KR20090028324A - Arsenic-resistant oenothera odorata characterized by inoculation of arbuscular mycorrhizae - Google Patents

Abstract

Oenothera odorata with improved arsenic resistanc, a cultivation method thereof and a oil composition for cultivating the Oenothera odorata are provided to reduce the arsenic toxicity of Oenothera odorata, and to overcome the lack of phosphorus and the rsenic toxicity of the soil polluted by abandoned mine tailing. Oenothera odorata improves arsenic resistance by being inoculated arbuscular mycorrhizae. the inoculated arbuscular mycorrhizae is glomus mosseae. a cultivation method of oenothera odorata comprises the steps of: subculturing arbuscular mycorrhizae and the host plant for cultivating arbuscular mycorrhizae, in a soil culture medium; preparing for culture bed soil including the root from which arbuscular mycorrhizae is infected by cutting the ground part the host plant for cultivating arbuscular mycorrhizae; and cultivating Oenothera odorata by sowing seeds of Oenothera odorata in the culture bed soil.

Description

Evening Evening Primrose flower, characterized by inoculation with endogenous mycorrhizal fungi (AMM, Aｒｂｕｓｃｕｌａｒ Ｍｙｃｏｒｒｈｉｚａｅ) {Arsenic-resistant Oenothera odorata selected by inoculation of Arbuscular Mycorrhizae}

The present invention relates to an evening primrose inoculated with endogenous mycorrhizal fungi, and more particularly to an evening primrose flower, characterized in that the inoculation with endogenous mycorrhizal fungi.

Soil remediation (Phytoremediation) technology is not an old technology, but is now rapidly expanding in research and application, especially in the United States. This technique can be applied to soils contaminated with heavy metals, metalloids, radioactive contaminants, and organic compounds. In order to apply the plant-based soil purification technique to the waste metal mine soil contaminated with heavy metal or metalloid, it is essential to overcome the heavy metal toxicity of the applied plant and the poor soil factor for plant growth. However, the existing plant purification method focuses only on the enhancement of the absorption of heavy metals through indoor experiments, and thus, there is no research in terms of stable sticking and sound growth of applied plants when applied to soil polluted soil. At present, many attempts at phytoremediation of arsenic pollution have not been made in Korea. In addition, no attempt has been made on soil purification techniques using endogenous mycorrhizal bacteria on site soils.

Among plants that adapt to contaminated soil, endogenous mycobacteria are often symbiotic. Mycorrhizal bacteria are not known to be purely isolated or cultured in artificial media (Lewis, 1973). Therefore, for artificial culture, it was necessary to use the plant root directly, and Mosse (1953) succeeded in artificial plant root culture for the first time. Subsequently, it was successful to separate spores by wet sieving (Gerdemann, 1955). Spores of endogenous mycorrhiza germinate and combine with the roots of plants to form symbiosis. In legumes, it is known that the supply of nitrogen is smooth when coexisting with endogenous mycorrhizal fungi (Carling, et al., 1978), and the formation of endogenous mycobacterium is essential under conditions that require large amounts of phosphoric acid (Knight et al., 1988). ). In comparison with plants invading endogenous mycorrhiza and non-invasive plants, it was observed that growth was promoted in plants invading endogenous mycorrhiza (Gerdemann, 1964; Gupta and Janardhanan, 1991). In particular, research has been conducted in relation to inorganic phosphate nutrients in endogenous mycobacteria (VAM) (James et al., 1981; Jensen, 1982). Phosphate supply to plants plays an important role in crops because they are related to other metal nutrients and copper, zinc, nitrogen, magnesium, potassium, calcium, etc. and promote plant growth (Ojala et al., 1983; Elmes and Mossse, 1984). Mosse, 1973; Saif, 1981). To absorb insoluble phosphate, the endogenous mycorrhiza produces phosphate enzyme on the surface of the mycelia, and the phosphates absorbed by the mycorrhizal fungi are converted into polyphosphate in arbuscles, vesicles, and their own mycelia. It is then decomposed into a form that can be absorbed by the plant in the arbuscule, and phosphoric acid is supplied to host plants (Callow et al., 1978). Endogenous mycorrhizal fungi are widely distributed in wild plants and forests in Korea, and limestone and limestone About 80-95% of plant infections were investigated in the zone of endogenous mycobacteria (Kim et al., 1989; Lee and Koo, 1983).

 Arsenic is a metalloid classified as a toxic and carcinogen. At present, there are many reports of contamination on arsenic, a metal, as well as heavy metals. Mining activities generate a lot of mine waste, and they are threatening the environment around them. In particular, soil and water pollution have emerged as a serious social problem due to infiltration into the surrounding environment due to leaching and erosion by the weathering of arsenic-containing minerals in the waste metal mines. Recovery of soil contaminated with these waste tailings is necessary.

Endogenous mycorrhizal fungi are known to play a role in reducing the toxicity of roots to heavy metals of host plants in soils contaminated with heavy metals. For cation heavy metals, the effects on endogenous mycorrhizal fungi and various host plants are known. However, only a few reports have been known about arsenic, and the mechanism is not clear. In particular, research on indigenous species of wildflowers is insignificant.

Therefore, the present inventors have made intensive studies to overcome the problems of the restoration of soil contaminated with waste tailings, and as a result, when planting and growing non-soak resistant enhanced evening primrose, characterized by inoculation with endogenous mycorrhizal fungi, The present invention was completed by overcoming arsenic toxicity reduction and phosphorus deficiency, inducing high biomass and stable lubrication to solve arsenic toxicity in soil contaminated with waste tailings.

Therefore, the main purpose of the present invention is to inoculate and infect the endogenous flowers of evening primrose, the domestic native weed species, endogenous mycorrhizal fungi of Glomales neck to increase biomass by nutrient absorption in arsenic-contaminated soil and reduce the toxicity by arsenic. It is to provide an evening primrose with enhanced non-soak resistance.

Another object of the present invention is to provide a method for purifying arsenic-contaminated soil by planting an evening primrose inoculated with arsenic resistance inoculated with the endogenous mycobacteria.

According to an aspect of the present invention, the present invention provides an evening primrose (Oenothera odorata), characterized in that the inoculated with endogenous mycorrhiza (AM, Arbuscular Mycorrhizae).

Mycorrhizae is a form in which the roots of plants coexist with fungi in the soil, absorbing inorganic salts and delivering them to host plants. In return, host plants maintain a symbiotic relationship that delivers carbohydrates to molds. Mycorrhizae is invasive to plant roots and interacts with plants, mainly related to plant growth, suppression of soil infectious pathogens (Caron et al., 1986), resistance to salts and toxic substances accumulated in soil, and drought and root Effects on damage damage reduction (Warterer and Coltman, 1989). The endogenous mycorrhizal fungi are characterized by the appearance of Glomaceae, which produces vesicles, and Gigasporaceae, which form Arbuscle, mainly in the cortex of the roots, resulting in Vesicular-Arbuscular Mycorrhizae (VAM). (Powell and Bagyarat, 1984). Mycorrhizal fungi are endogenous and exogenous mycorrhizal fungi, and occupy an ecologically important position in marginal soils in which plant growth is disadvantageous. In fact, seedlings inoculated with mycorrhiza can play a role in promoting growth and increasing crop productivity. Hyphae, roots infected by mycorrhizal, penetrate into the pores where the roots can't reach and supply the plant more efficiently.

In the evening primrose flowers of the present invention, the inoculated endogenous mycorrhizal fungi may include any endogenous mycobacteria that may be inoculated into the evening primrose flowers, but preferably, the Glomos eggs of the genus Glomus Vidilium species, glomos clalium species, glomos desulticola species, glomos ethenicatium species, glomos pack conditionis pollium species, glomos oligolithium species, glomos synthans, glomos sphsis species and glomos Moses A. species are preferred, and most preferably, G. mosseae, which is readily available on the market, can be easily inoculated against host plants, and can coexist with host plants to increase biomass and enhance resistance to arsenic (G. mosseae). ) Is good. 2, G. mosseae. BEG 107 shows that root colonization is possible in evening primrose and arsenic-contaminated soils.

According to an aspect of the present invention, the present invention comprises the steps of subcultured the endogenous mycobacteria and host plants for culturing the endogenous mycorrhizal strain in a soil medium; Cutting a ground portion of the host plant for endogenous mycorrhizal strain culture and preparing a culture soil including roots infected with the endogenous mycobacteria; It provides a cultivation method of evening primrose flowers, including the step of planting and seeding the seeds of evening primrose (Oenothera odorata) in the culture soil. The host plant for endogenous mycorrhizal strain culture refers to a host plant used to propagate the endogenous mycorrhiza as necessary before inoculation into the evening primrose. The host plant for culturing the endogenous mycorrhizal fungi may be any plant well infected with the root, such as shamrock (T. repens ), sudangrass, corn, wheat, etc., but preferably using shamrock (T. repens ). It is easy and cheap to infect and convenient to grow.

According to an aspect of the present invention, the present invention provides a soil composition for evening primrose flowers cultivated non-soak resistant, including roots infected with the endogenous mycobacteria. The endogenous mycorrhiza can be any endogenous mycobacteria that can be inoculated into host plants such as shamrock, sudangrass, corn, etc., but is preferably widely available and easily inoculated for host plants and symbiotic with host plants to obtain biomass. Endogenous mycobacteria of G. mosseae. BEG 107 (European Bank of Glomales), which can enhance and enhance resistance to arsenic.

According to one aspect of the present invention, the present invention provides a method for purifying arsenic-contaminated soil by planting an evening primrose flower with enhanced arsenic resistance including roots infected with the endogenous mycobacteria.

Evening primrose is a native native weed species that has been determined to be resistant to arsenic through vegetation surveys and indoor tolerance tests in domestic abandoned metal mine soils. In addition, infection was confirmed when this species was inoculated with the genus Arbuscular Mycorrhizae. The present invention, which promotes non-resistant resistance through inoculation of endogenous mycorrhiza into evening primrose flowers, prevents ecological disturbance caused by plants introduced from foreign countries and prevents the risk of human influx by feeding plants. Have Arsenic resistance is generally known to achieve resistance to arsenic through inhibition of absorption. Endogenous mycorrhiza is known to provide a barrier to the heavy metal toxicity of host plants. In particular, it is a conflict with the present study, but as for the metalloid As, it is known that mycorrhizal lowers the As concentration in the body through a filter role (Sharples et al., 2000). However, no arsenic inhibition was observed in this experiment. Evening Primrose flowers inoculated with endogenous mycorrhizal fungi in the present invention, after 8 weeks of cultivation, the above-ground biomass was increased to 323% (P <0.01), underground biomass 24% (P <0.01) compared to the control (Fig. 3). The mycorrhizal inoculation increased the growth of the host plant roots and increased the surface area, so that it could supply enough phosphorus nutrients deficient in mines. In the basement of evening primrose, the concentration of arsenic was increased to 240.14 mg · kg ⁻¹ rather than the sterilization treatment (FIG. 5). Even in the results of this experiment, Evening Primrose is considered to be caused by a high level of phosphorus content (Fig. 4) and the concentration dilution effect by biomass enhancement of the resistance to arsenic. It is believed that the underground arsenic content will remain high as well as other body detoxification (PC synthesis and antioxidant reactions). Plants resistant to arsenic maintain high levels of phosphorus at the ground level despite inhibition of phosphorus absorption (Wright et al. 2000). In Fig. 3, 4 and 5, the endocrine evening primrose flowers accumulate more arsenic in the roots than in the control, and the transition of arsenic to the ground is low, resulting in high levels of phosphorus in the ground. Maintaining biomass reduces arsenic toxicity.

T. repens , widely found in mining areas in Korea, is widely commercially available and can be sown directly into mine soil. In addition, inoculation to the endogenous mycorrhizal fungi and easy cultivation In the present invention, shamrock (T. repens ) was selected as a host plant for the endogenous mycobacterial strain culture. In addition, AM fungus (Glomus mosseae BEG 107) was selected as a strain of endogenous mycorrhizal fungi and treated with endogenous mycobacteria (10% (v / v)) in a mixed medium containing vermiculite and sand in a 1: 1 ratio. Subsequently, T. repens , a host plant for endogenous mycorrhizal strain culture, was subcultured. Increasing the volume of Medium by 10 times and cultivating while diluting and mixing roots confirmed infection. Vermiculite is a material heated by crushing rock mined from the Kolesk Peninsula, which is known to be cheaper and surpass black soil, and can hold more than 10 times the weight of its own weight and at any time needed. As much as the plant can replenish moisture. It is sold at a general agricultural product distributor and used for general cultivation experiments. In addition, in order to prepare a cultured soil containing infected roots inoculated with endogenous mycorrhizal fungi were cut out the ground portion of T. repens subcultured for three months and then prepared a cultured soil containing infected roots. Cut 100 roots into 1cm length, put them on a Petri dish with 1cm * 1cm grid line, and observe the number of root slices stained with trypan blue under a microscope. About 60% was observed (gridline-intersect method (Giovannetti and Mosse, 1980)).

Hereinafter, the present invention will be described in more detail with reference to Examples. Since these examples are only for illustrating the present invention, the scope of the present invention is not to be construed as being limited by these examples.

Example 1. Selection of host plants and endogenous mycorrhizal fungi (AM fungus) and cultured soil containing roots infected with endogenous mycobacteria

T. repens , widely found in mining areas in Korea, is widely commercially available and can be sown directly into mine soil. In addition, inoculation to the endogenous mycorrhizal fungi and easy cultivation In the present invention, shamrock (T. repens ) was selected as a host plant for the endogenous mycobacterial strain culture. Strains of endogenous mycobacteria were used as AM fungus (Glomus mosseae BEG 107) obtained from the European Bank of Glomales. The evening primrose flower was passaged by treating the endogenous mycorrhizal fungi (10% (v / v)) in a mixed medium of vermiculite and sand (1: 1 v / v). Increasing the volume of the medium by 10 times, and continuously diluting and mixing the roots identified with infection, and cultured for 3 months, and then cut and removed the ground portion of T. repens , the host plant for endogenous mycobacteria strain culture, and cultured the infected roots. Topsoil was used as a strain. Cut 100 washed roots into 1cm lengths, place them on a Petri dish with a 1cm * 1cm grid line, and count the number of root sections stained with trypan blue under a microscope. Before entering the experiment, the infection rate of mycorrhiza was about 60%.

Example  2. Growth medium Preparation

The collected arsenic contaminated soil was collected from the topsoil of Geumjeong Mine and its chemical properties are shown in <Table 1>. Arsenic-contaminated soil having the chemical properties of <Table 1> was air dried and passed through a 4 mm sieve eye to be used for pot experiments. 15 days with the addition of nutrients (476 mg N (NH4NO3), 186 mg K (K2SO4), and 19 mg Fe (FeCl2) [kg soil ^-1 ].) Was stabilized during. Treatments were made into two treatments, mycorrhizal infection and sterilization. Strains prepared for mycorrhizal infection were mixed with mine soil at 10% (v / v). The sterilized spheres were sterilized in an autoclave for 20 minutes at 121 ° C., and the sterilized strain filtrate (Whatman No. 2) was added to the sterilized mine soil again to prevent loss of nutrients.

<Table 1> Physicochemical Properties of Arsenic-Contaminated Geumjeong Mine Soils

Example  3. Evening Primrose (O. odorata Sowing and planting

The mixed soil prepared in the plastic pot (1.5 L) was filled with 1.2 kg, and 0.1 mm net was laid at the bottom of the pot to prevent soil loss. The density of the pot was adjusted to about 1.3 g cm ^-1 . Seeds of evening primrose (O. odorata) were sterilized with 10% H ₂ O ₂ for 10 minutes and pre-germination with saturated CaSO ₄ for 6 hours. Sixty seeds were sown in the infection and sterilization ports, and after germination, the evening primrose (O. odorata) was cultivated for 30 weeks. Cultivation was carried out at Korea University's glass greenhouse (weekly 24 ℃, nighttime 16 ℃), and the pot was cultivated for 8 weeks, maintaining 60% of the packaging capacity. The growth of the evening primrose flower (AM) and the uninfected primrose evening primrose (NAM) after 30 days after the germination of the seedling evening primrose germ root can be seen that the growth of the evening primrose flower (AM) is better . After 8 weeks, the ground and underground parts were separated and the fresh and dry weights were measured. Some of the fresh root was used to measure the infection rate of mycorrhiza.

Example  4. Plant Analysis

As a dry sample, As was quantified using ICP-AES (JY 138 Ultrace, Jobin Yvon, Edison, NJ, USA) after wet decomposition according to the method of Cai et al. (2000). It was quantified by molybdenum blue assay (Kitson and Mellon, 1944, Blochem. J., 37, 256). Root mycorrhizal infection rates were analyzed by trypan blue staining (Koske and Gemma, 1989) and gridline-intersect method (Giovannetti and Mosse, 1980). It was confirmed that the mycorrhiza was not infected in the sterilized treatment, and the infection rate of the evening primrose was 62.3% in the mycorrhizal infection treatment (FIG. 2). After 8 weeks of cultivation, the biomass analysis of the evening primrose (AM) above and below ground showed that the biomass of 323% (P <0.01) and 24% (P <0.01) were increased in the basement compared to the control (NAM), respectively. (FIG. 3). 4 and 5 show the concentrations of phosphorus and arsenic in the above and below ground of evening primrose according to the inoculation of endogenous mycorrhiza. As a result, the content of phosphorus and arsenic in evening primrose flowers increased during inoculation of endogenous mycorrhiza, especially in the underground, the concentration of arsenic increased to 240.14 mg * kg ^-1 rather than in sterilization treatment (Fig. 5).

According to the present invention, inoculation and infection of endogenous mycorrhizal fungi of the genus Glomus to the evening primrose, a native weed herb species, can enhance biomass and arsenic resistance in arsenic-contaminated soil.

In addition, according to the present invention when cultivated by planting a non-exophysiologically enhanced evening primrose, characterized in that the inoculation with endogenous mycorrhizal fungi, overcome the non-toxic toxicity and phosphorus deficiency in the existing evening primrose and induce high biomass and stable sticking Arsenic toxicity can be addressed in labyrinthed soils.

1 is a view showing the growth of evening primrose (AM) and uninfected evening primrose (NAM) infected with mycorrhiza after 30 days after germination of seeded evening primrose.

2 is a diagram showing the infection rate of evening primrose against the endogenous mycobacteria.

3 is a diagram showing the biomass of the above-ground and underground parts of Evening Primrose (AM) and uninfected Evening Primrose (NAM).

4 is a diagram showing the content of phosphorus in the above-ground and underground parts of Evening Primrose (AM) and uninfected Evening Primrose (NAM).

5 is a diagram showing the content of arsenic of the above-ground and underground parts of Evening Primrose (AM) and uninfected Evening Primrose (NAM).

Claims (7)

Evening-enhanced evening primrose (Oenothera odorata), characterized in that inoculated with the endogenous mycorrhizae (Arbuscular Mycorrhizae). The evening primrose according to claim 1, wherein the inoculated endogenous mycobacteria are genus Glomus. The evening primrose flower according to claim 1, wherein the inoculated endogenous mycorrhizal fungus is G. mosseae. Subcultured host plants for culturing endogenous mycobacteria and endogenous mycobacteria on soil medium; Preparing a culture soil comprising roots infected with endogenous mycorrhizal bacteria by cutting the ground portion of the host plant for endogenous mycorrhizal strain culture; A method for cultivating evening primrose flowers, comprising the step of planting seeds of evening primrose (Oenothera odorata) in the culture soil. [Claim 5] The method of cultivating evening primrose flowers according to claim 4, wherein the host plant for culturing endogenous mycorrhizal strains is shamrock (T. repens ). Soil composition for cultivating evening primrose flowers including roots infected with the endogenous mycobacteria bacteria. A method for purifying arsenic-contaminated soil, comprising planting and planting evening primrose flowers having improved arsenic resistance according to claim 1 in arsenic-contaminated soil.

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