MXPA01001609A - Method for producing plants with increased flavonoid and phenolic compound content - Google Patents

Abstract

The invention relates to a method for increasing flavonoid content in plants. Said method is characterized that a plant is produced using molecular genetic methods in which the activity of enzyme flavanon-3-hydroxylase is reduced.

Description

METHOD FOR PRODUCING PLANTS WITH AN ELEVATED CONTENT OF FLAVONOIDS AND PHENOLIC COMPOUNDS The present invention relates to a method for increase the content of flavonoids and phenolic constituents in plants, where a plant is generated, by molecular genetic methods, in which the activity of enzymatic flavanone 3-hydroxylase is reduced. Furthermore, in the method according to the invention, the activity of 3-hydroxylase of enzymatic flavanone is reduced completely or partially, permanently or temporarily, throughout the plant or in parts of the plant, by molecular biology methods ( for example antisense constructions, cosuppression, the expression of specific antibodies, or the expression of specific inhibitors). The invention also relates to plants with a high content of flavonoids and phenolic constituents, whose enzymatic activity of enzymatic flavanone 3-hydroxylase is reduced. On the other hand, the invention relates to the use of plants, generated by the method according to the invention, or parts of these plants as food products, supplements or to produce curative compositions, compositions that promote health or tonics (juices, a ^ w ^ - ^^. ^. a ^ L ,, ..i ^^^ - infusions, extracts, fermentation products) for humans or animals, and for the production of cosmetics. A variety of phenolic substances are found in plants, for example, caffeic acid, ferulic acid, chlorogenic acid, gallic acid, eugenol, lignans, coumarins, lignin, stilbenes (polydatin, resveratrol), flavonoids (flavones, catechins, flavanones, anthocyanidins , isoflavones), and polymethoxylated flavones. Therefore, generally also the Phenols are generally a constituent of a number of food products and stimulants derived from plants. Certain phenolic substances are of particular importance since, after having been taken with the food, they can exert an antioxidant effect in the human or animal metabolism (Baum, BO; Perun, AL Anti-oxidant effectiveness against structure.) Plast Engrs Trans 2: 250-257, (1962); Gardner, PT; McPhail, DB; Duthie, GG Rotational resonance spectroscopic test of electrons of antioxidant potential of teas in aqueous and organic medium. J. Sci. Food Agrie 76: 257-262, (1997); Rice-Evans, C. A .; Miller, N. J .; Pananga, G. Relationship of antioxidant activity-structure of flavonoids and phenolic acids. Free Radie Biol. Med. 20: 933-956, (1996); Salah, N .; Miller, N. J .; Paganga, G .; Tijburg, L .; Bol ell, G. P .; Rice-Evans, C. Flavonoides polyphenols as an aqueous phase radical scavenger and as chain breaking antioxidants. Arch Biochem Biophys 322: 339-346, (1995); Stryer, L. Biochemistry S. Francisco: Freeman, (1975); Vieira, 0; Escargueil-Blanc, I .; Meilhac, 0; Basile, J. P .; Laranjinha, J .; Almeida, L .; Salveyre, R .; Negre-Salvayre, A. Effect of dietary phenolic compounds on apoptosis of human cultured endothelial cells induced by oxidized LDL. Br J Pharmacol 123: 565-573, (1998)). In addition, polyphenols also have a plurality of effects on cellular metabolism. Among other things, they modulate the signal transduction enzymes of protein kinase C, protein tyrosine kinase and phosphatidylinositol kinase-3.

(Agullo, G., Gamet-Payrastre, L., Manenti, S., Viala, C. Remesy, C. Chap, H., Payrastre, B. The relationship between the flavonoid structure and the inhibition of phosphatidylinositol kinase-3. : a comparison with tyrosine kinase and inhibition of protein kinase C. Biochem Pharma 53: 1649-1657, (1997), Ferriola, P. C, Cody, V. Middleton, E. Inhibition of protein kinase C by flavonoids of plant, kinetic mechanisms and structure activity relationship, Biochem Pharmacol 38: 1617-1624, (1989), Cushman, M., Nagarathman, D., Burg, DL, Geahlen, RL, Synthesis and inhibitory activity of tyrosine protein kinase. of flavonoid analogues J Meed Chem 34: 798- ^^^ - ^ t ^ - ^^ - ^ l 06, (1991); Hagi ara, W Inoue, S .; Tanaka, T .; Nunoki, K .; Ito, M .; Hidaka, H. Differential effects of flavonoids as inhibitors of protein tyrosine kinases and serine / threonine protein kinases. Biochem Pharmacol 37: 2987-2992, (1988)) whose NO inducible synthase down regulates (Kobuchi, H., Droy-Lefaix, MT; Christen, Y .; Packer, L. Ginkgo biloba extract (EGb761): inhibitory effect on the production of nitric oxide in the macrophage cell line RAW 264.7, Biochem Pharmacol 53: 897-903, (1997)) and regulates the expression of, for example, interleukins and adhesion molecules (ICAM-1, VCAM-1) (Kobuchi, H .: Droy-Lefaix, M. T .; Christen, Y .; Packer, L.

Ginkgo biloba extract (EGb761): inhibitory effect on the production of nitric oxide in the macrophage cell line RAW 264.7. Biochem Pharmacol 53: 897-903, (1997); Wolle, 5 J .; Hill, R. R .; Ferguson, E .; Devall, L. J .; Trivedi, B. K .; Newton, R. S.; Saxena, U. Selective inhibition of cell-cell adhesion molecule-1 gene expression induced by Tumor Necrosis Factor by a novel flavonoid. The lack of effect on transcription factor NF-kB. Atherioscler Thromb Vasc Biol 16: 1501-1508, (1996)). Now it has been accepted that these effects are beneficial to prevent heart attacks, cardiovascular diseases, diabetes, a variety of certain cancers, tumors and other chronic diseases (Bertuglia, ? a? * A ^ a aj ^^ w ^ & ^ s ^ S. ^^^^ i ^^^^ Lji S.; Malandrino, S. ¿, Colantuoni, A. Effects of natural flavonoid delphinidin on diabetic microangiopathy. Aranei-Forsch / Drug Res 45: 481-485, (1995); Griffiths, K .; Adlercreutz, H .; Boyle, P .; Denis, L .; 5 Nicholson, R.I .; Morton, M. S. Nutrition and Cancer Oxford: Isis Medical Media, (1996); Hertog, M. G. L.; Fesrens, E. J. M .; Hollman, P. C. K .; Ratan, M. B .; Kromhout, D. Flavonoids diet antioxidants and the risk of coronary heart disease: the old study of Zutphen. The Lancet 342: 1007-1011, (1993); Kapiotis, S .; Hermann, M .; Held, I .; Seelos, C; Ehringer, H .; Gmeiner, B. M. Genistein, the angiogenesis inhibitor derived from diet, prevents the oxidation of LDL and protects the endothelial cells from atherogenic LDL damage. Arterioscler Thromb Vasc Biol 17: 2868-74, (1997); Stampfer, M. J .; Hennekens, C. H .; Manson, J. E .; Colditz, G. A .; Roener, B .; D Willet, W. C. The consumption of vitamin E and the risk of coronary heart disease in women. New Engl J Med 328: 1444-1449, (1993); Tijburg, L. B. M .; Mattern, T.; Folts, J. D .; Weisgerber, U. M .; Katan, M. B. Tea flavonoids and cardiovascular diseases: a review. Cpt Rev Food Sci Nutr 37: 771-785, (1997); Kirk, E. A .; Sutherland, P .; Wang, S.A .; Chait, A .; LeBoenf, R.C. Diet isoflavones reduce plasma cholesterol and atherosclerosis in C57BL / 6 mice but not in mice deficient LDL receptor. J Nutr 128: 954-9, (1998) -references-). A series of curative compositions, compositions that promote health or tonics whose effect is based on their content of phenolic substances that are therefore already being obtained from suitable plants (Gerritsen, ME; Carley, WW; Ranges, GE; Shen; , CP; Phan, SA; Ligon, GF; Perry, CA Flavonoids inhibit cytosine-induced endothelial cell adhesion protein gene expression Am J Pathol 147: 278-292, (1995); Lin, JK; Chen, Y C, Huang, YT, Lin-Shiau, SY suppression of protein kinase C and nuclear oncogene expression as possible molecular mechanisms of cancer chemoprevention by epigenin and curcumin J Cell Biochem Suppl 28-29: 39-48, 1997; Zi, X; Mukhtar, H.; Agarval, R. The novel cancer chemopreventive effects of a flavonoid antioxidant silymarin: the inhibition of mRNA expression of an endogenous tumor TNF alpha promoter Biochem Biophys Res Comm 239: 334-339, 1997. Furthermore, it is known that certain food products derived from plants or stimulants prepared therefrom have a positive effect on several diseases. Resveratrol, which is found in white wine, but in particular in red wine (in addition to other components), for example, acts against heart attacks, cardiovascular diseases and cancer (Gehm, B.D.; McAndrews, J.M .; Chien, P.-Y .; Jameson, J.L. Resveratrol, a polyphenolic compound found in grapes and wine, is an agonist for estrogen receptor. Proc Nati Acad Sci USA 94: 14138-14143, (1997); Jang, M .; Cai, L .; Udeam, G.O .; Slowing, K.V .; Thomas, C.F .; Beecher, C.W.W .; Fong, H.H.S; Farnsworth, N.R .; Kinghorn, A.D .; Mehtha, R.G .; Moon, R.C., Pezzuto, J.M. The cancer chemopreventive activity of resveratrol, a natural product derived from grapes. Science 275: 218-220, (1997). A similar action is also found in substances such as catechin, epicatechin-3-gallate, epigallocatechin, and epigallocatechin-3-gallate, all of which are found in tea leaves (Camellia sinensis). The drinks are made with desfermented tea leaves (green tea) in particular, they are health promoters (Hu, G., Han, Cachen, J. The inhibition of oncogene expression by green tea and (-) - epigallocatechin gallate in mice: Nutr Cancer 24: 203-209; (1995); Scholz, E; Bertram 'B .. Camellia sinensis (L.) O. Kuntze, Der Teestrauch [the tea shrubl., Z. Phytotherapie 1 7: 235-250 , (1995); Yu, R .; Jiao, JJ; Duh, JL; Gudehithlu, K .; Tan, TH; Kong, AN Activation of mitogen-activated protein kinases by green tea polyphenols: potential signaling pathways in regulation of the expression of the phase II enzyme gene mediated by the antioxidant response elements, e.Carcinigenesis 18: 451-456, (1997); Jankun, J .; Selman, SH; Swiercz, R. Green tea can prevent 1 cancer Na ture 387: 561, (1997). On the other hand, polymethoxylated flavones of the citrus fruit also shows a potential antitumor action (Chem, J .; Montanari, AM; Widmer, Ww. Two flavanones polymethoxilierte new a class of compounds with potential anticancer activity, isolated from tangerine peel oil solids dancy cold compressed J Agrie Food Chem 45: 364-368, (1997)). It is an object of the present invention to find a simple, inexpensive method for increasing the content of flavonoids and phenolic constituents in forage plants. It has been found that this object is achieved, starting from the physiological studies on growth regulators of the acylcyclohexandion group, by means of genetic engineering methods, which are now available, surprisingly, and with the help of plants that can be generated, which are characterized by a high content of healing constituents, that promote health or that tone. Acylcyclohexanediones such as prohexadione-Ca and trinexapac-ethyl (previously named: cimectacarb) are used as bioregulators to inhibit growth longitudinal of the plants. The reason for its action Bioregulatory is that they block the biosynthesis of gibberellins, which promote longitudinal growth. Due to their structural relationship with 2-oxobutyrate, they inhibit certain dioxygenases that require 2-oxobutaric acid as a co-substrate (Rademacher, W, Biochemical effects of plant growth retardants, in: Plant Biochemical Regulators, Gausman, HW (ed. .), Marcel Dekker, Inc., New York, pp. 169-200 (1991)). It is known that such compounds also bind in phenol metabolism and therefore are capable of causing in a variety of plant species, the production of antiocyanins that are inhibited (Rademacher, W et al., The mode of action of acylcyclohexanediones - a new type of growth retardant, in: Progress in Plant Growth Regulation, Karssen, CM, van Loon, LC, Vreugdenhil, D (eds.), Kluwer Academic Publishers, Dordrecht (1992)). Such effects on the balance of phenolic constituents are considered to be the cause for the secondary effect of prohexadione-calcium against fire blight (Rademacher, W et al., Prohexadione-Ca - a new plant growth regulator for apple with interisting biochemical features, Society of America, July 7-10, 1998, Chicago). A. Lux - Endrich (PHD Thesis, Technical University of Munich at Weihenstephan, 1998) finds in the course of its studies on the mechanism of action of prohexadione-calcium against bacterial fire, which prohexadione-calcium causes, in crops cell phones the content of phenolic substance that will increase during many occasions, during which it processes a series of phenols, which are found, which usually are not present. It was also found within these studies that prohexadione-calcium causes relatively high amounts of luteoliflavan and eriodicyol because they occur in the bud tissue of apples. Luteoliflavana does not normally occur in apple tissue, and eriodicyol occurs in small amounts only as the intermediary of flavonoid metabolism. However, the expected flavonoid catechin and cyanidin were not detected in the treated tissue, or only occurred in markedly reduced amounts (S. Rommelt et al., Paper presented at the 8 th International Worshop on Fire Blight, Kusadasi, Turkey, October 12 -15, 1998). Now it is accepted that prohexadione-Calcium, trinexapac-ethyl and other acylcyclohexandiones that inhibit the hydroxylases that depend on 2-oxoglutaric acid that play an important role in the metabolism of phenolic substances. You are mainly chalcone synthetase (CHS) and flavanone 3-hydroxylase (F3H) (W. Heller and G.

Forkmann, Biosynthesis, in: The Flavonoids, Harborne, JB (ed.), Chapman and Hall, New York, 1998). However, it can not be excluded that acylcyclohexanediones also inhibit other hydroxylases that depend on 2-oxoglutaric acid.

^ AA, -v.,. Go; Ap.ná ^^ wrt ^ which are not yet known. In addition, it seems obvious that a lack of catechin, cyanidin and other final products of flavannoid synthesis is recorded by the plant and that the activity of key enzyme phenylalanine ammonium lyase (PAL) is increased by a feedback mechanism. Due to the fact that CHS and F3H continue to be inhibited, however, these flavanoid end products can not be produced, and the result is an increased formation of luteoliflavan, eriodictyol and other phenols (Figure 1). Because the enzymatic activity of the enzymatic flavanone 3-hydroxylase (F3H) is reduced, the eriodictyol of flavonoids, the proanthocyanidins which are replaced on the C atom by hydrogen, for example, luteoforol, luteoliflavana, apigeniflavana and tricetiflavana, and homogeneous and heterogeneous oligomers and polymers of the aforementioned and structurally related substances are produced in larger amounts. The high concentrations of hydroxycinnamic acid of phenols (p-coumaric acid, ferulic acid, sinapic acid), salicylic acid or unbelliferone, including the homogeneous and heterogeneous oligomers and polymers formed therefrom, are found in plants after has reduced the enzyme activity of the enzymatic flavanone 3-hydroxylase (F3H).

Likewise, the concentrate of the chalcones, increases of for example, floretin, and of the stilbenes, such as, for example, resveratrol. Since enzyme activity of the enzymatic flavanone 3-hydroxylase is reduced, the concentration of the glycosides of the flavonoids, the phenolic compounds, the chalcones and the stilbenes is also increased. Starting from these findings and the hypotheses derived, the genetically modified forage plants were generated in which the activity of F3H was completely or partially reduced, permanently or temporarily, in the whole plant or in plant organisms or tissues Individuals, by means of antisense constructions, so that the content of curative, health promoting or toning substituents are improved in terms of quantity and quality. The method according to the invention for increasing the content of flavanoid and phenolic compounds by expressing flavonone 3-hydroxylase in antisense orientation can be successfully applied in the following forage plants, although the method is not restricted to the plants mentioned: vines, cherries, tomatoes, plums, sloes, blueberry, strawberries, citrus fruits (such as oranges, grapefruit), papaya, red cabbage, broccoli, brussels sprouts, cocoa, kale, carrots, parsley, celery, onions, garlic, tea, coffee, hops, soybeans, oily rape, oats, wheat, rye, Aronia melanocarpa, Ginko biloba. On the other hand, the invention relates to plants with a high content of flavonoid and phenolic constituents, generated by the method according to the invention and with a reduced enzymatic activity of the enzymatic flavanone 3-hydroxylase. As an alternative for generation plants whose flavanone 3-hydroxylase activity is reduced with the help of antisense technology, it is also possible to use other methods of molecular genetics which is known from the literature, such as cosuppression or expression. of specific antibodies, in order to achieve this effect. In addition, the invention relates to the use of plants, generated by the method according to the invention, or parts of these plants as food products, food supplements, or to produce curative compositions, health promoting compositions or tonics (juices, infusions, extracts, fermentation products) for humans and animals, and for the production of cosmetics. Surprisingly it has now been found that the plants that have been generated according to the invention, or parts of these plants or products produced therefrom (teas, extracts, fermentation products, juices and the like) have the following effects: ( 1) the antioxidant capacity in vi tro is improved (Electron Spin Resonance (ESR), LDL oxidation, total antioxidant capacity, NO elimination); (2) a modulation effect on enzymes, especially signal transduction enzymes (protein kinase C, protein tyrosine kinase, phosphatidylinositol kinase 3) is observed; (3) a modulation of redox-sensitive transcriptional factors (NF-kB, AP-1) is induced in endothelial cells, lymphocytes and soft muscle cells; (4) regulation of gene expression of target genes involved in the pathogenesis of inflammatory diseases (cytokines IL-1 and IL-8, protein 1 ma crophage chemoa ttractant (MCP-1), adhesion factors ICAM-1 is modulated and VCAM-1); (5) an antiagregatory effect is induced; (6) the synthesis of cholesterol in hepatocytes is inhibited; (7) there are antiproliferative / antineoplastic effects.

Example 1 Manipulation of a 3-hydroxylase gene from flavanone from Lycopersicon esculentum Mill.cv. Moneymaker The ripe tomato fruits of Lycopersicon esculentum Mill.cv. Moneymaker was washed, and dried, using a sterile knife and the pericarp was freed from the seeds, the central axis and wooded parts. The pericarp (approximately 50 g) was frozen in liquid nitrogen. Then, the material was ground in a mixer. In a pre-cooled mortar, the ground material was treated with 100 ml of homogenization medium and mixed. Then, the suspension was transferred into a centrifugal flask and drained through a sterile gauze. Then, 1/10 of 10% of the volume of SDS was added and the material mixed thoroughly. After 10 minutes on ice, a volume of phenol / chloroform was added, and the centrifuge flask was sealed and the contents mixed thoroughly. After centrifugation for 15 minutes at 4000 rpm, the supernatant was transferred into a new reaction vessel. This was followed by three more phenol / chloroform extractions and one chloroform extraction. Then, 1 volume of 3 M NaAc and 2.5 volumes of ethanol were added. The nucleic acids were precipitated overnight at -20 ° C. The next morning, the nucleic acids were pelleted in a refrigerated centrifuge (4 ° C) for 15 minutes at 10,000 rpm. The supernatant was discarded and the pellet was resuspended in 5-10 ml of 3 M NaAc cold. This washing step was repeated twice. The pellet was washed with 80% ethanol. When completely dried, the pellet was extracted in approximately 0.5 ml of sterile DEPC water, and the concentration of RNA was determined photometrically. 20 μg of total RNA was first treated with 3.3 μl of 3M sodium acetate solution, 2 μl of the magnesium sulfate solution of IM, and the mixture was placed with DEPC water at a final volume of 100 μl. Added to this a microliter of RNase-free DNase (Boehringer Mannheim), and the mixture, was incubated for 45 minutes at 37 ° C. After the enzyme has been removed by extraction with stirring with phenol / chloroform / isoamyl alcohol, the RNA was precipitated with ethanol and the pellet was extracted in 100 μl of DEPC water. 15 2.5 μg of RNA from this solution was transcribed into cDNA using a cDNA kit (Gibco BRL). Using the amino acid sequences that were derived from the cDNA clones that encode flavanone 3-hydroxylase, regions conserved in the main sequence were identified (Britsch et al, Eur. J. Biochem 217, 745-754 (1993), and these acted as the basis for the design of the degenerate PCR oligonucleotides using the SRWPDK peptide sequence (amino acids 147- 152 in the hybrid sequence Petunia FL3H PETHY), the oligonucleotide of 5 'was determined using and had the following sequence: '-TCI (A / C) G (A / G) TGG CC (A / C / G) GA (C / T) AA (A / G) CC-3. The sequence of the deduced oligonucleotide using the peptide sequence THQAVV (amino acid 276-281 in the hybrid sequence Petunia FL3H PETHY) was as follows: 5'-TCI (A / C) G (A / G) TGG CC (A / C / G) GA (C / T) AA (A / G) CC-3. The PCR reaction was carried out using the tTth polymerase by Perkin-Elmer following the manufacturer's instructions. 1/8 of the cDNA was used as a standard (corresponding to 0.3 μg of RNA). The PCR program was as follows: 30 cycles. 94 degrees 4 seconds 40 degrees 30 seconds 72 degrees 2 minutes 72 degrees 10 minutes The fragment was cloned into the pGEM-T vector of Promega following the manufacturer's instructions. The accuracy of the fragment was checked by sequencing. Using the Ncol and Pctl restriction cleavage sites, which are present in the vector polylinker pGEM-T, the PCR fragment was isolated, and the projections were blunted at the ends using T4 polymerase. This fragment was cloned into a pBinAR vector (blunted) Smal (Hdfgen and Willmitzer, Plant Sci 66: 221-230 (1990)) (see Figure 2). This vector contains the promoter ¿^^^^^^ you ^^ ^^^^^ you? Faith ^ te ste gjfc ^^^^^^^^^ g ^ A CaMV 35S (virus cauliflower mosaic) (Franck et al., Cell 21: 285-294 (1980)) and the octopine synthase gene termination signal (Gielen et al., EMBO J. 3: 835-846 (1984)). In plants, this vector mediates resistance to the antibiotic kanamycin 5. The resulting DNA constructs contained the PCR fragment in sense and antisense orientation.The antisense construct was used to generate transgenic plants Figure 2: Fragment A ( 529 bp) contains the promoter 35S CaMV (nucleotide 6909 to 7437 of cauliflower mosaic virus). Fragment B contains the fragment of the F3H gene in antisense orientation. Fragment C (192 bp) contains the signal terminating the octopine synthase gene. Manipulation of a longer cDNA fragment of flavanone 3-hydroxylase from Lycopersicon esculentum Mill.cv. Moneymaker using the 5 'RACE system. To exclude the generation of plants with an amount of flow balance mRNA reduced F3H is not successful, because of the small size of the PCR fragment F3H 20 used in the construction of antisense, a second antisense construct using a fragment F3H longer must be generated. To clone a longer F3H fragment, the 5 'RACE method (system for rapid extension of cDNA ends) was used. .aAsA.áMi »» ^ < ) & '^ Æ ia¡lfeg ^^ extension F3H PCR fragment by the method 5'RACE using the 5' RACE system for Rapid Amplification of cDNA ends, Version 2 ~ 0 by Life Technologies ™. The total mRNA was isolated from the mature tomato fruits of Lycopersicon esculentum Mili. cv. Moneymaker (see above). The first synthesis of cDNA strain was carried out following the manufacturer's instructions using GSP-1 (gene-specific primer) 5 '-TTCACCACTGCCTGGTGGTCC-3 After digestion of RNase, the cDNA was purified following the manufacturer's instructions using the system GlassMAX rotational by Life Technologies ™. Following the manufacturer's instructions, a homopolymer of cytosine was added to the 3 'end of cDNA F3H individual strain purified using deoxynucleotide transferase terminal. DNA F3H 5 'extended amplified using a second primer specific gene (GSP-2) which binds to the 5' end in the 3 'region of the recognition sequence GSP-1, and allowing it to perform this mode a "nested" PCR. The 5 'primer used was the' 5 'RACE shortened anchor primer which was provided by the manufacturer and which is complementary to the homopolymer dC termination of the cDNA. The cDNA amplified in this way, which was called F3He? Tended was cloned into the pGEM-T vector of Promega following the manufacturer's instructions. The identity of the cDNA was confirmed by sequencing. The F3Hextend? Do cDNA fragment was isolated using the restriction cut sites of Ncol and PstI, which are presented in the polylinker of the vector pGEM-T, and the projections were made blunt at the ends using T4 polymerase. This fragment was cloned into a pBinAR vector cut (blunt) (Hofgen and Willmitzer, (1990) (see Figure 3) .This vector contains the 35S CaMV promoter (cauliflower mosaic virus) (Franck et al., (1980) and the termination signal of the octopine synthase gene (Gielen et al., (1984). in plants, the average vector a resistance to antibiotic kanamycin. the constructions resulting DNA contained the PCR fragment in sense orientation and antisense The antisense construct was used to generate transgenic plants Figure 3: Fragment A (529 bp) contains the 35S CaMV promoter (nucleotides 6909 to 7437 of cauliflower mosaic virus) Fragment B contains the gene fragment F3H in antisense orientation Fragment C (192 bp) contains the octopine synthase gene termination signal. ^^ - ^ > ^ .i? B? ^ ^^ Í Example 2 The generation of Lycopersicon esculentum Mill.cv. Transgenic moneymaker which expresses a subfragment of flavanone 3-hydroxylase in antisense orientation. The method used was that of Ling et al. , Plant Cell Report 17, 843-847 (1998). Cultivation was carried out at approximately 22 ° C under a 16-hour light / 8-hour dark regime. Tomato seeds (Lycopersicon esculentum Mill.cv. Moneymaker) were sterilized by incubation for 10 minutes in sodium hypochlorite solution at 4% strength and subsequently washed 3-4 times with sterile distilled water and placed on supplemented MS medium with 3% sucrose, pH 6.1, for germination. After a germination time of 7-10 days, the cotyledons were ready for use in the transformation. Day 1: Petri dishes containing "MSBN" medium were covered with 1.5 ml of a tobacco suspension culture of approximately 10 days. The plates were covered with film and incubated at room temperature until the next day. Days 2: Sterile filter paper was placed on the plates covered with the tobacco suspension culture in such a way that no water bubbles formed. The cotyledons, which had been cut from side to side, They were placed on the flipped filter paper. The Petri dishes were incubated for 3 days in a culture chamber. Day 5: Agrobacterial culture (LBA4404) was sedimented for 10 minutes at approximately 3000 g and resuspended in MS medium so that the OD was 0.3. Cotyledon sections were placed inside this suspension and incubated for 30 minutes at room temperature with gentle agitation. Afterwards, the sections of cotyledons were dried a little on sterile filter paper and returned to their initial plates to continue cocultivation for 3 days in the culture chamber. Day 8: The sections of cocultivated cotyledons were placed on MSZ2K50 + ß and incubated during the following four weeks in the culture chamber. Then they were subcultured. The suckers that were formed were transferred to root induction medium. After the successful rooting, the vegetables were tested and transferred into the greenhouse. Example 3 Inhibition of cholesterol biosynthesis in cultures of primary rat epatocytes. Preparation of standard solutions een 10 and 20 mg of the lyophilized ripe tomatoes cv. "Moneymaker" containing A) only the native flavanone 3-hydroxylase gene (control) and B) as described in Example 2, a subfragment of the flavanone 3-hydroxylase in antisense orientation, is exactly weight and was treated with such amount of DMSO that resulted in a 10 mM stock solution of total flavonoids. Immediately before the tests will begin, dilutions of these standard solutions were prepared in the culture medium. Dilution steps of these standard solutions were carried out in the culture medium. 10-fold dilution steps were carried out een 10 ~ 4 and 10 ~ 8 M. Preparation of hepatocyte cultures Primary hepatocytes were obtained from the livers of male Spraque-Dawley rats (240-290 g) by perfusion with collagenase (Gebhardt et al., Arzneimittel-Forschung / Drug Res. 41: 800-804 (1991) 1990). The hepatocytes were cultured in collagen-coated Petri dishes (6-well plates, Greiner, Nurtingen) at a cell density of 125,000 cells / cm 2 in Williams E medium supplemented with 10% calf serum. More details, in particular on the culture medium, are found in Gebhardt et al., Cell Biol. Toxicol. 6: 369-372 (1990) and Mewes et al., Cancer Res. 53: 5135-5142 (1993). After 2 hours, the cultures were transferred to a medium free of . -. HE? Á6AA. ^. serum supplemented with 0.1 μM insulin. After an additional 20 hours, they were used in the tests. The test substances were tested in each case in three independent cultures of 2-f3 rats. Incubation of hepatocyte cultures with test substances A and B. To demonstrate that cholesterol biosynthesis is affected by test substances A and B, hepatocyte cultures were maintained for a total of 22 hours. They were then incubated with serum-free Williams E medium supplemented with 1C acetate (trace amount only) for 2 hours together with the test substances in the indicated concentrations. In each series of tests, a control was included. The methodology is described in detail by Gebhardt (1991) and Gebhardt, Lipids 28: 613-619 (1993). The trace amounts of 14C acetate are rapidly exchanged with the intracellular acetyl-CoA pool and therefore allow the incorporation of 14C acetate into the sterol fraction, which consists of >90% cholesterol, which will be determined in a problem-free way (Gebhardt, 1993). Analytical methods to influence cholesterol biosynthesis Incorporation of 14C acetate into the sterol fraction (non-hydrolysable lipids) was inhibited by the method of Gebhardt (1991). In the extraction used, in which Extrelut® columns (Merck, Darmstadt) were used, more than 95% of 1C acetate (and small amounts of other low molecular weight metabolites formed thereof) are removed. This test allows to make comparisons between the relative speed of synthesis of cholesterol and precursor sterols under the effect of the test substances (Gebhardt, 1993). Visual and microbial quality checks of hepatocyte cultures. Before and after the test incubation, all the cultures used were visually checked under the microscope for contamination with microorganisms and for the integrity of the cell monolayer. A recognizable change in cell morphology (particularly at high concentrations) was not observed in any of the samples. This largely excludes that the results of the test were affected by cytotoxic effects of the test substances. The tests of routine sterility on all crops revealed no signs of contamination with microorganisms. Results Samples A) of the tomatoes that were not genetically modified (control) showed no effect on cholesterol biosynthesis. In contrast, cholesterol synthesis was significantly inhibited by B samples tagM i .. ». of tomatoes containing a subfragment of flavanone 3-hydroxylase in antisense orientation. ^, ^ ¡^ ¿? T fe ^ ¿A ^ A ^.,

Claims (5)

1. CLAIMS 1. A method to increase the content of flavonoids and phenolic constituents in plants, where a plant is generated, by molecular genetic methods in which the activity of the enzymatic flavanone 3-hydroxylase is reduced.
2. 2. A method as claimed in claim 1 for increasing the content of flavonoids and phenolic constituents in plants, wherein the activity of the 3-hydroxylase of enzymatic flavanone is reduced completely or partially, permanently, or temporarily, throughout the plant or parts of the plant by molecular biology methods (eg, antisense constructs, or deletion, expression of specific antibodies or the expression of specific inhibitors).
3. 3. A method as claimed in claim 1 or 2, wherein the plants are vines, cherries, tomatoes, plums, sloes, blueberry, strawberries, citrus fruits, (such as oranges, grapefruit), papaya, red cabbage, broccoli, brussels sprouts, cacao, kale, carrots, parsley, celery, onions, garlic, tea, coffee, hops, soybeans, oleaginasa, oats, wheat, rye, Aronia melanocarpa, Ginkgo biloba.
4. 4. A plant with a high content of flavonoids and phenolic constituents generated by a method as claimed in any of claims 1-3, wherein the enzymatic activity of the enzymatic flavanone 3-hydroxylase is reduced.
5. 5. The use of plant or parts of these plants, generated by a method as claimed in any of claims 1-3, as food products, supplements, or to produce curative compositions, health promoting compositions or tonics ( juices, teas, extracts, fermentation products) for humans and animals, and for the production of cosmetics. • «^