KR20200027721A - A method for increasing the content of functional substances in plants using endophytic bacteria - Google Patents

Abstract

The present invention relates to a method for increasing the content of a functional substance in a plant by using endophytes having a symbiotic relationship with roots of the plant, and a plant cultivated thereby. The present invention includes a cultivation step of inoculating the plant roots with endophytes.

Description

A method for increasing the content of functional substances in plants using endophytic bacteria

The present invention relates to a method of increasing the content of a functional substance in a plant using root endogenous bacteria.

Cabbage is a vegetable that shows the largest consumption as a single crop among vegetables with an annual consumption of about 50 kg per capita. In addition, cabbage is an important vegetable due to the traditional food culture that favors kimchi in Korea as the main ingredient of kimchi. In addition, kimchi is a side dish that is noted around the world as a balanced nutrition source processed with many ingredients such as garlic, green onion, and salted fish. In addition, Chinese cabbage contains a large amount of glucosinolate, which is known to have anti-cancer and anti-inflammatory properties, and thus has better food value.

Glucosinolate (Glucosinolate) and its derivatives are sulfur metabolites rich in metabolites, and are one of the secondary metabolites present in cabbage plants. Glucosinolates are degraded to isothiocyanate and nitrile by myrosinase, a hydrolytic enzyme, when plant tissues are dissolved, and these hydrolysis products protect against pests It also exhibits several different biological activities, including substances and attractants. It is known that there are about 200 different glucosinolates in nature. Glucosinolates are aliphatic glucosinolates from methionine, indolyl glucosinolates from tryptophan, phenylalanine and tyrosine. It is divided into 3 types such as aromatic glucocinolate.

Glucosinolate contained in Chinese cabbage and vegetables has different contents or types depending on the type of vegetables. In addition, the cultivated Chinese cabbage varieties also differ in the type and content of glucosinolate depending on the model used in the cultivation process.

It is known that gluconasturtiin, one of the glucosinolates contained in Chinese cabbage, is hydrolyzed by myrosinase to be converted to phenylethylisothicyanate (PEITC), a type of isothiocyanate that exhibits anticancer activity. However, most of the glucosinolates of Chinese cabbage are alyphatic glucosinolates, which produce isodiocyanates that exhibit salt and cancer activity during the decomposition process, but sometimes they are hyperic, which can cause hyperthyroidism when consumed in large amounts during the decomposition process. It also contains glucosinolates, such as progoitrin or epiprogoitrin, which produce a goitric material. Accordingly, rapeseed such as canola was developed as a variety with a low progoitrin content, and rapeseed feed ingredients are also subjected to a microbial fermentation process to reduce the substance of koitrin.

On the other hand, since the content of glucostatin is not so high in the existing cabbage varieties, it is difficult to expect physiological activities such as anticancer effects through cabbage intake. Therefore, in order to obtain an anti-cancer effect through Chinese cabbage, which is routinely consumed, development of Chinese cabbage varieties having a high content of gluconstatin is required. Therefore, a turnip with a high gluconastatin content is used as a mating parent to cultivate a cabbage varieties with a high gluconastatin content, or a gene that gluconastatin is involved in synthetic regulation is searched for and used to raise a cabbage variety with a high content. It was also done.

In addition, various methods of increasing the content of isodiocyanate, a useful decomposition product derived from glucosinolate, have been introduced, but can be commercialized and accepted by general growers as a breeding plan to increase specific glucosinolate content. It is not easy to develop varieties with various high quality traits, and it takes a lot of time to develop.

Accordingly, the present inventors completed the present invention by confirming that the above-mentioned problems can be solved by using root endogenous bacteria as a method for increasing the content of a specific glucosinolate having high anticancer and anticancer properties by cultivation.

Korean Open Patent No. 10-2016-0183556

An object of the present invention is to provide a method for increasing the content of a functional substance in a plant, comprising the step of cultivating the plant root by inoculating endogenous bacteria.

Another object of the present invention is to provide a plant with an increased content of functional substances by the above method.

Another object of the present invention is to provide a food prepared using the above plant.

The present invention provides a method of increasing the content of a functional substance in a plant, comprising the step of cultivating the plant root by inoculating endogenous bacteria.

Creatures are symbiotic with many other organisms in the ecosystem. Plants also coexist with various bacteria in the soil and air, one of which is endophytes, which exist in the tissues of plants. Endogenous bacteria share the life cycle of plants without any negative symptoms in the tissues of plants, and also have useful effects on plants in the process of interacting with plants. In particular, it exhibits antimicrobial properties through various substances produced in the process of interacting with plants and their efficacy. Endogenous bacteria found in some herbaceous species protect plants from weeds and pathogens, and some endogenous bacteria are said to increase plant resistance to disease and environmental stress. In addition, endogenous bacteria produce anti-microbial and antioxidant substances, and in recent studies, 51% of newly reported bioactive substances were separated from endogenous bacteria, and this has attracted attention as an anticipation to solve the difficulty of exploring new substances.

While researching the useful use of these endogenous bacteria on plants, the present inventors found that the endogenous bacteria that symbiotic with the roots of the plants do not allow the plants to absorb or absorb substances with low absorption through the roots, thereby allowing the plants to develop new functional substances. Studies have shown that it helps to increase the content of synthetic or plant biosynthetic substances.

Specifically, the method of the present invention may be characterized in that substances that cannot be absorbed through the root of the plant move into the plant through the endogenous bacteria inoculated to the plant root. A path for movement of a material that is not absorbed from the soil to the plant root may be formed by an endosymbiotic symbiotic root. By absorbing the new material into the plant, the material can be converted into a new material in the metabolic process in the plant, and as a result, the plant may synthesize a new material that is not biosynthesized, or the content of the less existing material in the plant may increase. Can be.

Plants and endogenous bacteria that can use the plant cultivation method using the endogenous bacteria of the present invention are not limited, and endogenous bacteria known to symbiotic with the root of a specific plant can be used.

In a specific embodiment of the present invention, characterized by cultivating Chinese cabbage and crops by cultivating a bacterium that coexists on the roots of Chinese cabbage and crops, and cultivating Chinese cabbage and crops by applying organic matter rich in phenylalanine to the soil. After seeding the roots of the symbiotic spore solution on the seedling soil and seeding it on the roots, or after planting the cabbage and crops, cultivating the endogenous spore solution on the cultivated soil and cultivating the endosperm on the roots of the cabbage and crops to produce glucosinolate in the cabbage and crop bodies. It provides a method for increasing the content of glucostatin.

In the above embodiment, heteroconium that is symbiotic to the roots of Chinese cabbage and crops as an endogenous bacterium. chaetospira ), it was confirmed that the cabbage inoculated with heteroconium chaetospira absorbs phenylalanine, which is not absorbed by the cabbage without endogenous bacteria, and the phenylalanine is metabolized through glucosinolate in Chinese cabbage and crops. Can be converted to glucostatin.

More specifically, Chinese cabbage crops cultivated by the method of the present invention increase the content of total glucosinolate in the plant, but progoitrine or epipro among alyphatic glucosinolates, which may cause hyperthyroidism among glucosinolates. While reducing the level of goitlin, it can increase the level of glucostatin, which is known to have high anti-cancer and anti-inflammatory properties. In general, Chinese cabbage contains little glucona-stutin, but the cabbage grown through the present invention contains high glu-na-stutin, so it is possible to cultivate high-anti-cancer and anti-inflammatory cabbages.

The method of the present invention is a plant cultivation method capable of enhancing the content of a functional substance in a plant by using endogenous bacteria that do not affect the plant, solving the stability problems of the GMO crops produced through genetic manipulation, and It may be an environmentally friendly manufacturing method that can solve all of the shortcomings of a method for producing a mutant hybrid that exhibits a specific trait through artificial crossing.

The present invention, by inoculating endogenous bacteria in the root of the plant, enables the absorption of various substances that the plant does not absorb from the soil, thereby enhancing the content of useful functional substances in the plant and synthesizing new substances, It enables the cultivation of functional crops.

1 is a graph showing the content variation of glucosinolate types of 40 cabbage varieties and strains.
Figure 2 is a picture of the inoculation test of the bacteria that are endogenous to the cabbage root.
Figure 3 is an endogenous symbiotic Heteroconium on the roots of Chinese cabbage It is a hyphae picture of chaetospira
4 is a graph observing the initial root elongation and the width of the cotyledons of cabbage seedlings from various nitrogen sources such as aqueous phenylalanine solution.
Figure 5 is Heteroconium This is a picture of observing the growth of cabbage seedlings according to the supply of nitrogen sources such as phenylalanine and the presence of endosymbiotic symbiosis when inoculated with chaetospira .
6 shows Heteroconium This is an enlarged photograph focusing on unprocessed (no nitrogen source) and nitrate nitrate and phenylalanine as a result of observing the growth of cabbage seedlings according to the supply of nitrogen sources such as phenylalanine and endosymbiotic presence when chaetospira is inoculated.
7 is a schematic diagram of a nitrogen source mobility test to determine whether cabbage seedlings use a nitrogen source such as phenylalanine through an endogenous bacterium ( Heteroconium chaetospira ).
Figure 8 is a table using the soybean-derived organic material for cabbage cultivation soil and examining the growth of Chinese cabbage according to the inoculation of Heteroconium chaetospira .
9 is a table using an organic material derived from soybeans for cabbage cultivation soil and analyzing the phenylalanine content in cabbage leaves according to inoculation with endogenous bacteria ( Heteroconium chaetospira ).
10 is a graph using an organic material derived from soybeans for cabbage cultivation soil and analyzing the content of glucosinolate in cabbage leaves according to inoculation with endogenous bacteria ( Heteroconium chaetospira ).

Hereinafter, the present invention will be described in more detail based on examples. However, the following examples are provided only for easier understanding of the present invention, and the present invention is not limited to the following examples.

<Example 1> Glucosinolate content analysis of cultivars and strains

In order to measure the content of glucosinolate according to the variety and lineage of Chinese cabbage according to the present invention, an experiment was conducted as follows.

To investigate the growth characteristics of Chinese cabbage varieties grown in the highlands, 40 varieties, including 27 F1 varieties commercially available in Korea and 12 strains of Uiseong native and 12 breeding strains, were used. Glucosinolate was analyzed after cultivating each variety for 2 months on June 20, 2015 in the open field packaging.

The sample was freeze-dried, and 100 mg of 1 mL of 75% methanol was extracted by stirring in a water bath at 70 to 80 ° C for 60 minutes, and centrifuged at 4 ° C and 10,000 rpm for 10 minutes to repeat the process of collecting the supernatant twice. The liquid was collected. Crude glucosinolate in a Mini Bio-Spin chromato-graphy column (Bio-Rad Laboratories, Hercules, CA, USA) filled with 0.7 mL of DEAE Sephadex A 25 anion exchange resin activated with 0.1 M sodium acetate (pH 4.0) solution prior to analysis. Spilled extract. Sulfatase (Type H-1, EC 3.1.6.1, Sigma-Aldrich Co., 20units) was added to react enzymatically at room temperature for 24 hours, and then desulfo-glucosinolate in the column was extracted three times with 1.5 mL of tertiary distilled water and 0.2 Filtered with a μm syringe filter. Desulfo-glucosinolate was separated using HPLC (Agilent technologies, Santa Clara, CA, USA) with a C18 column (1.7 μm, 2.1 Х 100 mm, Waters Co.) and detected at 229 nm with a photodiode array (PDA) detector. All solvents were filtered with a filter of 0.2 μm, solvent A was 100% distilled water and solvent B was 20% acetonitrine (ACN). At this time, the flow rate was 0.2 μL / minuate, and the retention time was 25 minutes. Each desulfo-glucosinolate identified was quantified using a response factor for each substance after preparing a calibration formula using a sinigrin (Sigma-Aldrich Co.) standard.

1 is a graph showing the content variation of glucosinolate types of 40 cabbage varieties and strains.

As shown in FIG. 1, glucosinolate showed a difference in the content of glucosinolate between varieties, and among them, it was found that the content of gluconastatin was lower than other glucosinolates. Accordingly, as described above, it was predicted that the functional value would be further improved by increasing the content of glucostatin, which exhibits anti-cancer and anti-inflammatory properties, in various cultivars of Chinese cabbage.

<Example 2> Exploration and cultivation of endogenous bacteria symbiotic to the roots of Chinese cabbage and vegetables

Figure 2 is a picture of the inoculation test of the bacteria that are endogenous to the cabbage root.

Aspergillus flavipes (KACC, 43789) Neosartorya judged to be endogenous as shown in FIG. 2 Sp ( KACC 42968), Heteroconium chaetospira (KACC 41039) , three types are pre-sold at KACC and germinate cabbage seeds to determine their cabbage root endogenousity, so that they are placed on the medium where each bacteria is growing, and the roots of cabbage are observed under a microscope. Did.

Figure 3 is an endogenous symbiotic Heteroconium on the roots of Chinese cabbage It is a hyphae picture of chaetospira

Of these, as shown in Fig. 3, Heteroconium in Chinese cabbage root The endogenousness of chaetospira was confirmed. The culture for the growth of this bacteria was made in PDA medium (distilled water 1L + Potato infusion 4g, Dextrose 20g agar 15g (pH 5.6 ± 0.2)) and autoclave at 121 ℃ 1.2 ~ 1.5kg / cm ² 20 minutes autoclaved medium Inoculated at 20 ℃, can be used by culturing in a thermostat under dark conditions.

< Example 3 > Chinese cabbage in amino acid solution Seedling  Growth reaction and Endogenous bacteria  Amino acid availability of Chinese cabbage according to inoculation

In order to determine the cabbage growth reaction to amino acids according to the present invention and the availability of amino acids for inoculation of endogenous bacteria, inorganic nitrogen is used as the nitrogen source in the medium, potassium nitrate as the nitrate nitrate, ammonium phosphate monobasic as the ammonium nitrogen, and the amino nitrogen source Each of amino acid aqueous solutions such as valine, methionine, glutamine, glutamic acid, tryptophan, asparagine, and phenylalanine was prepared to observe the growth of cabbage seedlings. A filter paper was placed on the petri-dish, and each nitrogen source was supplied with an aqueous solution of 20 mg / L concentration, dentured cultivated cabbage seeds were cultured in a growth chamber controlled at 25 ° C, and after 2 weeks, the size of the cotyledon seedlings and the growth of the roots were investigated. Did. In addition, in order to find out the availability of amino acids by cabbage reaction according to inoculation of endogenous bacteria, inorganic nitrogen sources were the same as above, and amino acid aqueous solutions of amino acids such as valine, methionine, glutamine, glutamic acid, tryptophan, asparagine, and phenylalanine were used as amino acid nitrogen sources. Heteroconium in which a medium of potato dextrose agar (PDA) 15 g / L is endogenous to the cabbage root by making the nitrogen source at a concentration of 20 mg / L After inoculating the chaetospira bacteria, the cultivated cabbage seeds were dentured and cultured in a growth phase regulated to 25 ° C. After 2 weeks, the size of the cotyledon and the growth of roots of the cabbage seedlings were examined. After 2 weeks, the growth and roots of Chinese cabbage seedlings were observed.

4 is a graph observing the initial root elongation and cotyledon width of cabbage seedlings from various nitrogen sources such as aqueous phenylalanine solution.

As shown in Fig. 4, the cabbage seedling was initially raised to a certain level with a molecular sieve supplied from seeds. When supplying cabbage seedlings with an inorganic nitrogen source (ammonium) or amino acid at a concentration of N 20 mg / L, ammonium mononitrogen inorganic salts such as ammonium monophosphate or valine and methionine showed significant growth delay, whereas glutamic acid, In aqueous solutions such as tryptophan, asparagine, and phenylalanine, the growth was almost equal to that of untreated (distilled water). Although valine and methionine inhibited the growth of Chinese cabbage seedlings compared to untreated, as with ammonium monophosphate, tryptophan, asparagine, and phenylalanine were almost identical to those of untreated, so these amino acids absorbed the cabbage root directly. It was found that it was not used, but at least it did not impede growth.

Figure 5 is Heteroconium This is a picture of observing the growth of cabbage seedlings and the presence or absence of symbiosis of endogenous bacteria according to the supply of nitrogen sources such as phenylalanine when inoculating chaetospira .

6 shows Heteroconium This is an enlarged photograph focusing on untreated (no nitrogen source), nitrate nitrate, and phenylalanine as a result of observation of the growth of cabbage seedlings according to the supply of nitrogen sources such as phenylalanine and the presence or absence of endosymbiotic symbiosis when inoculated with chaetospira .

As shown in FIGS. 5 and 6, the medium in which HC was not inoculated in the supply medium of phenylalanine showed a form in which the roots were more likely to extend (these phenomena appear to be development to find nutrients), but the root was in the medium inoculated with endogenous bacteria. It grew into the medium, and the hyphae of endogenous bacteria were concentrated around the roots, so it could be judged to form a symbiotic relationship well. That is, it can be seen that the symbiotic relationship between cabbage seedlings and endogenous bacteria is formed according to mutual needs depending on the type of nitrogen source.

<Example 4> Nitrogen source mobility analysis by endogenous bacteria

7 is a schematic diagram of a nitrogen source mobility test to determine whether cabbage seedlings use a nitrogen source such as phenylalanine through an endogenous bacterium ( Heteroconium chaetospira ).

As shown in Fig. 7, according to the present invention, nitrogen nitrogen and C / N ratio of amino acids, such as amino acids, and C / N ratio of 10: 1 with glucose as carbon source, such as nitrogen nitrate, glutamine, phenylalanine, methionine, tryptophan, etc. As much as possible, fill the culture pot with glucose containing agar medium, put 2 PTFE filters (pore size 5μm) on it, fill the pure agar medium, and then endogenize Heteroconium. Chaetospira was inoculated and incubated for 1 week, and then the embryonic cabbage seeds were dentured thereon, and cultured for 2 weeks at 25 ° C. growth, and the growth of cabbage seedlings by nitrogen source according to the inoculation was compared.

Cabbage roots did not pass through the PTFE filter, but in all treatments Heteroconium The chaetospira hyphae passed through a PTFE filter and grew in the lower medium. In the treatment with no nitrogen source, after growing for about 2 weeks with its own nutrients, the cotyledon began to yellow. Heteroconium After 2 weeks of incubation for all treatments without inoculation with chaetospira , the roots did not grow well and decayed mostly. In the glutamine medium, the roots of Chinese cabbage seedlings were well penetrated into the medium during Heteroconium chaetospira inoculation, and the growth of Chinese cabbage seedlings was good. On the other hand, cabbage seedlings did not grow well in methionine medium, and most of them died, and the development of Heteroconium chaetospira in the lower medium was generally sluggish compared to other nitrogen source mediums. In tryptophan medium or phenylalanine medium, similar to glutamine medium, the seedlings of Chinese cabbage grew well. Heteroconium chaetospira bacteria also passed through the PTFE filter and grew well in the lower medium.

This is because the hydrophobic filter, PTFE filter, does not move amino acids such as water or phenylalanine dissolved in water to the upper medium, but when inoculated with endogenous bacteria, it passes through the PTFE filter and moves to the lower medium containing amino acid to use as a nitrogen source. It can be seen that the cabbage seedlings in the upper medium grow well. In other words, the root of Chinese cabbage seedlings does not directly absorb and use phenylalanine, but it means that it can be transported through endogenous bacteria and used as a nitrogen source in the body.

< Example 5 > Organic Use Endogenous bacteria  Growth reaction of cabbage according to inoculation and Glucosinolate  content

Heteroconium in order to grasp the growth of Chinese cabbage and the resulting amino acid and glucosinolate contents in the body after inoculating endogenous bacteria after applying organic matter, such as milk and soybean meal, to a field where cabbage is grown according to the present invention. chaetospira After cultivating the fungus, it is treated by bottom irrigation on the cabbage seedlings grown on the plug seedlings, and cultivated and grown in the rainforest house on June 22, 2017 by applying oilseed and soybean meal as organic matter. Content was investigated.

8 is a table in which soybean-derived organic matter is applied to the cabbage cultivation soil and cabbage growth according to inoculation with endogenous bacteria ( Heteroconium chaetospira ) is examined.

As shown in FIG. 8, the difference in growth between the two cabbages of Suho and Asian leaf ssam was slightly higher than that of Asian leaf ssam cabbage, but there was no statistically significant difference in the length or weight of the leaves. Did not appear.

In the endogenous inoculation, the number of lobes decreased in the inoculation of the bottom face compared to the untreated, and it decreased slightly in the weight, but it was confirmed that the inoculation of the endogenous bacterium did not significantly influence the growth of Chinese cabbage.

FIG. 9 is a table analyzing phenylalanine content in cabbage leaves according to inoculation of endogenous bacteria ( Heteroconium chaetospira ) by applying soybean-derived organic matter to cabbage cultivation soil.

As shown in FIG. 9, the content of phenylalanine in Chinese cabbage by inoculation with Heteroconium chaetospira was analyzed when applying organic materials containing Chinese cabbage varieties and soy components. Among the cabbage varieties, suho was significantly higher than that of leafy cabbage, and soybean meal, oilseed, and untreated in the order of soybean-based organic substances. On the other hand, when inoculated with endogenous bacteria, the phenylalanine content was statistically higher than that of the unvaccinated. There was also an interaction effect between varieties, organic matter, and endogenous bacteria. That is, it was found that the difference in phenylalanine content in cabbage cultivation is different depending on the variety, but is increased by the use of soybean-derived organics, and also by bacterial inoculation.

10 is a graph analyzing the content of glucosinolate in cabbage lobes according to inoculation of endogenous bacteria (Heteroconium chaetospira) by applying soybean-derived organic matter to the cabbage cultivation soil.

As shown in FIG. 10, when the content of glucosinolate was overall, the content of gluconapin and glucobrassicanapin was lower than that of Asian cabbage, and progoitrin and glucobrassicin showed similar content to both varieties. Gluconasturtiin hardly detected in both varieties, but it was found to be significantly higher in treatments inoculated with Heteroconium chaetospira with the use of oilseeds . At this time, the cultivars of sukhoi varieties in ganoderma use were slightly lower in gluconapin and glucobrassicanapin than those treated with no endogenous bacteria, and the gluconasturtiin content increased significantly, accounting for 74% of the total glucosinolate content 11.3 μmole.g-1, Asia Leaf ssam varieties showed 6.7 μmole.g-1, which is 81% of 8.3 μmole.g-1 due to the increase in gluconasturtiin content without any change in other glucosinolate content. The increase in gluconsturtiin content was greater in the guardian species and the total glucosinolate content was also higher.

In other words, when cultivated by using soybean-derived organic matter having a high phenylalanine content in cabbage cultivation soil and cultivating inoculated with endogenous bacteria symbiotic on the roots of cabbage, total glucosinolate increased compared to no treatment. It was found that the content of gluonastatin was significantly increased while the content of nolate was lowered.

Claims (8)

A method of increasing the content of a functional substance in a plant, comprising cultivating the plant root by inoculating endogenous bacteria.
According to claim 1,
The method is characterized in that substances that cannot be absorbed through the root of the plant move into the plant through an endogenous bacterium inoculated to the plant root, a method of increasing the content of the functional material in the plant.
The method of claim 2,
A method of increasing the content of a functional substance in a plant, further comprising adding to the plantation of the plant a substance that the plant cannot absorb through the root.
According to claim 1,
The plant is Chinese cabbage, and the endogenous bacteria are heteroconium chaetospira , a method for increasing the content of a functional substance in a plant.
According to claim 3,
A method for increasing the content of a functional substance in a plant, wherein the functional substance in a plant is glucosinolate.
The method of claim 5,
The glucosinolate is glucostatin, a method of increasing the content of a functional substance in a plant.
A plant with an increased content of functional substances in the plant, produced by the method of claim 1.
Food prepared using the plant of claim 7.

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