JPH07267812A - Method for infecting with symbiotic microorganism - Google Patents

Abstract

PURPOSE:To provide a method for infecting a regenerated seedling of a lawn grass with a large amount of symbiotic microorganisms efficiently and simply. CONSTITUTION:A callus is derived from a seed or a plant of a lawn grass. The callus is cultured in a liquid medium to give a regenerated seedling. Symbiotic microorganisms are inoculated into the liquid medium containing the regenerated seedling to provide a method for infecting the regenerated seedling with the symbiotic microorganisms.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for efficiently infecting a young plant regenerated from a callus of a liquid-cultured turfgrass with a symbiotic microorganism.

[0002]

2. Description of the Related Art Conventionally, various means have been tried to prevent the occurrence of diseases of turfgrass and protect the lawn grass from harmful insects, and spraying pesticides on turfgrass is the most effective method for controlling pests. It is one of the means. However, when the amount of pesticide applied is small, the effect of controlling insect pests cannot be sufficiently obtained, while when the amount of pesticide applied is too large, environmental damage occurs. Therefore, there is a demand for the development of a method of producing an effect of controlling insect damage against turfgrass by utilizing an effective microorganism that is effective and does not affect the environment.

Recently, many plants have symbiotic microorganisms,
It has been reported that these are involved in insect resistance and disease resistance of plants. In turfgrass, the presence of many types of symbiotic microorganisms has been reported, but among them, filamentous fungi of the genus Acremonium that are symbiotic with turfgrass called perennial ryegrass and tall fescue have strong insect resistance and disease resistance to plants. It is known to give. Therefore, in order to breed turfgrass that can effectively control pests and pests without using pesticides or by spraying a small amount of pesticide, development of a method for efficiently infecting turfgrass with symbiotic microorganisms such as filamentous fungus of the genus Acremonium is desired.

[0004] The filamentous fungus of the genus Acremonium has a property of being transmitted to the next generation through seeds. Utilizing this property, turfgrass seeds infected with Acremonium filamentous fungi are sold. However, since the proportion of perennial ryegrass and tall fescue originally infected with symbiotic microorganisms is about 10 to 30% of the total number of plant individuals, in order to obtain these seeds, acremonium from many plants is required. Only the plants infected with the filamentous fungi must be selected and produced by multiplying the selected individuals, which requires vast land and long time.

Further, this method cannot be applied to grass grass species that are not originally infected with Acremonium filamentous fungi such as bentgrass. On the other hand, there are also two known methods for artificially infecting plants with Acremonium filamentous fungi. One is a method of injuring a plant and applying a filamentous fungus of the genus Acremonium. However, this method has a drawback that it is inefficient because it is a fine work to be done manually and is not suitable for mass production. The other is a method of inoculating individual-cultured callus with filamentous fungus of the genus Acremonium and then regenerating the plant body. However, although this method is easy to work, it is not suitable for mass production because the growth rate of callus is slow.

[0006]

DISCLOSURE OF THE INVENTION An object of the present invention is to provide a method for efficiently and easily infecting a turfgrass plant with a symbiotic microorganism capable of producing a substance for controlling insect pests. And

[0007]

[Means for Solving the Problems] As a result of intensive studies based on the above problems, the present inventors inoculate a liquid medium containing seedlings regenerated from liquid-cultured turfgrass callus with a symbiotic microorganism. As a result, we succeeded in efficiently and easily infecting a large amount of symbiotic microorganisms, and completed the present invention.

That is, according to the present invention, callus is induced from seeds or plants of turfgrass, the callus is cultured in a liquid medium to obtain a regenerated seedling, and symbiotic in a liquid medium containing the regenerated seedling. A method for infecting a symbiotic microorganism, which comprises infecting a regenerated seedling with a symbiotic microorganism by inoculating the microorganism. Here, the symbiotic microorganisms are microorganisms that live together with plant tissues in the plant body, that is, microorganisms that provide insect repellent control substances instead of obtaining nutrients necessary for life from plant tissues. Means

Examples of turfgrass include perennial ryegrass, tall fescue, bentgrass and the like, and examples of symbiotic microorganisms include microorganisms belonging to the genus Acremonium. Hereinafter, the present invention will be described in detail. The method for obtaining turfgrass seedlings is as follows. 1) Callus induction Plants of grass (roots, foliage, seeds, etc.) are sterilized and placed on a solid medium to induce callus. Examples of the callus induction solid medium include conventionally known media such as Murashige-Skoog's medium (MS medium) and N6 medium.

It is possible to add plant hormones to these media and, if necessary, further add carbon sources, inorganic components, vitamins and the like. As the carbon source, disaccharides such as sucrose, maltose and lactose, monosaccharides of glucose, fructose and galactose sugar, starch, or a mixture of two or more of these sugar sources in an appropriate ratio can be used.

Examples of the inorganic component include inorganic salts containing elements such as phosphorus, nitrogen, potassium, calcium, magnesium, sulfur, iron, manganese, zinc, boron, copper, molybdenum, chlorine, sodium, iodine and cobalt. Specifically, potassium nitrate, sodium nitrate, ammonium nitrate, sodium dihydrogen phosphate, potassium chloride, calcium chloride, ammonium chloride, sodium sulfate, ferrous sulfate, ferric sulfate, manganese sulfate, copper sulfate, molybdenum. Examples thereof include compounds such as sodium acid salt, molybdenum trioxide, potassium iodide, zinc sulfate, boric acid and cobalt chloride.

Examples of plant hormones include indole acetic acid (IAA), naphthalene acetic acid (NAA) and 2,4-
Examples thereof include auxins such as dichlorophenoxyacetic acid (2,4-D) and cytokinins such as benzyladenine (BA), kinetin and zeatin. Examples of vitamins include biotin, thiamine (vitamin B1), pyridoxine (vitamin B6), pyridoxal, pyridoxamine, calcium pantothenate, ascorbic acid (vitamin C), inositol, nicotinic acid, nicotinamide riboflavin (vitamin B2) and the like. To be

Examples of amino acids include glycine, phenylalanine, alanine, glutamic acid, cysteine,
Examples include lysine and the like. Generally, each of the above components comprises about 0.1 μM to about 100 mM of inorganic component, about 1 to about 100 g / L of carbon source,
The plant hormone is used in an amount of about 0.1 to about 10 mg / L and vitamins and amino acids of about 0.1 mg / L to about 150 mg / L, respectively.

In the present invention, any of liquid medium and solid medium containing agar, gellite, agarose and the like can be used, but in callus induction, gellite, agar, agarose etc. are usually contained as a gel material in an amount of 1 to 10%. Preferred solid medium is. As a preferred medium for callus induction, 30 g / L sucrose in MS medium, 5 mg / L 2,4-D as auxin and 0.5 mg as cytokinin are used.
A solid medium containing / L BA and 3 g / L gellite as a gel material can be mentioned. And includes 25 cc of the solid medium
Place 20 plants on a 10 cmφ x 2 cm deep dish, 28 ℃,
Callus can be obtained by culturing in the dark for 1 month. 2) Callus Passage and Proliferation The callus obtained in 1) can be passaged and propagated by shaking culture in a liquid medium.

As the liquid medium for callus passage / growth, N6 medium is used in which the gel material of the solid medium is removed and the inorganic salt and hormone concentrations are slightly diluted. In this case, 30 g / L sucrose, 1 mg / L 2,4-D as auxin and 0.5 mg / L BA as cytokinin are preferably contained. Then, 1 g of callus was placed in a 200 cc wide-mouth flask (medium 40 cc), cultured at a temperature of 28 ° C., a shaking speed of 120 rpm in the dark, and subcultured and proliferated every week. 3) Regeneration of seedlings The callus obtained in 2) is placed in a liquid medium for regeneration and the seedlings are redifferentiated by shaking culture. In order to redifferentiate the seedlings, the auxin concentration may be lowered.

Therefore, as the redifferentiation liquid medium for seedlings, the callus passage medium is made to have a lower concentration of inorganic salts so as to remove auxin or to give NAA having a small effect, and 30 g / L
It is preferable to contain sucrose, 0.4 mg / L NAA, and 0.5 mg / L BA. Callus 500 in a 100cc flask (medium 20cc)
Place about mg on the bed, temperature 28 ℃, shaking speed 120 rpm, 4000L
After culturing for 3 weeks at x, full day length, changing the medium to double volume (40 cc) and culturing for 3 weeks, redifferentiated seedlings are formed.

On the other hand, as a microorganism belonging to the genus Acremonium which is a symbiotic microorganism, Acremonium loli (Acre
filamentous fungi such as monium lolii) and Acremonium coenophialum. Since these microorganisms are microorganisms that are symbiotic with perennial ryegrass, tall fescue, etc., for example, after surface sterilizing these turfgrass seeds or plants, they can be separated by placing them on a solid medium and culturing. it can.

For surface sterilization, ethanol, an aqueous solution of sodium hypochlorite or the like is used, and seeds or plants are immersed in this. The concentration of ethanol is 70%, and the effective chlorine concentration of the sodium hypochlorite aqueous solution is preferably 1 to 2%. The soaking time is preferably 15 to 25 minutes each for ethanol and sodium hypochlorite aqueous solution for seeds, and 1 to 5 minutes each for ethanol and sodium hypochlorite aqueous solution for plants.

As the separation medium, potato saccharose solid medium used for culturing microorganisms, N6 medium used for tissue culture and the like are suitable. A suitable culture temperature is 25 ° C. When culturing is performed under these conditions for 4 to 8 weeks, symbiotic microorganisms are separated. When the amount of the separated symbiotic microorganism is not necessary for infection, it is necessary to transfer it to a potato saccharose liquid medium or the like, if necessary, and to culture by shaking at 25 ° C. for 1-2 weeks. It is also possible to obtain the amount of symbiotic microorganisms.

To infect a regenerated seedling with a symbiotic bacterium, 1 to 20 mg of the obtained symbiotic microorganism is added to a medium containing the regenerated seedling obtained as described above per 1 ml of the medium.
It is preferable to inoculate so that the amount is 5 to 10 mg. The symbiotic microorganisms used for inoculation can be obtained by collecting the cells cultured as described above by centrifugation or directly using a medium containing the symbiotic microorganisms.

The symbiotic microorganisms do not have the ability to penetrate the epidermis of plants and infect the plants like the plant pathogenic microorganisms. Therefore, even if the cultured symbiotic microorganism is applied to the surface of the plant, the symbiotic microorganism does not infect the plant. However, when inoculated with the symbiotic microorganisms by this method, the epidermis of the regenerated seedlings is fragile, and since the epidermis does not exist at the boundary between the callus and the regenerated seedlings, the symbiotic microorganisms invade and infect the seedlings. It becomes possible.

As a result, plants originally infected with the above-mentioned symbiotic microorganisms, such as perennial ryegrass and tall fescue, can be infected with the symbiotic microorganisms at a higher rate and are not originally infected with the above-mentioned symbiotic microorganisms. It is possible to infect a plant such as bentgrass with a symbiotic microorganism.

[0023]

The present invention will be described in more detail with reference to the following examples. However, the present invention is not limited to these examples. [Example 1] Perennial ryegrass, tall fescue,
Callus induction solid medium (composition:
MS medium, 30 g / L sucrose, 5 mg / L 2,4-D,
0.5 mg / L BA, 3 g / L gellite) was placed on the plate and cultured for 1 month, and the callus obtained by dedifferentiating the seed was obtained by liquid passage medium (composition: N6 medium, 30 g / L sucrose, 1 mg / L). L
2,4-D, 0.5 mg / L BA) was subcultured every one week.

Next, 500 mg of each callus derived from seeds by the above method was placed in a liquid regeneration medium (composition: N6 medium, 30 g / L sucrose, 0.4 mg) placed in a 100 cc flask.
/ LNAA, 0.5mg / L BA) Placed in 20ml, illuminance 4000L
The cells were cultured for 3 weeks under the conditions of x, full-day length, shaking speed of 120 rpm, and temperature of 28 ° C. After culturing for 3 weeks, the medium was replaced with 40 ml, and the cells were further cultivated for 3 weeks.

As a result, regenerated seedlings regenerated into about 5 mm in the culture solution were obtained. In this culture solution, 1 ml of a medium containing the symbiotic microorganisms Acremonium loli and strain PE-001
5 mg per inoculation. At this time, the time (required time) required for the work of inoculating the strain PE-001 was also measured for 20 regenerated seedlings of perennial ryegrass. After inoculation with the strain, shaking culture was further performed at a temperature of 25 ° C. for 1 week.

After completion of the culture, it was confirmed whether or not the regenerated seedlings were infected with the symbiotic microorganisms. That is, sampling the plant from the flask, cutting out the plant tissue, by microscopic observation perennial ryegrass, tall fescue,
The presence of symbiotic microorganisms was confirmed in each grass grass species of bentgrass. Then, the regenerated plant was further grown for 1 month in a solid medium (medium containing only inorganic salts) to confirm whether or not symbiotic microorganisms were present in the plant. As a confirmation method, the plant tissues of each of the 20 regenerated plants were observed with a microscope to examine the presence or absence of symbiotic microorganisms.

As a result, in any grass grass species,
It was confirmed that symbiotic microorganisms were infected. The results are shown in Table 1. Perennial ryegrass and tall fescue, which are the hosts of the symbiotic microorganisms of the genus Acremonium, were infected with the symbiotic microorganisms at a high rate. In addition, even in bentgrass, which is not the host of the symbiotic microorganisms of the genus Acremonium, some cases were found to be infected with the symbiotic microorganisms.

Furthermore, the time required to inoculate 20 individuals of perennial ryegrass with the strain PE-001 was an extremely short time of 1 minute (Table 2). [Comparative Example 1] On the other hand, seedlings that were not regenerated from callus (seed-derived seedlings) were scratched with a fine needle, and a symbiotic microorganism, Acremonium loli, strain PE
-001 was applied (the time required to inoculate 20 individuals of perennial ryegrass with strain PE-001 was also measured). After the application, the plants were further grown for 1 month, and it was observed whether or not the plants were infected with the symbiotic microorganisms.

The results are shown in Table 1.

[0030]

[Table 1]

As is clear from Table 1, the infection rates of both perennial ryegrass and tall fescue were lower than those of Example 1, and there was no infected individual in bentgrass. Furthermore, it took 45 minutes to inoculate 20 individual perennial ryegrass strains PE-001, which was a long time as compared with Example 1 (Table 2).

[0032]

[Table 2]

[0033]

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a method for efficiently and easily infecting a regenerated young plant of turfgrass with a symbiotic microorganism capable of producing a pest control substance. Further, it is possible to provide a method for infecting a plant other than the original host of the symbiotic microorganism with the symbiotic microorganism.

Claims (3)

[Claims] 1. A callus is induced from a turfgrass seed or plant body, the callus is cultured in a liquid medium to obtain a regenerated seedling, and a symbiotic microorganism is inoculated into the liquid medium containing the regenerated seedling. Thus, a method for infecting a symbiotic microorganism, which comprises infecting a regenerated seedling with a symbiotic microorganism. 2. The method for infecting a symbiotic microorganism according to claim 1, wherein the turfgrass is perennial ryegrass, tall fescue or bentgrass. 3. The method for infecting a symbiotic microorganism according to claim 1, wherein the symbiotic microorganism is a microorganism belonging to the genus Acremonium.