein the said genetic modification causes a reduction in the activity of the enzyme plastidial phosphoglucomutase. 10 In particular it is also recognised that tomato fruit of the invention would be well suited to incorporation in tomato products, e.g. tomato purees and pastes, soups and sauces, to increase their vitamin C content. Furthermore this invention can very advantageously be used to increase the 15 vitamin C level of tea. In a preferred embodiment of the invention the methodologies outlined above are used to increase the content of vitamin C in peas. As would be appreciated by the skilled person the 20 mutations identified as being appropriate for previously described embodiments of the present invention would also be applicable here. In this respect the invention also relates to a process for 25 the production of peas for human consumption with increased vitamin C comprising the steps wherein; (i) pea seeds that have been exposed to one or more genetically mutagenic compounds are grown and the progeny are selected on the basis of achieving an 30 increased vitamin C content; WO 99/53041 PCT/EP99/02046 - 13 (ii) said selected peas are then incorporated by way of a breeding programme into a commercially suitable seed line which maintains the increased vitamin C content; 5 (iii) seeds from (ii) are then further grown and harvested; (iv) optionally the harvested product is blanched and frozen. 10 In step (i) of this process the selection for increased vitamin C content can be made by any suitable means. Typically the phenotypical wrinkled seed characteristics of the seed or reduced starch content can be used as an 15 indication of increased vitamin C content. Alternatively the selection of high vitamin C progeny in (i) may be made by using the plastidial phosphoglucomutase 20 gene sequence, disclosed in WO-A-98/01574, as a probe in a DNA preparation analysis. In this instance a reduction in the amount of oligonucleotide material that encodes active plastidial phosphoglucomutase would be indicative of increased vitamin C. 25 Further vitamin C assay techniques as outlined in the examples could also be used as the basis for selection of high vitamin C progeny. 30 Preferably the mutagenic compounds used in step (i) induces a mutation that reduces the activity of plastidial WO 99/53041 PCT/EP99/02046 - 14 phosphoglucomutase further preferred this is a mutation at the rug 3 locus. It is believed to be within the capacity of the skilled person in the art to choose any suitable mutagenic compound. Preferably the mutagenic compound is 5 selected from ethyl methanesulfonate or methylnitrosourea. The genetically mutagenic compound of step (i) preferably causes a genetic modification capable of increasing the content of sucrose or any intermediate compound in, or 10 precursor to the biosynthetic pathway from sucrose to starch to produce said increase in vitamin C. In step (ii) it is preferable that the peas with increased vitamin C that are selected in step (i) are incorporated 15 into one or more suitable commercial seed lines which would result in high quality vining peas. Preferably said commercial seed lines are selected from the group comprising Novella, Avola, Harrier, Sancho. 20 In step (iii) it is preferable that the seeds that are to be consumed as vining peas are harvested at low tenderometer readings as this has been shown by way of the present invention to induce the greatest increase in the amount of vitamin C in the seeds. More preferably the peas are 25 harvested between 80 and 140 tenderometer units, most preferably between 100-110 tenderometer units. Said vining peas may optionally then be blanched and frozen 'in-pack' or alternatively frozen and subsequently packaged. 30 It is apparent from figure 3 that even after blanching peas WO 99/53041 PCT/EP99/02046 - 15 of the present invention maintain a higher level of vitamin C than their commercial alternatives. Peas that are to be consumed as in a dried state may be 5 harvested at higher tenderometer readings. It is appreciated that the process of the invention in steps (i) to (iii) would also be highly applicable for the modification, selection and production of other commercial 10 plant lines with high vitamin C. In a particularly preferred embodiment this process may be alternatively applied, by the skilled person, to the production of high vitamin C content tomatoes.

WO 99/53041 PCT/EP99/02046 - 16 Examples Example 1: Comparative SIM line experiments. 5 Comparisons between plants of the genotype r, rb, rug3, rug4 and rug5 and wild-type round-seeded plants have been made. Experiments involving such comparisons have utilised near isogenic pea lines, largely differing only in respect of a 10 single locus. These near isogenic lines were derived by backcrossing the original seed induced mutant lines back to their parents for a number of generations. Seeds were sown in a glasshouse which was maintained in a 15/10 0 C day/night cycle with a minimum photoperiod of 16 hours per day at an 15 intensity of approx. 300:E at plant level (HQI lamps; Wotan Powerstars, Osram, Wembley, UK). Relative humidity was 75%. The production of flowers was carefully monitored and the development stage corresponding to one day post anthesis was 20 deemed to be the time at which the outer petals of the flower had opened and were roughly perpendicular to the inner petals. At this stage, flowers were tagged and the date recorded. 25 On a single occasion, pea seeds which were 17, 21 and 25 days after anthesis were harvested and immediately frozen in liquid nitrogen. Vitamin C, sucrose and starch were measured in these samples. 30 The lines used were; wild type (BC1/18RR) r-f (SIM 56 BC 1997) WO 99/53041 PCT/EP99/02046 - 17 rb-e (SIM 16 B6 1997) rug3-1 (SIM 1 B6F3 1997) rug3-b (SIM 32B6F3 1998) rug4-b (SIM 91 B6F3 1997) 5 rug5-a (SIMB6 1997) Measurement of content of sugars: 0.5-2 g pea seed material was extracted in 25 ml boiling 80 10 % ethanol (5 min), the supernatant was removed and the extraction in boiling 80 % ethanol repeated twice. The residue was ground in 25 ml 80% ethanol, then centrifuged 10 min, 4500 rpm. The supernatants were combined in a round bottomed flask and reduced to a small volume using a rotary 15 evaporator (30 'C). Assay were carried out in accordance with the methods as described in Methods in Enzymology 174, 518-551. 20 Sucrose was measured by change in OD 340 nm in a 1 ml reaction as follows; 25mM Hepes, pH 7.5, 1 mM MgCl 2 , 0.4mM NADP, 0.5mM ATP, 2.8 units glucose 6-phosphate dehydrogenase (EC 1.1.1.49 from yeast), 2.8 units hexokinase (EC 2.7.1.1 from yeast), 5.6 units hexose phosphate isomerase (EC 5.3.1.9 25 from yeast), 20 units invertase (EC 3.2.1.26 from yeast). Sequential addition of the enzymes allows measurement of glucose, fructose and sucrose in a single assay. Data inserted in table 1. 30 WO 99/53041 PCT/EP99/02046 - 18 Measurement of Vitamin C content: This method was based on that described in Plant Physiology 5 (1995) 109, 1047-1057. For the extraction pea material (0.5-1g) was ground with 3.5 fold (v/w), MPA (metaphosphoric acid) /EDTA (ethylenediaminetetraacetic acid) (5% (w/v)/lmM) in a pestle 10 and mortar at 4 0C. The homogenate was incubated on ice for 30 min, then centrifuged for 2 min in a microcentrifuge. Vitamin C was then measured in the supernatant. Vitamin C was determined by following the decrease in 15 absorbance at 265nm, after addition of ascorbate oxidase. An aliquot of the sample (30 pl) was added to (2.7 ml) 1 M sodium phosphate buffer, pH 6.0, in a 3-ml cuvette, and OD 265 determined. 12 units ascorbate oxidase (EC 1.10.3.3 from Curcubita spec.) were added and the decrease in OD 265 20 determined. The assay was calibrated using standard solutions of ascorbate (0-1 mM in MPA/EDTA). Data represented in table 1. Measurement of starch content: 25 The residue from sucrose assays was resuspended in 15 ml water and dispersed, then incubated at 1000C for 3 hr to solubilise the starch. The sample was then allowed to cool, and 10 ml sodium acetate buffer (0.5M, pH 4.5) and 2 ml (20 30 U) amyloglucosidase (EC 3.2.1.3 from Aspergillus niger) added. The sample was then incubated overnight (16hr) at 55 WO 99/53041 PCT/EP99/02046 - 19 C. The sample was quantitatively transferred to a 100 ml volumetric flask. A portion of this extract was then filtered through a piece of 541 Filter paper. Glucose was then measured in this filtrate. A scaled down version of 5 this process was used when sample sizes were small. Data represented table 1.

WO 99/53041 PCT/EP99/02046 - 20 Results table 1: SI lie DAI SUCROSE % STRC % mg/100g ....

_... 17 50.14 nd 19 38.29 2.29 6.03 21 52.1 2.47 3.94 25 42.4 1.87 5.98 45.73 5.32 17 53.2 2.58 1.69 18 nd 2.05 1.95 21 39.7 2.12 1.61 24 35.7 2.97 1.35 25 nd 3.47 2.18 42.87 1.76 17 nd 2.26 0.46 18 47.1 2.07 0.84 21 46.2 2.55 0.99 242.43 2.82 25 38. 3.501.59 43.83 1.34 rug3a 18 nd 3.27 0.19 19 56.5 2.63 0.10 21 52.5 5.52 0.09 25 42.3 1.74 0.46 50.43 0.21 21 47. 4.390.36 25 31. 3.780.36 47.37 0.74 rug_4_17_6.2_2.551.41 21___52.5 _ __2.60___2.27 24 24.6 2.49 3.26 25 33. 3.314.64 39.23 2.90 rg517 Nd 2.47 1.44 18 52. 2.531.53 21 44.5 2.80 1.78 46.27 1.70 WO 99/53041 PCT/EP99/02046 - 21 Table 1 shows comparative data for a range of near isogenic pea lines with the mutations at the loci indicated, as compared with wild type pea seeds. 5 Example 2: Comparison between standard commercial varieties and varieties in which a mutation at the rug3 locus has been introduced. 10 Breeding programme: As outlined in WO-A-98/01574 the rug3 mutant lines used in the above work, while useful for experimental purposes, are 15 not suitable for commercial use. A breeding programme was therefore undertaken to produce pea plants of agronomically acceptable character, with the rug3 mutation. Details are as follows: 20 1. The first crossing was undertaken early in 1993 between the five rug3rug3 SIM lines and two normal (Rug3Rug3 or ++) varieties as parents; Harrier, a Unilever bred registered variety, and Novella a leading commercial variety. 25 2. The crosses were carried out as a half diallel (i.e. each of the SIM lines onto each of the two varieties, without concern over which was used as the male or female). 30 WO 99/53041 PCT/EP99/02046 - 22 3. The (Fl) seed from the crossed flowers was harvested and Fl plants grown in insect proof glasshouses. The plants were allowed to self and the F2 seed collected during 1993. 5 4. The seed was sorted into wrinkled (rug3rug3--,--rr, or rug3rug3rr) (-- indicates either dominant wild type or heterozygote) or round (containing at least one dominant copy of Rug3 and R). The wrinkled seed were sown in the 10 field in spring 1994, supported by wires, and standard pedigree selection of F2 plants was done. 5. During August 1994 five F3 seed were taken from each of those plants selected from the above as having 15 acceptable agronomic characters. 6. The F3 seed was checked for starch content. This was done by drilling a small quantity of dust from a cotyledon of each seed, and testing for starch by the 20 addition of iodine solution. In this way the wrinkled but starchy rrRug3Rug3 lines should be rejected but the test on large numbers was imprecise and some miscategorisation probably occurred. The putative rug3rug3 lines were put into four very approximate 25 groups (<1, <5, <10 and 10+% starch) by comparisons of the colour density with the SIM standards. 7. The remaining 765 F3 seed, all now known to be probable rug3rug3 and potentially acceptable agronomically were 30 sown in insect proof glasshouses in October 1994.

WO 99/53041 PCT/EP99/02046 - 23 8. When ripe, four F4 seed were taken from each of the surviving F3 plants, and sown in a large pot in the same glasshouse. 5 9. When ripe (May 1995) as many seed as possible up to 100 were taken from each pot and sown as a square meter F5 plot in the field. 10. The standard method of assessing maturity by 10 tenderometer is not applicable to small plots. Hence at a date decided by the experience of the breeder, looking at and feeling the fullness of the pods, small samples were taken for the sugar analysis. 15 11. The samples were frozen to -18 degrees C, and sugar content was analysed by a modified Hexakinase/Glucose-6 Phosphate dehydrogenase hexose analytical technique. 12. On the basis of these tests and agronomic assessment, 45 20 lines were selected for development, these covering a range of increased sweetness levels. The dry seed of these lines was harvested in August 1995. 13. Fifty seed of each selected line were grown up in New 25 Zealand. 14. In March 1996 the multiplied seed (F7, but a mixture of F4 derived lines) was returned to the UK, added to the remnant F6 seed from the 1995 plots, and sown as two 30 plots of 6.75 sq m. One plot was harvested in July 1996 for vining and freezing, with the other being left to WO 99/53041 PCT/EP99/02046 - 24 dry seed harvest to produce further seed to be used for larger scale trials. 15. Lines were selected on the basis of wrinkled seed and 5 agronomy and confirmed as rug3 based on the starch content of the mature dry seed. The dry starch analysis gave the largest range of values and clearly separated the lines with and without homozygous rug3. (Methods detailed in WO-A-98/01574) 10 16. Some lines were backcrossed into both parents to confirm genotype, this confirmed results based on the starch level of the dried seed. 15 Analysis of pea composition in commercial seed lines: Peas were harvested at a range of tenderometer readings (as indicated in table 2) and their composition was analysed. 20 Measurement of sucrose content: Whole seeds were harvested throughout development and fresh weights recorded. The weights of the seeds ranged from 25 approximately 20mg to 600mg from both wild type and rug3rug3 plants. The seeds were freeze-dried and soluble sugars extracted by boiling in 5ml 80% v/v ethanol followed by grinding to a fine paste. After pelleting the solids by centrifugation at 2000g for 10 minutes, the supernatant was 30 removed and evaporated to dryness. More 80% ethanol was added to the pellet and the process was repeated twice more.

WO 99/53041 PCT/EP99/02046 - 25 The supernatants from each stage were pooled and evaporated to dryness to leave a final pellet containing all of the soluble sugars. Extracts were analysed by ion chromatography 5 under the following conditions: Chromatograph: Dionex 4000i (BIO LC) Column: Guard; CarboPac PAl 50mm x 4mm i,d.(l0Fm). 10 Main; CarboPac PAl 250mm x 4mm i,d.(l0Fm). Column temp. 25 0 C Injection vol. 25ml Mobile phase: 150mM NaOH isocratic (continually 15 degassed with He) at a flow of lml/min. Detector: Pulsed Amperometric (PAD), with gold working electrode and silver reference. 20 Detector settings: Range; 3KnA Applied Potentials; El: + 0.05V.(480ms) E2: + 0.60V.(120ms) E3: - 0.60V.(60ms) 25 Data represented in table 2. Measurement of Vitamin C content: This analysis was carried out on freshly vined peas in 30 addition to blanched-frozen peas. The latter data thereby representing a simulation of the typical commercial WO 99/53041 PCT/EP99/02046 - 26 processing steps that frozen peas for human consumption would normally undergo. A 50g macerated sample was blended in 200ml, 5% 5 metaphosphoric acid using Ultra Turrax, then filtered. 25ml of filtratre was reduced to pH 0.6 with iml 50% sulphuric acid. 2.9ml formaldehyde (37-41%) was then added and solution swirled to mix. 10ml aliquots were then transferred to 25ml test tubes. Eight minutes after adding 10 the formaldehyde, solution titrated against 0.08% 2,6 dichlorophenol-indophenol. Data represented in table 2. 15 Measurement of starch content: This was carried out as described in example 1. Data represented in table 2. 20 WO 99/53041 PCT/EP99/02046 - 27 Results table 2: .Variety TR Vi C Vitamin Sucrose'sac Ratio peas blInche .g/10 d starch mag /100g 1Q92 84 37.6 27.8 5.34 0.8 6.68 98 32.6 27.3 6.30 1.1 5.73 118 26.6 21.2 5.25 1.1 4.77 142 27.8 21.5 5.15 1.4 3.68 1115 82 30.9 21.8 5.35 0.2 26.75 90 29.4 19.9 5.84 0.2 29.2 111 24.2 19.1 6.13 0.4 15.33 118 24.2 19.1 5.16 0.4 12.9 123 24.9 16.4 5.47 0.4 13.68 24821 88 32.0 24.5 7.18 0.1 71.8 105 28.3 20.7 7.05 0.1 70.5 143 23.3 16.9 6.22 0.1 62.2 168 26.4 20.3 6.39 0.1 63.9 2495 87 39.0 28.2 6.99 0.1 69.9 101 30.9 N/D N/D N/D N/D 132 29.2 18.7 6.85 0.1 68.5 165 27.6 19.1 6.53 0.1 65.3 Avola 87 27.8 17.8 4.50 1.7 2.65 103 24.7 17.8 4.94 2.0 2.47 107 26.5 17.4 4.66 2.2 2.12 130 22.1 17.1 5.08 2.9 1.75 155 20.3 15.2 4.29 4.1 1.05 H&rrier 82 24.9 21.1 4.24 1.9 2.23 92 29.1 24.4 4.70 2.1 2.24 100 26.0 21.5 4.72 2.2 2.15 S 140 21.0 16.1 3.70 3.8 0.97 Nella'& 83 28.1 15.9 4.54 1.9 2.39 96 26.5 19.4 4.89 2.4 2.04 115 24.4 20.0 4.85 3.0 1.62 136 20.0 15.4 4.07 4.2 0.97 5 N/D: no data Statistics WO 99/53041 PCT/EP99/02046 - 28 Analysis of variance was used to compare control and rug3 groups of samples Comparison of std varieties with rug3 lines: 5 Vitamin C p=0.21, sucrose, p=0.0001, starch, p=0.0001 Vitamin C significantly higher in rug3 lines at 95 % level. Variety 1092 = Harrier crossed with SIM 1 1115 = Harrier crossed with SIM 41 10 2482 = Novella crossed with SIM 43 2495 = Sancho crossed with SIM32 Example 3: Tomatoes with increased Vitamin C 15 Tomatoes of the CB 4

S

2 line, which are known to express reduced sucrose invertase were investigated to assess the effects of such a characteristic on the sucrose and vitamin C content. CB 4

S

2 are derived from a cross between FM6203 20 (Lycopersicon esculentum) which accumulate hexose sugars and Lycopersicon chmelewskii which is a wild type green fruited tomato with a sucrose accumulatory trait. A series of crossings and back crossings was undertaken wherein marker assisted breeding techniques were used to identify the 25 maintenance at the sucrose accumulation trait. A comparison was drawn with a conventional commercial tomato line FM6203. 30 Tomato plants were grown in a glass house and analyses of the vitamin C content and sugars were made a various stages in the fruit ripening process. The content of vitamin C, sugars and starch were analysed 35 according to the methods described in example 1. Data represented in table 3.

WO 99/53041 PCT/EP99/02046 - 29 Results table 3:. ... . . ................ . 19.00 0.0' 10.25 11.33 -Green 2.46 2.6 2.39 CBeen 23.93 4.61 6.92 7.45 0.23 3.79 2.50 2.29 FM6203 16.27 0.0 15.18 16.05 Break 1.36 3.24 1.77 CB4S222.12 13.7 8.16 8.99 r 2.36 6.18 1.90 2.04 F62~3 14.61 0.0 14.23 14.95 0.84 0.62 0.74 "Turnin 20.87 13.51 5.14 5.51 0.13 4.22 0.60 1.13 W63 12.45 0.0 19.98 18.95 1.92 1.14 1.59 25.72 11.68 5.47 5.66 4.62 3.98 0.14 0.02 FM 21.86 0.0 17.65 17.72 2.73 0.7 0.96 CBd 25.31 11.28 5.79 6.52 1.69 2.23 0.96 1.71 FM62(3 18.74 0.0 19.32 21.07 2.45 1.52 0.23 Ripe 27.46 18.90 4.53 5.00 Red* 0.86 1.81 1.75 1.51 e p<O.05 5 The low invertase tomatoes consistently contained significantly higher vitamin C at all stages of maturity.

Claims (18)

1. Use in a plant of a genetic modification capable of increasing the content of sucrose or any intermediate 5 compound in, or precursor compound to the biosynthetic pathway from sucrose to starch to produce an increase in vitamin C content in said plant.

2. Use in a plant of a genetic modification to increase 10 vitamin C content according to claim 1, wherein the said genetic modification has an effect of increasing the content of one or more intermediates in the sucrose starch pathway selected from the group comprising UDP glucose, glucose-l-phosphate, glucose-6-phosphate, 15 fructose-6-phosphate or ADP glucose.

3. Use in a plant of a genetic modification to increase vitamin C content according to claim 2 wherein the genetic modification is capable of increasing the 20 content of one or more intermediates selected from the group comprising UDP glucose, glucose-l-phosphate or glucose-6-phosphate.

4. Use in a plant of a genetic modification to increase 25 vitamin C content according to claim 1 wherein said modification causes a reduction in activity of one or more cellular enzymes selected from the group comprising starch branching enzyme, ADP glucose pyrophosphophylase, plastidial phosphoglucomutase, starch synthase or 30 sucrose invertase. WO 99/53041 PCT/EP99/02046 - 31

5. Use in a plant of a genetic modification to increase vitamin C content according to claim 4, wherein said plant is selected from the group comprising pea, tea, spinach, potato, beans, carrot, tomato, pepper, borlotti 5 beans, citrus fruit, Brassicaceae, maize, berries.

6. Use in a plant of a genetic modification to increase vitamin C content according to claim 4, wherein the plant is selected from the group comprising pea, 10 spinach, tea, tomato.

7. Use in a plant of a genetic modification to increase vitamin C content according to claim 4 wherein the plant is tomato. 15

8. Use in a plant of a genetic modification to increase vitamin C content according to claim 7 wherein tomato fruit from said plant are used in the preparation of pastes, pur6es or sauces. 20

9. Use in a plant of a genetic modification to increase vitamin C content according to claim 4 wherein the plant is pea. 25

10. Use in a plant of a genetic modification to increase vitamin C content according to claim 9 wherein said genetic modification is a mutation at a r locus, a rb locus, a rug3 locus or a rug5 locus. 30

11. Use in a plant of a genetic modification to increase vitamin C content according to claim 9, wherein the WO 99/53041 PCT/EP99/02046 - 32 genetic modification is a mutation at the r locus, the rb locus, or the rug3 locus.

12. Use in a pea plant of a genetic modification capable of 5 increasing the content of sucrose or any intermediate compound in, or precursor compound to the biosynthetic pathway from sucrose to starch to produce an increase in vitamin C, wherein the said genetic modification causes a reduction in the activity of the enzyme plastidial 10 phosphoglucomutase.

13. Use in a plant of a genetic modification to increase vitamin C content according to claim 9, wherein the genetic modification is a mutation at the rug3 locus. 15

14. Process for the production of peas with increased vitamin C for human consumption comprising the steps; 20 (i) pea seeds that have been exposed to one or more genetically mutagenic compounds grown and the progeny are selected on the basis of achieving an increased vitamin C content; (ii) said selected peas are then incorporated by way of 25 a breeding programme into a commercially suitable seed line which maintains the increased vitamin C content; (iii) seeds from (ii) are then further grown and harvested; 30 (iv) optionally the harvested product is blanched and frozen. WO 99/53041 PCT/EP99/02046 - 33

15. Process for the production of peas with increased vitamin C according to claim 14, wherein the genetic modification increases the content of sucrose or any 5 intermediate compound in or precursor compound to the biosynthetic pathway from sucrose to starch.

16. Process for the production of peas with increased vitamin C for human consumption according to claim 13, 10 wherein the genetic modification in (i) reduces plastidial phosphoglucomutase activity in said plant.

17. Process for the production of peas with increased vitamin C according to claim 14, wherein the genetic 15 modification in (i) comprises a mutation at the rug3 locus.

18. Process for the production of peas with increased vitamin C according to claim 14, wherein the genetic 20 modification is incorporated into the seed lines selected from the group comprising Novella, Harrier, Avola, Sancho 25