KR101512652B1 - Method for enhancing resistance against fungal disease of grape by treating signal molecules - Google Patents

Abstract

The present invention relates to a method for increasing the stilbene or gamma-aminobutyric acid (GABA) content of grapes comprising the step of treating a signaling substance or hydrogen peroxide in a grape, a method for increasing the content of stilbene or GABA produced by the method A method for increasing tolerance to fungal diseases of grapes, including the step of treating increased grapes, signaling substances or hydrogen peroxide in grapes, increasing the content of stilbenes or GABA in grapes containing plant signaling substances or hydrogen peroxide as active ingredients The present invention provides a composition for enhancing tolerance to a mold or a disease of a grape.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a method for enhancing tolerance to fungal diseases of grapes by treatment with a signaling substance,

The present invention relates to a method of enhancing tolerance to fungal diseases of grapes by treatment with signal transduction materials, and more particularly to a method of treating grape stilbene or hydrogen peroxide, A method for increasing the content of gamma-aminobutyric acid (GABA), a method for treating grape mold disease comprising treating staphylococcal or GABA-rich grape produced by the method, signaling substance or hydrogen peroxide in the grape , A plant signaling substance or a composition for enhancing stilbene or GABA content of grape containing hydrogen peroxide as an active ingredient or a composition for enhancing tolerance to fungal diseases of grape.

Plant defense mechanisms are also induced not only by pathogens, but also by abiotic stresses such as ultraviolet rays and signaling substances. These signaling substances, which are abiotic stressors, include respiratory inhibitors, antibiotics, Heavy metals and elicitors. Plant defense responses are mediated through a complex network of signaling pathways, including endogenous signaling pathways including salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) , Salicylic acid, and hydrogen peroxide are important regulators that mediate disease resistance. Ethylene, methyl jasmonic acid, and salicylic acid are important signaling molecules that induce local or systemic disease resistance responses in plants, and an artificial external treatment of these signaling substances induces resistance-related genes that are activated by pathogenic infection. Phytoalexin is a low-molecular secondary metabolite. It is an antimicrobial substance that accumulates after infection with pathogens. It also produces plants in response to pathogenic bacteria, heavy metals, UV, AlCl _3, and other abiotic stresses. Phytoalexin produced in grapes contains viniferins and pterostilbene (trans-3, trans-3, 4-dihydroxyphenyl), which are biochemically related compounds with simple stilbene, trans-3,5,4'- trihydroxystilbene, 5 dimethoxy-4'-hydroxystilbene).

Grape is one of the most important fruit trees in the world, but it is susceptible to many pathogens such as germs, fungi and viruses during cultivation. Gray molds in grapes are susceptible to maturity during late summer or high relative humidity and develop after harvest, causing serious problems in many crops such as tomatoes, strawberries, cucumbers, bulbs, cut flowers, .

The gray mold of the grape is caused by B. cinerea , which mainly affects flowers and fruits, especially in the flowering stage, which is a serious factor in the production of grapes depending on the region . Induction of disease resistance-related genes by treatment of signaling substances or accumulation of phytoalexins such as stilbene compounds is expected to enhance resistance to these pathogens. In the present invention, three signaling substances and hydrogen peroxide And gabbana to induce disease resistance genes and to inhibit the development of gray mold disease by accumulation of various stilbene compounds and GABA substances.

Korean Patent Publication No. 2003-0021976 discloses resveratrol-fortified grapes that amplify the content of resveratrol, a natural anticancer substance derived from grapes, and a production method thereof. Korean Patent No. 0582737 discloses a resveratrol- Production methods have been disclosed. However, as in the present invention, the method for producing grapes having enhanced resistance to fungal diseases and the grapes therefrom have not been disclosed.

SUMMARY OF THE INVENTION The present invention has been made in view of the above-described needs, and it is an object of the present invention to provide a method of spraying grape leaves with Ethepton, methyl jasmonic acid (MJ) or salicylic acid (SA) stilbene) and GABA (gamma-aminobutyric acid) were increased, and the tolerance to gray mold of grapes was also increased, thus completing the present invention.

In order to solve the above-mentioned problems, the present invention provides a method for increasing stilbene or GABA (gamma-aminobutyric acid) content of grapes, comprising the step of treating the grape with a signaling substance or hydrogen peroxide.

The present invention also provides stilbenes or grapes with increased GABA content produced by the method.

The present invention also provides a method for increasing tolerance to fungal diseases of grapes comprising the step of treating signaling substances or hydrogen peroxide in grapes.

The present invention also provides a composition for increasing stilbene or GABA content of grape containing plant signaling substance or hydrogen peroxide as an active ingredient.

The present invention also provides a composition for enhancing tolerance to a fungal disease of grape containing a plant signaling substance or hydrogen peroxide as an active ingredient.

The present invention is based on the finding that spraying of grape with ethephon, methyl jasmonic acid (MJ) or salicylic acid (SA) signaling agent or hydrogen peroxide during the cultivation process results in an increase in the content of stilben and GABA in the grape, And it was confirmed that there is an excellent effect on the increase of tolerance. Therefore, the grapes produced by the production method of the present invention can contain a high content of stilbenes and GABA having anticancer and antimicrobial activity, thereby making it possible to produce a functional food and a pharmaceutical composition containing the same as an effective ingredient, which is very useful in the food industry and the pharmaceutical industry The present invention can provide a grape plant having enhanced tolerance to grape mold disease, which can greatly contribute to an increase in the productivity of the grape industry.

, 대조구(물 처리),

1000 ppm 에테폰 처리,

10 mM H₂O₂ 처리,

0.5 mM 메틸자스몬산 처리,

1 mM 살리실산 처리. 수직 바는 SEs를 나타낸다 (n=9).
도 3은 신호전달물질을 처리한 거봉 포도나무 잎에서의 GABA 함량 변화를 나타낸다. Cont, 물 처리; MeJ, 메틸자스몬산; SA, 살리실산.Figure 1 shows the expression analysis of the defense-related genes in Campbell and vine leaves treated with the signaling substance. After a period of time had elapsed, total RNA was isolated and cDNA was synthesized and analyzed by RT-PCR. Beta-actin gene was used as a control to confirm the equivalence of RNA. (CHI) chalcone isomerase (CHS), chalcone synthase (CYP), cytochrome p450, FLS, flavonol synthase, GST, glutathione- S- transferase, TLP, thaumatin-like protein, PRP, proline rich protein lipoxygenase, STSY, stilbene synthase, cell wall, cell wall protein, Mei5, meiosis 5, PGIP, polygalacturonase-inhibiting protein,
Figure 2 shows the inhibition of gray mold fungal disease on vine leaves inoculated with Vortex sinceria after treatment with signaling substance and hydrogen peroxide. (A) The campbell, (B) The barb.

, Control (water treatment),

1000 ppm of tepone treatment,

10 mM H ₂ O ₂ process,

0.5 mM methyl jasmonate treatment,

Treatment with 1 mM salicylic acid. The vertical bars represent SEs (n = 9).
FIG. 3 shows the change in the GABA content in the jabon vine leaves treated with the signaling substance. Cont, water treatment; MeJ, methyl jasmonic acid; SA, salicylic acid.

In order to accomplish the object of the present invention, the present invention provides a method for increasing stilbene or gamma-aminobutyric acid (GABA) content of grapes comprising treating a grape with a signaling substance or hydrogen peroxide.

In a method according to an embodiment of the present invention the signaling substance can be ethylene, ethephon, methyl jasmonic acid (MJ) or salicylic acid (SA), preferably ethephon, methyl jasmonic acid (MJ) (SA), but is not limited thereto.

In the method according to one embodiment of the present invention, the treatment concentration of the signal transduction material is preferably 450 to 1100 ppm of TEPE, 0.08 to 0.6 mM of methyl jasmonic acid (MJ) or 0.4 to 1.1 mM of salicylic acid (SA) May be 4 to 11 mM, but is not limited thereto.

In the method according to one embodiment of the present invention, the signal transduction material or hydrogen peroxide can be sprayed or sprayed onto the grape leaves, but is not limited thereto. The method of spraying or spraying the signal transmitting substance or hydrogen peroxide on the grape leaves may be any method known in the art and is not particularly limited to a specific method.

In the method according to one embodiment of the present invention, the grape may be Kyoho, Delaware, Campbell Early, Seri Da, MBA, Mauro or Niagara, ≪ / RTI > or grapevine grapes.

In a method according to an embodiment of the present invention, the stilbene may be, but is not limited to, resveratrol, piceatannol or piceid.

Stilbene is a kind of phytoalexin, one of the secondary metabolites derived from the phenylpropanoid pathway. The physiological activity of the compound is related to antioxidant activity, and the radical scavenging ability of stilbene It has a correlation with the number of hydroxyl groups (-OH), and has a stronger antioxidative activity than resveratrol, a hydroxylated derivative, pisatanol, resveratrol.

The present invention also provides grapes with increased stilbene or GABA (gamma-aminobutyric acid) content produced by the method.

GABA is a substance that induces a defense reaction against reactive oxygen stress in the plant. It removes the accumulation of hydrogen peroxide and induces a mechanism to resist external stress such as insect attack. The increase in GABA content in plants is indicative of resistance to nematodes and insects, and the increase in reactive oxygen species produced by pathogen infestation in plants is associated with an increase in GABA content in the body.

The grape according to one embodiment of the present invention may be Kyoho, Delaware, Campbell Early, Seri Da, MBA, Mauro or Niagara, preferably Campbell or Geobong grapes But is not limited thereto.

The present invention also provides a method for increasing tolerance to fungal diseases of grapes comprising the step of treating signaling substances or hydrogen peroxide in grapes.

In the method according to one embodiment of the present invention, the fungal disease may be, but is not limited to, a gray mold disease caused by V. vivax.

The present invention also provides a composition for enhancing stilbene or GABA (gamma-aminobutyric acid) content of grapes containing plant signaling substances or hydrogen peroxide as active ingredients. The composition includes an active ingredient, a plant signaling substance or hydrogen peroxide, and the plant signaling substance or hydrogen peroxide can be sprayed or sprayed on the grape leaves to increase the content of stilbene or GABA.

In a composition according to an embodiment of the present invention the signaling substance may be ethylene, ethephon, methyl jasmonic acid (MJ) or salicylic acid (SA), preferably ethephon, methyl jasmonic acid (MJ) (SA), but is not limited thereto.

The present invention also provides a composition for enhancing tolerance to a fungal disease of grape containing a plant signaling substance or hydrogen peroxide as an active ingredient. The composition comprises an effective component, a plant signaling substance or hydrogen peroxide, and the plant signaling substance or hydrogen peroxide can be sprayed or sprayed onto a grape leaf to increase tolerance to fungal diseases of the grape.

In a composition according to an embodiment of the present invention the signaling substance may be ethylene, ethephon, methyl jasmonic acid (MJ) or salicylic acid (SA), preferably ethephon, methyl jasmonic acid (MJ) (SA), but is not limited thereto.

In the composition according to an embodiment of the present invention, the fungal disease may be, but is not limited to, a gray mold disease caused by V. vivax.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Materials and methods

Various signaling materials treatment of vine leaves

(500 ppm, 1000 ppm), H ₂ O ₂ (5 mM, 10 mM), methyl jasmonic acid (MJ, 0.1 mM, 0.5 mM) and salicylic acid (SA, 0.5 mM, 1 mM) were divided into two concentrations and sprayed sufficiently. After spraying, the grape leaves were sampled at 0, 1, 6, 24, 48 and 72 hours, respectively, and stored at -80 ° C. Total RNA was extracted and used for RT-PCR. Ethepton, hydrogen peroxide and methyl jasmonic acid were dissolved in distilled water and diluted to the respective concentrations. Salicylic acid was dissolved in ethanol and diluted with distilled water.

gun RNA  Separation and RT - PCR  analysis

Total RNA was isolated from grape leaves and first strand cDNA was synthesized from 1 μg of total RNA using PrimeScript ^™ 1 ^st strand cDNA synthesis kit (TaKaRa Bio Inc., Japan) and used as a template for PCR reaction. Primers were prepared based on the nucleotide sequences related to beta-actin and 16 defense reactions (Table 1). PCR was performed with primary denaturation (94 ° C., 5 min), amplification reaction 35 times (denaturation at 94 ° C. for 45 sec, annealing at 55 ° C. for 45 sec, amplification at 72 ° C. for 1 min.) And final amplification (7 min, 72 ° C.) And the PCR product was identified on agarose gel (1%).

Inhibition of Gray Mold Fungal Disease by Signal Transduction

The cells were incubated for 5 days in a PDA irradiated with fluorescent lamps in an alternating manner for 12 hours, and the gray mold fungus ( Botrytis cinerea ) were collected. (10 ⁶ spores / ml) was added to 0.24% potato dextrose broth (containing 0.8% NaCl), and 20 μl of spore suspension was added to the back of the vine leaf evenly wounded with a pencil tip Was inoculated onto the scar on the back of the grape leaf using Eppendorf repeater (pipet). The mycelial inoculation was performed by inoculating the agar block containing the mycelium of the gray mold fungus cultured in the PDA for 5 days. The number of necrotic spots was measured after 4 days at 25 ℃ in the laboratory of grape leaves inoculated with pathogenic bacteria.

GABA (? aminobutyric acid ) And HPLC  analysis

The extraction of GABA was performed by adding liquid nitrogen to 1 g of the vine leaf treated with 10 ml of 3% TCA (trichloroacetic acid) and grinding for 3 minutes. The extract was centrifuged at 25,000 × g for 20 minutes Respectively. The supernatant was collected and stored at -20 ° C and injected into HPLC to analyze GABA content. Qualitative and quantitative analysis of GABA was performed using HPLC / FLD.

Stilben ( Stilbene ) ≪ / RTI > HPLC  analysis

To extract the stilbene compound, liquid nutrient was added to 1 g of the treated vine leaves and the mixture was thoroughly crushed. 4 ml of 80% (v / v) methanol was added, and the mixture was again grinded for 3 minutes. And centrifuged. The supernatant was collected and stored at -20 ° C and injected into HPLC to analyze stilbene content. The stilbene compound was analyzed using an HPLC-mass spectrophotometer (model 2695 HPLC, model 3100 MS, Waters, USA). The analytical method was Choi et al. (2011 Kor. J. Hort. Sci. Technol. 29: 374-381 ) Were performed under the same conditions.

Example  1. Expression of genes related to grape defense by signal transduction

RT-PCR was performed using a total of 38 primers shown in Table 1. In order to find genes specifically expressed, RT-PCR was performed by mixing the cDNAs first, and the individual genes were selected and RT-PCR was performed again. Among the eight selected genes in the Campbell grape, the expression of CYP (cytochrome p450) and TLP (thaumatin-like protein) increased in chitosan, chalcone synthesis (CHS) The expression of chitinase-like protein (LOP), lipoxygenase (LOX), proline-rich protein (PRP) and thaumatin-like protein (TLP) increased, and CHS (chalcone synthesis) , Polygalacturonase-inhibiting protein (PGIP), and sirtuin. Chalcone isomerase (CHI), chalcone synthesis (CHS), cytochrome p450, FLS (flavonol synthase), glutathione S- transferase (GST) TLP (thaumatin-like protein), and PRP (proline-rich protein) showed a decrease in gene expression (Fig. 1). The expression of three genes including cold inducible protein (CI), LOX (lipoxygenase) and STSY (stilbene synthase) increased in the case of treatment with methyl jasmonic acid. Mei5 (cell wall protein, meiosis 5) , PGIP (polygalacturonase-inhibiting protein) and STSY (sirtuin, stilbene synthase) (Fig. 1). Overall, genes such as CYP, serotonin, STSY and TLP tended to be strongly expressed.

Example  2. Inhibition of grape-gray mold disease by treatment with signaling substance

In order to investigate the effect of various signal transduction agents on the occurrence and inhibition of gray mold, we sprayed two concentrations of Ethepton, hydrogen peroxide, methyl jasmonic acid or salicylic acid in Campbell and Jubon vine leaves, The lesion formation and lesion size were examined. The occurrence of gray mold was inhibited by the treatment of signaling substances in the Campbellies and hermaphrodites (Fig. 2).

It was shown that the growth of mycelial growth was suppressed in all cases of spore suspension or inoculation regardless of whether it was wounded or not. The size of the lesion was 1.4-9 times larger in Campbell, and 1.2-7 times larger in Seabong than in the case of scarring and spore suspension inoculated. In case of inoculation, Mycorrhizal growth was higher by 1.2-3 times than that of non - wounded inoculation.

Example  3. Stilben  The compounds and GABA  Content analysis

The content of stilbene compounds was determined by spraying latex, hydrogen peroxide, methyl jasmonic acid, or salicylic acid at low and high concentrations, respectively, on the grape leaves. Each compound was identified by the retention time as an index in the SIR chromatogram of the corresponding m / z. The content of each compound was compared with that of trans-resveratrol, (Kor, J. Hort. Sci. Technol. 29: 374-381). The contents of stilbene compounds of Campbell and Geobong were slightly higher than those of resveratrol (piceatannol). Especially, the content of trans- Piceid was found to be the highest among the five compounds. (Table 2 and Table 3). In the present invention, it is considered that the increase of the stilbene compound by the signaling substance treatment is related to the inhibition of mycelial growth of the gray mold.

In the present invention, the contents of the three signaling substances of low concentration and high concentration and the stilbene compound by the hydrogen peroxide treatment are shown in Tables 2 and 3. Among the stilbene compounds, both of Campbell and Geobong showed a relatively high content of trans / cis-physeide, which is a glycoside form having a lower physiological activity than resveratrol or pychethanol, and the aglycone type trans / cis-resveratrol Was detected. Especially, Fisshide had more trans-type than cis-type, and Trans-Fisshide content of Campbell was higher than Geobong. Lorenz et al. (2003 Nitric Oxide 9: 64-76) found that the content of resveratrol hydroxide derivative, fisethanol, was higher than that of Campbell, Phosphatasol has a stronger antioxidant activity than resveratrol.

The change in GABA content by signal transduction treatment is shown in FIG. GABA content was higher than that of untreated control in all signal transduction materials, and GABA content after 24 hours of treatment was higher than that after 48 hours. Especially, it was found that GABA content increased rapidly after 24 hours in the treatment with Ettenon, and GABA content was higher in salicylic acid treatment among the three signaling substances and hydrogen peroxide treatment.

In the present invention, when the plant was treated with a signaling substance and hydrogen peroxide, the content of GABA in the vine leaves was increased and the occurrence of grape gray mold was decreased in the plants with increased contents. In other words, by the signaling substance and hydrogen peroxide treatment, it is considered that resistance to disease is increased by activation of various defense reaction genes in plants, accumulation of stilbene compounds and accumulation of GABA.

The increase of antioxidant activity of the stilbene compounds after treatment with the signal transduction substance in the grape leaves is considered to enhance the function of the grape and also to improve the disease resistance by the phytoalexin substance. In the present invention, three signaling substances and hydrogen peroxide were treated on grape leaves to induce the expression of disease resistance-related genes, and the content of stilbene phytoalexin was increased to inhibit mycelial growth of grape mold fungi. It is considered that this signaling substance treatment inhibits the generation of various pathogens in the grapes or that the increase of the content of stilbene compounds having antioxidant activity may be used as a health functional substance.

<110> Industry-Academic Cooperation Foundation, Yeungnam University <120> Method for enhancing resistance against fungal disease of grape          by treating signal molecules <130> PN12254 <160> 38 <170> Kopatentin 2.0 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 aatatcccac tcttgccg 18 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 cctggaggat catagttg 18 <210> 3 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 atctcactct tctcatgc 18 <210> 4 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 tacctggagg atcatagt 18 <210> 5 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 cttgtctcta cgcttctc 18 <210> 6 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 aacacgaacc gagttacg 18 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 acgtcgttgc tttcttgctt 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 aaccaatctg gggagtttgt 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 acgatcccat atgcaccact 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 tccactgccc acattacaga 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 tcctcacaag ctgatgcaag 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 gaaagacagc cggacaagac 20 <210> 13 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 aaccaggaca cagatcgt 18 <210> 14 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 aggtacaatt gctcctgg 18 <210> 15 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 cttgtctcta cacttctc 18 <210> 16 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 gtatggactt tcgcctct 18 <210> 17 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 gcaacatatt cagggatc 18 <210> 18 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 18 attgaaattg agttgata 18 <210> 19 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 19 ccagagtgac agatatta 18 <210> 20 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 20 gccctggccg aagttcct 18 <210> 21 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 21 cacgagaaac cctggaag 18 <210> 22 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 22 gtcgaatagc ttcaatgc 18 <210> 23 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 23 gcatggcact ctgctggtac c 21 <210> 24 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 24 ggggattggt agtccaaggt c 21 <210> 25 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 25 ggatccatac aagtaccgtc c 21 <210> 26 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 26 caaggaccct ccaattctcc tg 22 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 27 gatctgtctg gggaaatggc 20 <210> 28 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 28 gcttctccaa tcccttaacc c 21 <210> 29 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 29 ggcgatcaaa gtccatggta g 21 <210> 30 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 30 gcttctccaa tcccttaacc c 21 <210> 31 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 31 gaagtcgtgg ctgtggatct g 21 <210> 32 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 32 cagcccaaat cagcttcctt tc 22 <210> 33 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 33 cgaggtccga aacaactg 18 <210> 34 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 34 ggtctttgtg tgcaacaa 18 <210> 35 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 35 gtcaaccaat gcacctac 18 <210> 36 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 36 ggtggatcat cctgtgga 18 <210> 37 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 37 acgagaaatc gtgagggatg 20 <210> 38 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 38 attctgcctt tgcaatccac 20

Claims (9)

A method for increasing the gamma-aminobutyric acid (GABA) content of grapes comprising the step of treating the grape with hydrogen peroxide. delete delete delete delete delete delete delete delete

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