JP3133605B2 - How to increase the vitamin C content of vegetable fruits - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel method for increasing the vitamin C content of a harvest by inoculating a cucumber mosaic virus, particularly an attenuated cucumber mosaic virus, into a vegetable fruit seedling and performing cultivation management by a conventional method. About.

[0002]

2. Description of the Related Art In recent years, quality improvement of vegetable fruits has been regarded as important from the viewpoint of high added value, and research on quality breeding and cultivation techniques related thereto has become more and more active with the needs of the times. Studies aimed at increasing the content are no exception.

[0003] Among the essential vitamins for the human body, there are vitamins A, B1, C, K, etc., which use agricultural crops as a main source. Among them, vitamin C almost entirely depends on vegetables and fruits, and oranges, cabbage, Chinese cabbage, radish, tomato, and the like have a high vitamin C content by item.

[0004] Vegetables and fruits have been breeded for the purpose of improving the vitamin C content. In particular, in recent years, simple and rapid instrumental analysis methods have been developed one after another. There is a good momentum at the center of breeding improvement aimed at increasing the vitamin C content.

[0005] In order to improve the quality of the harvest of vegetable and fruit, attention is being paid to research on cultivation techniques such as a brix nine system and a drain bed system in which light is often applied to the harvest and the cultivation is managed with a slight lack of moisture. is there.

[0006]

However, the method of increasing the vitamin C content of vegetable fruits by breeding techniques is based on the interaction between genotype and environment because many of these quality-related traits are quantitative traits. As in the case of high yield, it is difficult to select varieties, and it is difficult to breed varieties that increase vitamin C in crops.

In addition, in tomatoes and the like, a high pigment gene (such as a high pigment gene) is effective for improving the vitamin C content.
The possession line tends to be inferior in early growth and yield, and 195
Although breeding has been undertaken in various parts of the world since the 0s, it has not been possible to overcome these problems and to develop practical varieties with high vitamin C content and excellent cultivation.

[0008] Furthermore, a cultivation method for improving the quality of fruits such as tomatoes is a drainbet method in which cultivation is performed with a small amount of irrigation, or a sheet is placed in a planting hole and planted thereon to suppress rooting. The Brix Nine system, which suppresses the growth of fruits, focuses on increasing the sugar content of the fruit,
The increase in vitamin C content is incidental, and a cultivation method for increasing the vitamin C content of a crop is not yet known.

Therefore, the present invention does not require special cultivation equipment and materials, and a cultivation method in view of the circumstances in which conventional techniques for increasing the vitamin C content of vegetable fruits have not yet been established. The purpose is to improve the vitamin C content of vegetable fruits.

[0010]

The present invention is characterized in that attenuated cucumber mosaic virus (NDM2) containing satellite RNA selectively isolated from the natural world is inoculated into vegetable fruit seedlings and cultivation management is carried out by a conventional method. This is a method for increasing the vitamin C content of vegetable fruits.

Hereinafter, the present invention will be described in detail.

Plants that are infected with the cucumber mosaic virus include solanaceae, cucurbitaceae, asteraceae, cruciferous family, rose family, lily family, etc.
There are zero or more species. (Reference: Dictionary of plant virus)

Among them, vegetables that can be expected to increase the vitamin C content by inoculating cucumber mosaic virus include cabbage, Chinese cabbage, radish, cucumber, eggplant, asparagus, udo, cauliflower, spinach, and other vegetables And as fruits,
Examples include tomato, watermelon, melon, peach, apple, citrus, and other fruits.

As the cucumber mosaic virus used in the present invention, any cucumber mosaic virus can be mentioned, but an attenuated cucumber mosaic virus which has a weak side effect of the virus and has no influence on cultivation is preferable.
Attenuated cucumber mosaic virus can be selected from among the nucleic acid components that make up this virus, including those that contain satellite RNA, which is a nucleic acid component that controls disease symptom reduction. Preferred for making mosaic virus. The cucumber mosaic virus may not have satellite RNA.

Hereinafter, a method for preparing an attenuated cucumber mosaic virus will be described. First, 1 gram of tomato leaves infected with a cucumber mosaic virus that does not show necrotic symptoms is
A buffer solution (0.1 M phosphate buffer near neutrality) was added, and 10 ml of the ground solution obtained by grinding was added to tomato 100
After inoculating the cotyledons or true leaves of the strain and growing the virus for about 1 to 2 weeks, 150 g of tomato-infected leaves are collected and frozen at an ultra-low temperature of -80 ° C. After crushing this infected leaf,
The same amount of chloroform as 300 ml of 0.5 M citrate buffer (PH 6.5) containing 0.1% thioglycolic acid was added, and the mixture was ground with a Waring blender to obtain a ground liquid containing virus particles. .

This triturated liquid is centrifuged at 2,000 × g for 10 minutes, 120 ml of the upper layer (aqueous layer) is recovered, 12 g of 10% by weight powdered polyethylene glycol is added and dissolved, and the mixture is allowed to stand for 40 minutes. To precipitate and facilitate precipitation.

This solution was centrifuged at 9,500 × g for 20 minutes, and the resulting precipitate (virus particles) was recovered.
A 0.05 M citrate buffer (pH 7.0) containing 5% Triton X-100 is added for washing and dissolved.

This was centrifuged at 12,000 × g, and the supernatant containing the virus particles was collected.
The mixture is centrifuged at Xg for 45 minutes, and the obtained precipitate is suspended in a 10 mM phosphate buffer to prepare an attenuated cucumber mosaic virus.

It is preferable to inoculate the seedlings of the vegetables and fruits with the attenuated cucumber mosaic virus, because it is easy to infect the seedlings and the young seedlings of the seedlings.

The method of inoculating the attenuated cucumber mosaic virus is as follows. An arbitrary attenuated cucumber mosaic virus suspended in a buffer solution (0.1 M phosphate buffer near neutrality) is sprayed on the leaf surface of the seedling and attached. From above, a method in which a roller having an abrasive attached to the surface is pressed and rotated to inoculate (see JP-A-4-330005), or an attenuated cucumber mosaic virus solution in which the abrasive is suspended, Either a method of spraying and inoculating vegetable fruit seedlings with a spray gun at a constant pressure or a method of rubbing a suspended attenuated cucumber mosaic virus solution with a cotton swab, finger, glass stick or the like may be used.

One to two weeks after inoculation with the attenuated cucumber mosaic virus, infection of an appropriate number of strains is confirmed by ribonucleic acid (RNA) analysis, ELISA analysis and the like of the cucumber mosaic virus.

Cultivation management such as seedling raising, fertilization, and application of chemicals is performed according to a usual cultivation management method corresponding to each target crop.

According to the present invention, as described above, by inoculating a cucumber mosaic virus into a vegetable fruit seedling, the cultivation can be controlled as usual, and the vitamin content is hardly affected on the vegetable fruit sugar content and lycopene content. Only the C content can be increased.

Next, the satellite R selectively separated from the natural world
Attenuated cucumber mosaic virus containing NA (NDM2)
The method for preparing is described.

In 1988, from Kanto to Koshinetsu, 50
From a field of crops, 300 leaves of viral symptoms were collected from plants such as Solanaceae, Cucurbitaceae, Asteraceae, and Brassicaceae, and 10 times the amount of phosphate buffer (neutral 0.1 M phosphate buffer) was added and the mixture was ground. The solution was inoculated into tobacco (xanthin nc), and one week later, each ELISA test was carried out. As a result, 144 strains were confirmed to be infected with cucumber mosaic virus. did.

In order to select an attenuated cucumber mosaic virus from among them, infected leaves of 144 strains of tobacco were inoculated on 10 tomato lines each, and one week later, ribonucleic acid (RNA) analysis was performed. Cucumber mosaic virus was detected from 11 lines. Thereafter, a symptom investigation was performed on them, and strains in which necrosis and mosaic disease developed and strains having no infectivity were removed, and an attenuated cucumber mosaic virus having minimal viral symptoms and a large amount of virus multiplication was selected.

Since the selected attenuated cucumber mosaic virus was not genetically uniform and contained a plurality of virus particles having different virus symptoms, 500 strains of tomato were inoculated with this virus and strains having the same virus symptoms were obtained. Only, and only those strains in which the presence of satellite RNA is continued are collected to prepare a virus inoculum,
The operation of inoculating the tomato again and performing the same selection operation to prepare the virus inoculum was repeated 10 times, the viral symptoms were light, and the attenuated cucumber mosaic virus containing satellite RNA stably even when subcultured with the tomato ( NDM2)
A strain was created.

In addition to the above, attenuated cucumber mosaic virus is disclosed in JP-A-5-3789, "Attenuated cucumber mosaic virus using cloned satellite RNA" or JP-B-62-37956, "Satellite RNA".
Method for Creating Attenuated Virus of Cucumber Mosaic Virus Using E. coli ". Hereinafter, the present invention will be described more specifically with reference to examples.

[0029]

EXAMPLE 1 In order to compare the vitamin C content of fruits in the open-field cultivation of summer and autumn tomatoes, an abrasive (Kishida Chemical Co., Ltd.) was applied to 1.5-leaf seedlings of a raw tomato (variety: Momotaro T-93). Mixed with carborundum 600 mesh)
A prepared solution of attenuated cucumber mosaic virus (NDM2) at a concentration of 0 microgram / milliliter was applied to a mini painter.
(Nippon Wagner Spretic Co., Ltd.) and sprayed with carpet rollers from above the seedlings to inoculate them.

Next, 10 days later, 100 strains whose presence of satellite RNA was confirmed by ribonucleic acid (RNA) analysis were analyzed.
100 strains and 100 non-inoculated strains were planted in two places with different soil conditions in the paddy field conversion field and the field of the fifth year of tomato continuous cropping, respectively.

[0031] Subsequent spraying of drugs, hormonal treatment, attraction,
Cultivation management methods such as sprouts were performed in accordance with general cultivation standards. Harvesting was performed from late July to late September, and during that period, component analysis was performed on mature peaches for comparison. Table 1 shows the results of the paddy field conversion.

[0032] * The cultivation outline was sowing on April 8, inoculation on April 26, temporary planting on May 2, and regular planting on May 30. ** Analysis of fruits: August 26, September 2, September 8, 9
The test was performed four times on March 15, and the average value was shown. The number of analyzed fruits per one time was 15 each. *** Vitamin C was analyzed by indophenol titration.

Table 2 shows the results of another field in the fifth year of continuous tomato cultivation.

[0034] * The outline of cultivation is sowing on April 8, inoculation on April 26, temporary planting on April 30, and regular planting on May 23. ** Fruit analysis is July 28, August 11, August 26
Dates, September 2, September 8, and September 15 were performed six times, and the average values were shown. The number of analyzes per one time was 10 each. *** Vitamin C was analyzed by indophenol titration.

From the results of Tables 1 and 2, it can be seen that in the present invention, even if the field conditions are different, only the vitamin C content can be increased without affecting the sugar content, lycopene content and the like of the tomato fruit.

[0036]

[Example 2] Next, in summer cultivation of tomatoes in summer and summer, using a variety different from that of Example 1 (variety: Saturday) in order to compare the vitamin C content of the fruits in the 0.5 leaf leaf season Seedlings were inoculated with an attenuated cucumber mosaic virus (NDM2) preparation at a concentration of 50 micrograms / milliliter mixed with an abrasive (Carborundum 600 mesh) using a cotton swab.

Next, after 14 days, 50 strains whose presence of satellite RNA was confirmed by ribonucleic acid (RNA) analysis,
Then, 50 non-inoculated strains were planted in the field.

[0038] Subsequent drug application, hormonal treatment, attraction,
Cultivation management, such as sprouts, was made in accordance with general cultivation standards, and harvesting was performed from late July to mid-September. Table 3 shows the results.

[0039] * The cultivation outline is sowing on April 9, inoculation on April 27, temporary planting on April 30, and regular planting on June 14. ** Fruit analysis was performed three times on August 19, September 9, and September 15, and the average value was shown. The number of analyzed fruits per one time was 15 each. *** Vitamin C was analyzed by indophenol titration.

From the results shown in Table 3, in the present invention, even if the cultivation type and cultivar of tomato were changed, the vitamin C content was not affected without affecting the sugar content, lycopene content, etc. of the fruit, as in the results of Example 1. It can be seen that can be increased.

[0041]

[Example] Next, in a house suppression cultivation of tomato, in order to compare the vitamin C content of the fruit, a raw tomato (cultivar:
House Momotaro) on the seedlings of the 0.5-leaf leaf stage, use an abrasive (car
A preparation of attenuated cucumber mosaic virus (NDM2) at a concentration of 50 micrograms / milliliter mixed with Borundum 600 mesh) was inoculated with a cotton swab.

Next, after 14 days, 100 strains in which the presence of satellite RNA was confirmed by ribonucleic acid (RNA) analysis were determined.
100 strains and 100 uninoculated strains were planted in the field.

[0043] Subsequent spraying of drugs, hormonal treatment, attraction,
Cultivation management, such as sprouts, was made in accordance with general cultivation standards, and harvesting was conducted from late September to mid-December, during which period component analysis was performed on mature peaches for comparison. Table 4 shows the results.
Shown in

[0044] * The outline of cultivation is June 20 for sowing, July 2 for inoculation, July 5 for temporary planting, and August 20 for constant planting. ** Fruit analysis was performed twice on November 12 and December 6, and the average value was shown. The number of analyzed fruits per one time was 15 each. *** Vitamin C was analyzed by indophenol titration. *** Lb / a was measured with a color difference meter manufactured by Nippon Denshoku Industries Co., Ltd.

From the results shown in Table 4, it can be seen that the present invention increases the vitamin C content even in the house suppression cultivation of tomato.

[0046]

Example 4 In house suppression cultivation of crab tomato,
In order to compare the vitamin C content of fruits using two varieties of Momotaro and House Momotaro in the same field, attenuated at a concentration of 50 microgram / milliliter obtained by mixing an abrasive (Carborundum 600 mesh) with seedlings at the cotyledon stage. A cucumber mosaic virus (NDM2) preparation was inoculated with a cotton swab.

Next, 10 days later, 50 strains whose presence of satellite RNA was confirmed by ribonucleic acid (RNA) analysis,
Then, 50 non-inoculated strains were planted in the field.

[0048] Subsequent drug spraying, hormone treatment, attraction,
Cultivation management, such as sprouts, was made in accordance with general cultivation standards, and harvesting was conducted from late September to mid-December, during which period component analysis was performed on mature peaches for comparison. Table 5 shows the results for Momotaro.

[0049]

Table 6 shows the results of the other type, House Momotaro.

[0051] * The outline of cultivation is June 7 for sowing, June 21 for inoculation, June 26 for temporary planting, and July 20 for regular planting. ** Analysis of the fruit was performed twice on October 29 and November 10, and the average value was shown. The number of analyzed fruits per one time was 15 each. *** Vitamin C was analyzed by indophenol titration. *** Lb / a was measured with a color difference meter manufactured by Nippon Denshoku Industries Co., Ltd.

From the results shown in Tables 5 and 6, it can be seen that the present invention stably increases the vitamin C content even when comparing two varieties in tomato house suppression cultivation.

[0053]

[Effects of the present invention]

According to the present invention, as described above, the cucumber mosaic virus is inoculated to vegetable fruit seedlings, and cultivation management is carried out in a conventional manner, and vitamin C is hardly affected by the sugar content and lycopene content of the crop. The effect of increasing only the content is exhibited. In addition, it was difficult to breed varieties with high vitamin C content that are suitable for cultivation in breeding technology. C content can be improved. Furthermore, the cultivation method for improving the quality of the crop in the cultivation technique requires expensive equipment and materials, and it was not possible to improve only the vitamin C of the crop by such a method. Does not require any special cultivation equipment, materials, or cultivation methods, does not require any conventional cultivation methods, does not require any cost, and also has the effect of increasing the vitamin C content of the crop.

Continuation of the front page (56) References JP-A-61-177985 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) A01G 7/00 A01G 63/00

Claims (1)

(57) [Claims] 1. A satellite RNA selectively separated from nature.
A method for increasing the vitamin C content of vegetable fruits, comprising inoculating a vegetable fruit seedling with attenuated cucumber mosaic virus (NDM2) containing