DE19927568A1 - Increasing resistance of crop plants to chemical stress, especially herbicides, by engineering a reduction in activity of flavanone-3-hydroxylase - Google Patents

Abstract

Method for increasing the resistance of crop plants to chemical stress, particularly where caused by insufficiently selective or inappropriately applied herbicides, by genetically engineering plants to have reduced activity of flavanone-3-hydroxylase (I). An Independent claim is also included for plants produced this way.

Description

The present invention is a method for increasing the resistance of crops to chemical Stress, triggered in particular by insufficiently selective or un Properly applied herbicides, characterized in that molecular genetic methods a plant is produced in which reduces the activity of the enzyme flavanone 3-hydroxylase is.

The inventive method is characterized in that the enzyme flavanone-3-hydroxylase by molecular genetic Ver drive (eg antisense constructs, co-suppression, the Expres sion of specific antibodies or the expression of specific Inhibitors) in whole or in part, permanently or temporarily, throughout the plant or in parts of the plant in it Activity is reduced.

The resistance force is the according to the invention Processes produced plants especially against glyphosate, Glu fosinate-ammonium and compounds of the formula I, against cyclohexene non-herbicides such as sethoxydim, cycloxydim, tepraloxydim or Cle increased foxydim and bromoxynil.

Furthermore, the invention relates to plants with increased cons standing power against insufficient selective and improper ap plicated herbicides by the method of the invention by expression of a flavanone 3-hydroxylase in antisense orien were produced and their enzymatic activity of the Flavanone 3-hydroxylase is reduced.

The productivity of crops can be varied in many ways be reduced by stress factors. To call here are below other: viral diseases, bacterial and fungal pathogens, damaging insects, nematodes, snails, game bites, heat, Cool, cold, lack of water, too high water content of the soil, Bo salinisation, radiation intensity too high, competition for light, Water and nutrients through accompanying flora, too high ozone content in the air surrounding the plants, plant-damaging emissions, z. B. from industrial companies or motor vehicles, improper or not optimally applied herbicidal applications, especially in fruit and wine crops, treatments with herbicides, insecti ciders, fungicides, bioregulators or foliar fertilizers  ger selectivity, foliar applications of pesticides or fertilizer during intense sunlight.

Most of the stressors mentioned can be commonly referred to as "Chemi shear stress. "Here are only limited Scope given for countermeasures. To name is here in particular the use of antidots to minimize herbi cider-induced damage to crops. The use of antidotes However, limited to certain herbicidal classes of active ingredients and is only important for Gramineae. Antidotes for Totalherbi Zide like glyphosate or glufosinate-ammonium are not for that Practice available as their use is not satisfactory leads.

Object of the present invention was accordingly the finding of Procedures that help the resilience of Kulturpflan against chemical stressors, in particular against herbicides, in wider scope is improved.

Based on physiological studies with growth Regulators from the group of acylcyclohexandione were now Surprisingly, genetic engineering methods available with the Help produce crops that are against a series of chemical stressors, in particular herbicides, resistant to are available.

Acylcyclohexanediones such as Prohexadione-Ca and Trinexapac-ethyl (former name: Cimectacarb) are used as bioregulators for the inhibition of plant elongation. Their bioreguatory effect is due to the fact that they block the biosynthesis of length-growth-promoting gibberellins. Due to their structural similarity to 2-oxoglutaric acid, they inhibit certain dioxygenases which require 2-oxoglutaric acid as co-substrate (Rademacher, W, Biochemical effects of plant growth retardants, in: Plant Biochemical Regulator, Gaus man, HW (ed.), Marcel Dekker, Inc., New York, pp. 169-200 (1991)). It is known that such compounds also intervene in the metabolism of phenols and thus in several Pflan zenarten can cause an inhibition of Anthocyanbildung (Rade macher, W et al., The mode of action of acylcyclohexanediones - a new type of growth retardant, in Progress in Plant Growth Regu lation, Karssen, CM of Loon, LC, Vreugdenhil, D (eds.), Kluwer Academic Publishers, Dordrecht (1992)). Such effects on the phenolic content are reported as the cause of the side-effect of prohexadione-Ca against fire blight (Rade macher, W et al., Prohexadione-Ca - a new plant growth regulator for apple with interesting biochemical features, poster on the 25 ^th Annual Meeting of the Plant Growth Regulation Society of America, 7-10 July 1998, Chicago). A. Lux-Endrich (Dissertation Technical University of Munich in Weihenstephan, 1998) found in the course of their studies on the mechanism of action of prohexadione-Ca against fire blight that there is a multiple increase in the content of phenolic substances in cell cultures of apple by Pro hexadione-Ca and that a number of otherwise non-existent phenols occurs. In the context of these investigations it was further found that relatively high levels of luteoliflavan and eryodyctiol occur in shoot tissue of apple under the influence of prohexadione-Ca. Luteoliflavan is not normally found in apple tissue, and erythodyctiol occurs only in small amounts as an intermediate in flavonoid metabolism. However, the expected flavonoids catechin and cyanidin were not detectable in the treated tissue or occurred only in significantly reduced amounts (S. Römmelt et al., Lecture 8th International Workshop on Fire Blight, Kusadasi, Turkey, 12-15 Octo about 1998).

It can be taken for granted that prohexadione-Ca, Trinexapac ethyl and other acylcyclohexanediones inhibit 2-oxoglutaric acid-dependent hydroxylases, which are important in the metabolism of phenolic sub stances. These are primarily flavanone-3-hydroxylase (F3H) (W. Heller and G. Forkmann, Bio-synthesis, in: The Flavonoids, Harborne, JB (ed.), Chapman and Hall, New York, 1988). However, it can not be excluded who the that acylcyclohexanediones inhibit other, previously unknown, 2-oxoglutaric acid-dependent hydroxylases. It should also be apparent that a lack of catechol, cyanidin or other ren end products of flavonoid synthesis is trated by the plant and that the activity of the key enzyme phenylalanine ammonium lyase (PAL) is increased via a feedback mechanism. However, the persistence of F3H inhibits the formation of these flavonoid endproducts and increases the production of luteoliflavan, eriodyctiol, and other phenols ( Figure 1).

By reducing the enzyme activity of the enzyme flavanone-3-hy droxylase (F3H) are the flavonoids eriodictyol, proanthocyani dines which are substituted on the C-atom 3 by hydrogen, z. B. Lu teoforol, luteoliflavan, apigeniflavan and tricetiflavan, as well as homogeneous and heterogeneous oligomers and polymers from the mentioned and structurally related substances increasingly formed.

Increased concentrations of phenols hydroxycinnamic acids (p-coumar acid, ferulic acid, sinapinic acid), salicylic acid or umbellife ron, including the homogeneous and het formed from them  Rogenic oligomers and polymers are used after reduction of the enzyme activity of the enzyme flavanone-3-hydroxylase (F3H) in plants detected.

Based on these findings and the derived Hypo theses were produced by genetically modified crops, in which flavanone 3-hydroxylase is completely replaced by anti-sense constructs or partially, permanently or temporarily, throughout Plants or in individual plant organs or tissues in ih activities were reduced, so that here the content of Phenols and thus the resistance of the novel Plants against chemical stress was increased.

Particularly in the case of the transgenic plants in which the Flava non-3-hydroxylase activity was reduced by molecular genetic methods, increased resistance to chemical stress, triggered in particular by insufficiently selective or improperly applied herbicides, was found. This effect was observed especially when the herbicides glyphosate, Glufo sinate-ammonium, compounds of the formula (I), cyclohexenone Herbi ciden such as sethoxydim, cycloxydim, Tepraloxydim or Clefoxydim and in Bromoxynil.

in which the variables have the following meanings:
R ¹ , R ^{2 are} hydrogen, nitro, halogen, cyano, C ₁ -C ₆ -alkyl, C ₁ -C ₆ -haloalkyl, C ₁ -C ₆ -alkoxy, C ₁ -C ₆ -haloalkoxy, C ₁ -C ₆ Alkylthio, C ₁ -C ₆ -haloalkylthio, C ₁ -C ₆ -alkylsulphinyl, C ₁ -C ₆ -haloalkylsulfinyl, C ₁ -C ₆ -alkylsulfonyl or C ₁ -C ₆ -haloalkylsulfonyl;
R ^{3 is} hydrogen, halogen or C ₁ -C ₆ -alkyl;
R ⁴ , R ^{5 are} hydrogen, halogen, cyano, nitro, C ₁ -C ₄ -alkyl, C ₁ -C ₄ -alkoxy-C ₁ -C ₄ -alkyl, di- (C ₁ -C ₄ -alkoxy) -C C ₁ -C ₄ -alkyl, di- (C ₁ -C ₄ -alkyl) amino-C ₁ -C ₄ -alkyl, [2,2-di- (C ₁ -C ₄ -alkyl) -hydrazino-1] C ₁ -C ₄ -alkyl, C ₁ -C ₆ -alkyliminooxy-C ₁ -C ₄ -alkyl, C ₁ -C ₄ -alkoxycarbonyl-C ₁ -C ₄ -alkyl, C ₁ -C ₄ -alkylthio-C C ₁ -C ₄ -alkyl, C ₁ -C ₄ -haloalkyl, C ₁ -C ₄ -cyanoalkyl, C ₃ -C ₈ -cycloalkyl, C ₁ -C ₄ -alkoxy, C ₁ -C ₄ -alkoxy-C ₂ - C ₄ alkoxy, C ₁ -C ₄ haloalkoxy, hydroxy, C ₁ -C ₄ alkylcarbonyloxy, C ₁ -C ₄ alkylthio, C ₁ -C ₄ haloalkylthio, di (C ₁ -C ₄ alkyl ) -amino, COR ⁶ , phenyl or benzyl, where the latter two substituents may be partially or completely halogenated and / or may carry one to three of the following groups: nitro, cyano, C ₁ -C ₄ alkyl, C ₁ -C ₄ -haloalkyl, C ₁ -C ₄ -alkoxy or C ₁ -C ₄ -haloalkoxy;
or
R ⁴ and R ⁵ together form a C ₂ -C ₆ alkanediyl chain which may be substituted one to four times by C ₁ -C ₄ alkyl and / or substituted by oxygen or an optionally C ₁ -C ₄ alkyl Nitrogen can be interrupted;
or
R ⁴ and R ⁵ together with the associated carbon form a carbonyl or a thiocarbonyl group;
R ^{6 is} hydrogen, C ₁ -C ₄ -alkyl, C ₁ -C ₄ -haloalkyl, C ₁ -C ₄ -alkoxy, C ₁ -C ₄ -alkoxy-C ₂ -C ₄ -alkoxy, C ₁ -C ₄ - Haloalkoxy, C ₃ -C ₆ alkenyloxy, C ₃ -C ₆ alkynyloxy or NR ⁷ R ⁸ ; Hydrogen or C ₁ -C ₄ -alkyl;
R ^{8 is} C ₁ -C ₄ -alkyl;
XO, S, NR ⁹ , CO or CR ¹⁰ R ¹¹ ;
YO, S, NR ¹² , CO or CR ¹³ R ¹⁴ ;
R ⁹ , R ^{12 are} hydrogen or C ₁ -C ₄ -alkyl;
R ¹⁰ , R ¹¹ , R ¹³ , R ^{14 are} hydrogen, C ₁ -C ₄ -alkyl, C ₁ -C ₄ -haloalkyl, C ₁ -C ₄ -alkoxycarbonyl, C ₁ -C ₄ -haloalkoxycarbonyl or CONR ⁷ R ⁸ ;
or
R ⁴ and R ⁹ or R ⁴ and R ¹⁰ or R ⁵ and R ¹² or R ⁵ and R ¹³ together form a C ₂ -C ₆ alkanediyl chain, one to four times by C ₁ -C ₄ alkyl may be substituted and / or interrupted by oxygen or an optionally C ₁ -C ₄ alkyl substituted nitrogen;
R ^{15 is} a linked in the 4-position pyrazole of the formula II

in which
R ^{16 is} C ₁ -C ₆ alkyl;
ZH or SO ₂ R ¹⁷ ;
R ^{17 is} C ₁ -C ₄ -alkyl, C ₁ -C ₄ -haloalkyl, phenyl or phenyl which is partially or completely halogenated and / or carries one to three of the following groups:
Nitro, cyano, C ₁ -C ₄ -alkyl, C ₁ -C ₄ -haloalkyl, C ₁ -C ₄ -alkoxy or C ₁ -C ₄ -haloalkoxy;
R ^{18 is} hydrogen or C ₁ -C ₆ -alkyl
mean;
where X and Y are not simultaneously oxygen or sulfur;
and their agriculturally useful salts.

The following list of compounds with herbicidal activity indicates possible active ingredients in which also in plants in which the enzyme activity of flavanone-3-hydroxylase by antisense Kon constructs is wholly or partially reduced, a higher resistance against the herbicide concerned can be observed , The list is not limited to these substances.
b1 1,3,4-thiadiazoles:
buthidazole, cyprazole
b2 amides:
allidochlor (CDAA), benzoylprop-ethyl, bromobutide, chlorothiamide, dimepiperate, dimethenamid, diphenamid, etobenzanide (benzchlomet), flamprop-methyl, fosamine, isoxaben, monalide, naptalame, pronamide (propyzamide), propanil
b3 aminophosphoric acids:
bilanafos, (bialaphos), buminafos, glufosinate-ammonium, glyphosate, sulfosate.
b4 aminotriazoles:
amitrol
b5 anilides:
anilofos, mefenacet
b6 aryloxyalkanoic acids:
2,4-D, 2,4-DB, clomeprop, dichloroprop, dichlorprop-P, dichloroprop-P (2,4-DP-P), fenoprop (2,4,5-TP), fluoroxypyr, MCPA, MCPB , mecoprop, mecoprop-P, napropamide, napropanilide, tri clopyr
b7 benzoic acids:
chloramben, dicamba
b8 benzothiadiazinones:
bentazone
b9 Bleacher:
clomazone (dimethazone), diflufenican, fluorochloridone, flu poxam, fluridone, pyrazolate, sulcotrione (chlormesulone)
b10 carbamates:
asulam, barban, butylate, carbetamide, chlorobufam, chloropropham, cycloate, desmedipham, diallate, EPTC, esprocarb, molates, orbencarb, pebulate, phenisopham, phenmedipham, pro pham, prosulfocarb, pyributicarb, sulfallate (CDEC), terbu carb, thiobencarb (benthiocarb), tiocarbazil, triallate, ver nolate
b11 quinolinic acids:
quinclorac, quinmerac
b12 chloroacetanilides:
acetochlor, alachlor, butachlor, butenachlor, diethyl ethyl, dimethachlor, metazachlor, metolachlor, pretilachlor, propachlor, prynachlor, terbuchlor, thenylchloro, xylachlor
b13 cyclohexenones:
alloxydim, caloxydim, clethodim, cloproxydim, cycloxydim, sethoxydim, tralkoxydim, 2- {1- [2- (4-chlorophenoxy) propyloxymino] butyl} '3-hydroxy-5- (2H-tetrahydrothiopyran-3-yl) -2 cyclohexen-1-one
b14 dichloropropionic acids:
dalapon
b15 Dihydrobenzofurans:
ethofumesates
b16 Dihydrofuran-3-one:
flurtamone
b17 Dinitroaniline:
benefin, butraline, dinitramine, ethalfluralin, fluchloralin, isopropalin, nitralin, oryzalin, pendimethalin, prodiamine, profluralin, trifluralin
b18 dinitrophenols:
bromofenoxime, dinoseb, dinoseb acetate, dinoterb, DNOC
b19 diphenyl ether:
acifluorfen-sodium, aclonifen, bifenox, chloronitrofen (CNP), difenoxuron, ethoxyfen, fluorodifen, fluoroglycofen-ethyl, fomesafen, furyloxyfen, lactofen, nitrofen, nitrofluorfen, oxyfluorfen
b20 dipyridylenes:
cyperquat, difenzoquat-methylsulfate, diquat, paraquat di chloride
b21 ureas:
benzthiazuron, buturon, chlorobromuron, chloroxuron, chlortoluron, cumyluron, dibenzyluron, cycluron, dimefuron, diuron, dymron, ethidimuron, fenuron, fluorometuron, isoproturon, isouron, carbutilate, linuron, methabenzothiazuron, metobenzuron, metoxuron, monolinuron, monuron, neburon, siduron, tebu thiuron, trimeturon
b22 imidazole:
isocarbamid
b23 imidazolinone:
imazamethapyr, imazapyr, imazaquin, imazethabenz-methyl (ima zame), imazethapyr
b24 oxadiazoles:
methazole, oxadiargyl, oxadiazon
b25 oxiranes:
tridiphane
b26 phenols:
bromoxynil, ioxynil
b27 phenoxyphenoxypropionic acid ester:
clodinafop, cyhalofop-butyl, diclofop-methyl, fenoxaprop ethyl, fenoxaprop-p-ethyl, fenthiapropethyl, fluazifop-butyl, fluazifop-p-butyl, haloxy-pent-ethoxy-ethyl, haloxy-pent-methyl, haloxy-stop-p-methyl, isoxapyrifop, propaquizafop, quizalofop ethyl, quizalofop-p-ethyl, quizalofop-tefuryl
b28 phenylacetic acids:
chlorfenac (fenac)
b29 phenylpropionic acids:
chlorophenprop-methyl
b30 protoporphyrinogen IX oxidase inhibitor:
benzofenap, cinidon-ethyl, flumiclorac-pentyl, flumioxazine, flumipropyne, flupropacil, fluthiacet-methyl, pyrazoxyfen, sulfentrazone, thidiazimin
b31 pyrazole:
nipyraclofen
b32 pyridazine:
chloridazon, maleic hydrazide, norflurazon, pyridate
b33 pyridinecarboxylic acids:
clopyralid, dithiopyr, picloram, thiazopyr
b34 pyrimidyl ethers:
pyrithiobacic acid, pyrithiobac-sodium, KIH-2023, KIH-6127
b35 sulfonamides:
flumetsulam, metosulam
b36 Sulfonylureas:
amidosulfuron, azimsulfuron, bensulfuron-methyl, chlorimuron ethyl, chlorosulfuron, cinosulfuron, cyclosulfamuron, ethamet sulfurone methyl, ethoxysulfuron, flazasulfuron, halosulfuron methyl, imazosulfuron, metsulfuron-methyl, nicosulfuron, pri-misulfuron, prosulfuron, pyrazosulfuron-ethyl, rimsulfuron, sulfometuron-methyl , thifensulfuron-methyl, triasulfuron, tribenuron-methyl, triflusulfuron-methyl
b37 Triazines:
ametryn, atrazine, aziprotryn, cyanazine, cyprazine, desme tryn, dimethamethryn, dipropetryn, eglinazine ethyl, hexazi non, procyazine, prometon, prometryn, propazine, hexetonone, simazine, simetryn, terbumeton, terbutryn, terbutylazine, trie tazine
b38 triazinones:
ethiozine, metamitron, metribuzin
b39 triazole carboxamides:
triazofenamid
b40 Uracile:
bromacil, lenacil, terbacil
b41 Various:
benazolin, benfuresate, benzenside, benzofluor, butamifos, cafenstrole, chlorthal-dimethyl (DCPA), cinmethylin, dichlobenil, endothall, fluorbentranil, mefluidide, perfluidone, pi perophos

The inventive method can be successfully used in the Kul wheat, barley, rye, oats, rice, maize, millet, Sugar cane, banana, tomato, tobacco, pepper, potato, rape, Zuc pitcher, soy, cotton and around fruit trees from the family of Rosacea, such as apple, pear, plum, plum, peach, nectar rine and cherry as well as grapevines, but not others named plants.

In greenhouse experiments, the inventively increased Resistance to a range of herbicides. At the Tests were carried out in plastic pots of approx. 300 ml Volume on loamy sand with about 3% humus content kulti fourth. The different herbicides were at one shoot length of about 12 cm applied. A visual determination of each Degree of damage occurred about 20 days after herbicide treatment development.  

example 1 Cloning of the gene of a flavanone-3-hydroxylase Lycopersicon esculentum Mill.cv. Moneymaker

Ripe tomato fruits from Lycopersicon esculentum Mill.cv. Money maker were washed, dried and sterilized by means of a sterile Blade the pericarp of seeds, middle columnar and wooden parts freed. The pericarp (about 50 g) was dissolved in liquid nitrogen frozen. The material was then crushed in a blender kleinert. The crushed material was in a pre-cooled Mortar mixed with 100 ml homogenization medium and mixed. The suspension was then transferred to centrifuge beakers by it was pressed through sterile cheesecloths. Subsequently was 1/10 vol 10% SDS added and mixed well. After 10 minutes on ice, 1 volume of phenol / chloroform was added, the centrifuge closed and well mixed. After 15 minutes Zen tritugation at 4000 rpm, the supernatant was in a new Reakti transferred onsgefäß. It was followed by three more phenol / chloro form extractions and a chloroform extraction. Hereinafter 1 volume of 3M NaAC (Na acetate) and 2.5 volumes of ethanol were added. The precipitation of the nucleic acids was carried out overnight at -20 ° C. At the The next morning, the nucleic acids were at 10,000 for 15 minutes rpm in the refrigerated centrifuge (4 ° C) pelleted. The supernatant was Discard and resuspend the pellet in 5-10 ml of cold 3 M NaAc diert. This washing step was repeated twice. The pellet was washed with 80% ethanol. That completely dried Pellet was dissolved in about 0.5 ml of sterile DEPC (diethylpyrocarbonate) Absorbed water and the RNA concentration photometrically be Right.

20 μg total RNA were first mixed with 3.3 μl 3M sodium acetate solution solution, 2 .mu.l of 1M magnesium sulfate solution and 100 .mu.l end Volume filled up with DEPC water. This was a microliter Rnase-free DNase (Boehringer Mannheim) and 45 min at 37 ° Degree incubated. After removing the enzyme by shaking out with Phenol / chloroform / isoamyl alcohol was the RNA ge with ethanol drops and the pellet is taken up in 100 μL DEPC water. 2.5 μg RNA from this solution were analyzed by means of a cDNA kit (Gibco BRL) in rewritten cDNA.

Using amino acid sequences derived from cDNA clones encoding Flava none-3 hydroxylase, conserved regions in the primary sequence could be identified (Britsch et al., Eur. J. Biochem., 217, 745-754 (1993), which is incorporated herein by reference) The basis for the design of degenerate PCR oligo nucleotides The 5 'oligonucleotide was determined using the peptide sequence SRWPDK (amino acid 147-152 in the sequence FL3H PETHY from Petunia hybrida) and had the following sequence:
5'-TCI (A / C) G (A / G) TGG CC (A / C / G) GA (C / T) AA (A / G) CC-3.

The sequence of using the peptide sequence DHQAVV (Amino acid 276281 in the sequence FL3H PETHY from Petunia hybrida) oligonucleotide read as follows: 5'-CTT CAC ACA (C / G / T) GC (C / T) TG (A / G) TG (A / G) TC-3.

The PCR reaction was carried out using the Perkin-Elmer tTth polymerase according to the manufacturer's instructions. 1/8 of the cDNA was used as template (corresponding to 0.3 μg RNA). The PCR program was:

AL = L <30 cycles 94 degrees 4 sec 40 degrees 30 sec 72 degrees 2 min 72 degrees 10 min

The fragment was according to manufacturer's instructions in the vector pGEM-T cloned by Promega.

The correctness of the fragment was checked by sequencing. The PCR fragment was isolated using the restriction sites Ncol and PstI present in the polylinker of vector pGEM-T and the protruding ends were transformed using T4 polymerase and blunt-ended. This fragment was cloned into a Smal (blunt) cut vector pBinAR (Hofgen and Willmitzer, Plant Sci. 66: 221-230 (1990)) (see Figure 2). This contains the 35S promoter of the CaMV (flower kohl mosaic virus) (Franck et al., Cell 21: 285-294 (1980)) and the termination signal of the octopine synthase gene (Gielen et al., EMBO J. 3: 835-846 (1984)). This vector mediates in plant resistance to the antibiotic kanamycin. The resulting DNA constructs contained the PCR fragment in sense and antisense orientation. The antisense construct was used to generate transgenic plants.

Fig. 2: Fragment A (529 bp) contains the 35S promoter of CaMV (nucleotides 6909 to 7437 of cauliflower mosaic virus). Fragment B Fragment of the F3H gene in antisense orientation. Fragment C (192 bp) contains the termination signal of the octopine synthase gene.

Cloning of a larger cDNA fragment of flavanone-3-Hydro xylase from Lycopersicon esculentum Mill.cv. Moneymaker under Use of the 5'RACESystem.

To rule out that the production of plants with reduce mRNA steady-state amount of F3H due to the low Size of the F3H PCR fragment used in the antisense construct not successful, should be a second antisense construct be generated using a larger F3H fragment.

For the purpose of cloning a larger fragment of F3H was the 5'RACE method (System for Rapid amplification of cDNA ends) applied.

Extension of the F3H PCR fragment by the 5'RACE method using the 5'RACE System for rapid amplification of cDNA ends, version 2 ~ 0 from Life Technologies ™.

From ripe tomato fruits by Lycopersicon esculentum Mill.cv. Moneymaker total RNA was isolated (see above).

The cDNA first strand synthesis was according to the manufacturer Use of the GSP-1 (Gene Specific Primer) 5'-TTCAC CACTGCCTGGTGGTCC-3 '. Following a Rnase Ver the cDNA was synthesized using the GlassMAX spin system of Life Tecgnologies ™ according to the manufacturer's instructions.

At the 3 'end of the purified single-stranded F3H cDNA using the terminal deoxynucleotide transferase ge according to the manufacturer's instructions added a cytosine homopolymer.

Amplification of the 5 'extended F3H cDNA was performed under Use of a second gene specific primer (GSP-2) in the Area 3 'before the GSP-1 recognition sequence binds and thus one "nested" PCR enabled. As 5'Primer was the manufacturer supplied "5'RACE abrided anchor primer" used the komple to the homopolymeric dC tail of the cDNA.

The thus amplified and designated as F3H _extended cDNA fragment was cloned according to the manufacturer's instructions in the vector pGEM-T Promega.

The identity of the cDNA was confirmed by sequencing.

The F3H _extended cDNA fragment was isolated using the restriction sites Ncol and PstI present in the poly left of the vector pGEM-T and the protruding ends were transformed using the T4 polymerase and blunt ends. This fragment was cloned into a Smal (blunt) cut vector pBinAR (Hofgen and Willmitzer, 1990) (see Figure 3). The sera contains the 35S promoter of CaMV (cauliflower mosaic virus) (Franck et al., 1980) and the termination signal of the octopine synthase gene (Gielen et al., 1984). This vector mediates resistance to the antibiotic kanamycin in plants. The resulting DNA constructs contained the PCR fragment in sense and antisense orientation. The antisense construct was used to generate transgenic plants.

FIG. 3: Fragment A (529 bp) contains the 35S promoter of CaMV (nucleotides 6909 to 7437 of cauliflower mosaic virus). Fragment B Fragment of the F3H gene in antisense orientation. Fragment C (192 bp) contains the termination signal of the octopine synthase gene.

Example 2

Production of Transgenic Lycopersicon esculentum Mill.cv. Moneyma ker which is a partial fragment of flavanone-3-hydroxylase in antisense Express orientation.

The method according to Ling et al., Plant Cell Report 17, 843-847 (1998). Cultivation takes place at approx. 22 ° C under a 16 h light / 8 h dark regime.

Tomato seeds (Lycopersicon esculentum Mill. Cv. Moneymaker) wur by incubation in 4% sodium hypochlorite for 10 minutes incubated, then 3-4 times with sterile distillate washed water and on MS medium with 3% sucrose, pH 6.1 designed for germination. After a germination period of 7-10d the cotyledons could be used for the transformation.

Day 1: Petri dishes containing the medium "MSBN" were mixed with 1.5 ml of a Overlaid about 10d old tobacco suspension culture. The plates were covered with foil and until the next day at Raumtem incubated.

Day 2: On the tobacco suspension culture coated plat In this case, sterile filter paper was applied without air bubbles. Thereon the cross-cut cotyledons receded with the top side placed on the bottom. The Petri dishes were cultured for 3 days room incubated.  

Day 5: The culture of agrobacteria (LBA4404) was determined by Zentri Fungation sedimented at about 3000 g for 10 min and in MS medium resuspended so that the OD is 0.3. In this suspension the cotyledons were added, which under light shake were incubated for 30 minutes at room temperature. to closing the cotyledons were on sterile Filterpa pierced a bit and returned to its original location for the continuation of cocultivation for 3 days in culture Renraum laid.

Day 8: The cocultivated cotyledons were assayed for MSZ2K50 + β and incubated in the culture room for the next 4 weeks. Then the subcultivation took place.

Forming shoots were placed on root induction medium.

After successful rooting the plants could be tested and be transferred to the greenhouse.

Example 3

Increasing the resistance in genetically modified Tomatoes contain a flavanone 3-hydroxylase in antisense orientation contained in relation to glyphosate.

Not genetically modified and genetically modified Tomato plants of the variety "Moneymaker" were grown in the greenhouse breeds. The genetically modified tomato plants ex primed the gene flavanone 3-hydroxylase in antisense orientation tion. Both the non-genetically modified plants as well as the genetically modified plants were with treated with different concentrations of glyphosate. there showed that the plants containing the flavanone 3-hydroxylase gene in antisense orientation contained a high resistance have glyphosate.

Claims (9)

1. A method for increasing the resistance of Kulturpflan zen against chemical stress, triggered in particular by un sufficiently selective or improperly applied herbicides, characterized in that a plant is produced by molecular genetic methods, in which the activity of En zyms flavanone-3-hydroxylase reduced is. 2. Method for increasing the resistance of crop zen against chemical stress according to claim 1, characterized gekenn records that the enzyme flavanone-3-hydroxylase by Moleku largenetic methods (eg antisense constructs, Co-Sup pressions, the expression of specific antibodies or the Expression of specific inhibitors) in whole or in part permanently or temporarily, throughout the plant or in Dividing the plant is reduced in its activity. 3. Process according to claims 1 and 2, characterized net, that the crops are wheat, barley, Rye, oats, rice, corn, millet, sugarcane, banana, tomato, Tobacco, peppers, potatoes, rapeseed, sugar beet, soybeans, cotton and fruit trees from the Rosacea family, such as apple, Pear, plum, plum, peach, nectarine and cherry as well as grapevines. 4. Process according to claims 1-3, characterized in that that the resistance to the herbicidal active ingredient Gly phosate is increased. 5. Process according to claims 1-3, characterized that the resistance to the herbicidal active ingredient Glu fosinate-ammonium is increased.   6. Process according to claims 1-3, characterized in that the resistance to compounds of the formula I it is increased
in which the variables have the following meanings:
R ¹ , R ^{2 are} hydrogen, nitro, halogen, cyano, C ₁ -C ₆ -alkyl, C ₁ -C ₆ -haloalkyl, C ₁ -C ₆ -alkoxy, C ₁ -C ₆ -haloalkoxy, C ₁ -C ₆ Alkylthio, C ₁ -C ₆ -haloalkylthio, C ₁ -C ₆ -alkylsulphinyl, C ₁ -C ₆ -haloalkylsulfinyl, C ₁ -C ₆ -alkylsulfonyl or C ₁ -C ₆ -haloalkylsulfonyl;
R ^{3 is} hydrogen, halogen or C ₁ -C ₆ -alkyl;
R ⁴ , R ^{5 are} hydrogen, halogen, cyano, nitro, C ₁ -C ₄ -alkyl, C ₁ -C ₄ -alkoxy-C ₁ -C ₄ -alkyl,
Di- (C ₁ -C ₄ alkoxy) C ₁ -C ₄ alkyl, di (C ₁ -C ₄ alkyl) -amino, C ₁ -C ₄ -alkyl, [2,2-di- (C ₁ -C ₄ -alkyl) hydrazino-1] -C ₁ -C ₄ -alkyl, C ₁ -C ₆ -alkyliminooxy-
C ₁ -C ₄ -alkyl, C ₁ -C ₄ -alkoxycarbonyl-C ₁ -C ₄ -alkyl,
C ₁ -C ₄ -alkylthio-C ₁ -C ₄ -alkyl, C ₁ -C ₄ -haloalkyl,
C ₁ -C ₄ -cyanoalkyl, C ₃ -C ₈ -cycloalkyl, C ₁ -C ₄ -alkoxy,
C ₁ -C ₄ -alkoxy-C ₂ -C ₄ -alkoxy, C ₁ -C ₄ -haloalkoxy, hydroxy, C ₁ -C ₄ -alkylcarbonyloxy, C ₁ -C ₄ -alkylthio, C ₁ -C ₄ -haloalkylthio, Di- (C ₁ -C ₄ -alkyl) amino, COR ⁶ , phenyl or benzyl, where the latter two substituents may be partially or fully halogenated and / or may carry one to three of the following groups:
Nitro, cyano, C ₁ -C ₄ -alkyl, C ₁ -C ₄ -haloalkyl, C ₁ -C ₄ -alkoxy or C ₁ -C ₄ -haloalkoxy;
or
R ⁴ and R ⁵ together form a C ₂ -C ₆ alkanediyl chain which may be substituted one to four times by C ₁ -C ₄ alkyl and / or substituted by oxygen or an optionally C ₁ -C ₄ alkyl Nitrogen can be interrupted;
or
R ⁴ and R ⁵ together with the associated carbon form a carbonyl or a thiocarbonyl group;
R ^{6 is} hydrogen, C ₁ -C ₄ -alkyl, C ₁ -C ₄ -haloalkyl,
C ₁ -C ₄ -alkoxy, C ₁ -C ₄ -alkoxy-C ₂ -C ₄ -alkoxy,
C ₁ -C ₄ haloalkoxy, C ₃ -C ₆ alkenyloxy, C ₃ -C ₆ alkynyloxy or NR ⁷ R ⁸ ;
Hydrogen or C ₁ -C ₄ -alkyl;
R ^{8 is} C ₁ -C ₄ -alkyl;
XO, S, NR ⁹ , CO or CR ¹⁰ R ¹¹ ;
YO, S, NR ¹² , CO or CR ¹³ R ¹⁴ ;
R ⁹ , R ^{12 are} hydrogen or C ₁ -C ₄ -alkyl;
R ¹⁰ , R ¹¹ , R ¹³ , R ^{14 are} hydrogen, C ₁ -C ₄ -alkyl, C ₁ -C ₄ -haloalkyl, C ₁ -C ₄ -alkoxycarbonyl, C ₁ -C ₄ -haloalkoxycarbonyl or CONR ⁷ R ⁸ ;
or
R ⁴ and R ⁹ or R ⁴ and R ¹⁰ or R ⁵ and R ¹² or R ⁵ and R ¹³ together form a C ₂ -C ₆ alkanediyl chain which is substituted one to four times by C ₁ -C ₄ alkyl may be interrupted and / or interrupted by oxygen or an optionally C ₁ -C ₄ alkyl-substituted nitrogen;
R ^{15 is} a linked in the 4-position pyrazole of the formula II
in which
R ^{16 is} C ₁ -C ₆ alkyl;
ZH or SO ₂ R ¹⁷ ;
R ^{17 is} C ₁ -C ₄ -alkyl, C ₁ -C ₄ -haloalkyl, phenyl or phenyl which is partially or completely halogenated and / or carries one to three of the following groups:
Nitro, cyano, C ₁ -C ₄ -alkyl, C ₁ -C ₄ -haloalkyl,
C ₁ -C ₄ alkoxy or C ₁ -C ₄ haloalkoxy;
R ^{18 is} hydrogen or C ₁ -C ₆ -alkyl
mean;
where X and Y are not simultaneously oxygen or sulfur; and their agriculturally useful salts. 7. Process according to claims 1-3, characterized in that that the resistance to cyclohexenone herbicides like Sethoxydim, Cycloxydim, Tepraloxydim or Clefoxydim increased becomes. 8. Process according to claims 1-3, characterized in that that the resistance to the herbicide bromoxynil increases becomes. 9. Plant with increased resistance to insufficient selective and improperly applied herbicides provides by a method according to claims 1 to 3, da characterized in that the enzymatic activity of the En zyms flavanone-3-hydroxylase is reduced.