JP2000139251A - Production of eggplant seedling free from virus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing an eggplant seedling free from virus, enabling the elongation of a shoot, the establish of a growth point culture technology for eggplants and the production of the seedling by culturing the tissue of an eggplant containing a growth point and then transplanting the cultured eggplant tissue to the cut surface of the stem of a Solanaceae plant as a stock. SOLUTION: A Solanaceae plant (preferably tomato) as a stock is cultured preferably by a method for culturing the plant in a good air-permeable container under the irradiation of light for 12 hr/d at 25 deg.C. On the other hand, a tissue containing a growth point is picked from an eggplant and then cultured preferably by a method for culturing the tissue under the irradiation of light for 12 hr/day for one to two weeks at 25 deg.C. The stem of the Solanaceae plant as the stock is cut preferably at the lower hypocotyl portion, and a tissue containing the growth point is transplanted to the cut surface, followed by elongating the shoot from the growth point. The seedling is raised from the elongated shoot. A time for cutting the stock is preferably two to three weeks after the seed was sowed, a time when two to four main leaves are developed.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for producing eggplant seedlings by vegetative propagation. This method enables the production of virus-free eggplant seedlings.

[0002]

2. Description of the Related Art Conventionally, vegetative propagation plants have been propagated by cuttings, grafts, and the like. However, once the proliferating mother strain is infected with the virus, there is a problem that it is transmitted endogenously to proliferating progeny. Was. In recent years, with the development of tissue culture technology, virus-free growth and culture propagation by growing point culture have become possible, and the production technology of virus-free seedlings combined with cuttings and grafts has become practical for some species such as carnations and sweet potatoes. ing. Among these plant seedling production techniques, growth point culture is an essential technique for virus-free production, and is particularly important for plants in which virus infection greatly affects the quality and yield of the final product. Furthermore, growth point culture can achieve a rejuvenating effect. However, virus-free growth point culture may be very difficult depending on plant species.

[0003] In solanaceous plants, tomato has already been established with growth point culture and culture propagation techniques, and it is possible to produce vegetatively propagated seedlings. However, in eggplant (Solanum melongena L.), growth point culture is difficult. There were no reports on them. Also, culture growth was not established. Therefore, no technology for producing virus-free seedlings has been established.

[0004]

As described above, if the growth point cultivation technique in eggplant can be established, virus-free seedlings can be produced and the seedlings can be rejuvenated. An object of the present invention is to establish such a highly useful growth point culture technique in eggplant.

[0005]

Means for Solving the Problems The inventors of the present invention have conducted intensive studies to solve the above-mentioned problems, and as a result, after culturing a tissue including a growth point for a certain period, transplanting the tissue to a cut surface of a stock obtained by cutting the stem. By doing so, it was found that the shoot could be elongated, and the present invention was completed.

That is, the present invention is a method for producing eggplant seedlings, comprising the following steps (1) to (4). (1) Growing solanaceous plants as rootstocks (2) Extracting tissues containing growing points from eggplants and culturing them (3) Cutting stems of solanaceous plants as rootstocks, Transplant the tissue containing the growth point on the cut surface and extend the shoot from the growth point. (4) Grow a seedling from the elongated shoot

Further, the present invention is a method for producing eggplant seedlings, comprising the following steps (1) to (7), wherein the steps (5) and (6) are performed at least once. (1) Growing solanaceous plants as rootstocks (2) Extracting tissues containing growing points from eggplants and culturing them (3) Cutting stems of solanaceous plants as rootstocks, A tissue containing a growing point cultured on a cut surface is transplanted, and a shoot is extended from the growing point. (4) A grown shoot is grown and then divided. (5) A solanaceous plant using the divided shoot as a rootstock (6) Growing and grafting the grafted shoots (7) Growing seedlings from the split shoots

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention relates to (1) cultivation of a rootstock plant, (2) extraction and cultivation of a growing point, (3) shoot induction from a growing point, and (4) seedling growth from a shoot. , Respectively. Hereinafter, the present invention will be described for each step. (1) Breeding of rootstock plants The rootstock plants are not particularly limited as long as they belong to the Solanaceae family, but are preferably plants belonging to the genus Solanum or Lycopersicon, and the eggplants (Solanum) are preferred. melongena L.), Hiranas (Solanum integr)
ifolium Poir.) or tomato (Lycopersicon esculent)
um Mill.) is particularly preferred. Most preferred is tomato.

[0009] Plants to be rootstocks can be grown according to the so-called rootstock rootstock growing method. That is, seeds subjected to surface sterilization treatment are sown on a medium, germinated in a culture container, and grown under appropriate conditions. Here, the medium to be used may be any medium as long as it is generally used for plant tissue culture, for example, MS to which sucrose or agar is added.
A medium or the like can be used. Culture conditions are not particularly limited, but preferably are performed at 25 ° C. under illumination for 12 hours / day. The culture vessel used at this time is not particularly limited,
A container having good air permeability is preferable.

[0010] By cutting the stem of the plant thus grown at an appropriate time, a plant can be obtained as a rootstock. The time for cutting the stem is not particularly limited, but is preferably a time when 2 to 4 true leaves have been developed for 2 to 3 weeks after sowing. Also,
The cutting of the stem is preferably performed immediately before transplanting the tissue containing the growth point. The cutting position of the stem is not particularly limited, but is preferably the hypocotyl. However, in some cases, the epicotyl part may be more preferable.

(2) Extraction and cultivation of growth point The plant from which the growth point is extracted may be any plant belonging to eggplant. The method of extracting the growth point is not particularly limited, and can be performed, for example, as follows. From the growing plant, a stem containing a growing point near the tip of the branch is collected, and excess leaves are removed. This is lightly sprayed with 70% ethanol for surface sterilization. This sterilization treatment can be omitted. The growing point is removed under a sterile condition using a sharp scalpel under a stereoscopic microscope. Although the growth point of the eggplant is located at the base of the axillary bud, it is covered with hairy mushrooms, so it is difficult to see. The size of the tissue near the explanted growth point is suitably about 0.3 to 0.5 mm, but is not limited thereto. If it is confirmed that the target plant is not infected with the virus and the purpose is merely to rejuvenate, the size of the neighboring tissue including the explanted growth point can be further increased.

The excised growth points are placed on a medium and cultured. The medium used here may be a known medium for plant tissue culture such as MS medium or a medium to which a plant growth regulator is added. Culture conditions are not particularly limited, but preferably 25 ° C,
Perform under 12 hours / day lighting. The culture period is not particularly limited, but is preferably about 1 to 2 weeks after placing.

(3) Induction of Shoot from Growth Point As a result of the cultivation, the growth point slightly enlarges and starts to take on a green color. Depending on the medium used, calligraphy of nearby tissues may be observed along with the growth of the growth point and greening. At this time, the tissue bottom including the enlarged growth point is cut with a scalpel or the like, and the cut surface is attached to the cut surface of the stock in the test tube. When the growth point is not large and small, it is possible to put it directly on the cutting surface of the stock without cutting the bottom surface. In this way, the growing point elongates to form a shoot 1 to 3 weeks after transplanting the growing point to the rootstock. If shoots of the rootstock themselves are formed, they are aseptically removed.

(4) Raising seedlings from shoots Plants are induced from shoots according to a conventional method. For example, the obtained shoot is cut from the base, transplanted to a plant tissue culture medium and continued to be cultured, thereby promoting rooting and obtaining a plant body. After acclimation treatment of the obtained plant under certain conditions, normal cultivation in a greenhouse or the like becomes possible. In addition, it is also possible to transfer the shoots elongated in the rootstock without cutting the shoots and transplanting them to a new medium, together with the rootstocks.

After the shoots obtained by placing the growing point on a rootstock are cut and transplanted to a new medium to induce a rooting plant, the upper part of the shoot continues to grow. The grown rooting plant is capable of normal nodal propagation, but its growth rate is extremely slow. Then, the obtained shoots are cut at each node and grafted to a stock prepared in the same manner as described above, whereby the shoot propagation efficiency can be significantly improved. In the case of this graft, it is desirable to use an auxiliary device for fixing the scion and the rootstock so that they can easily survive. The shoots propagated in this way can be transplanted to a rooting medium to promote rooting, or acclimated in the state of grafted seedlings to obtain eggplant virus-free plants. It is also possible to graft the grown shoots to the rootstock again to achieve regrowth. At this time, if plants having excellent disease resistance and the like are used for the rootstock, the subsequent cultivation management becomes very efficient.

[0016]

EXAMPLES [Example 1] Raising rootstocks in test tubes of solanaceous plants Eggplant varieties "Taitaro" (Takii Seed Co., Ltd.), tomato varieties "Strong Yoneju" (Takii Seed Co., Ltd.) and "Shin Mate""
The seeds of (Sakata Seed Co., Ltd.) are treated with antiformin having an effective chlorine concentration of 1% for 15 minutes, sterilized on the surface, and then sterilized with water.
Washed twice. Two seeds of each seed were aseptically inoculated into a solid medium for plant tissue culture containing various nutrients, which was sterilized in an autoclave. The medium used here was an MS medium containing 3% sucrose, adjusted to pH 5.8, and added with 0.8% agar. Agripot (Kirin Brewery Co., Ltd.) was used as a culture vessel, and 50 ml of a medium was dispensed per vessel. After aseptic seeding, the cells were grown in a culture room under illumination at 25 ° C for 12 hours / day. Approximately three weeks after the start of cultivation, the plants reached a growth stage that could be used as rootstocks in test tubes. This plant is
Immediately before transplanting the micro-tissue piece containing the growth point shown in Example 2, the hypocotyl was cut with a sharp scalpel and used as a rootstock in a test tube.

Example 2 Extraction of Eggplant Growing Points In order to test for vegetative propagation, actively growing branches are selected from eggplant material mother plants cultivated in a greenhouse, and about 5 to 20 cm from the tip of the branches. Branches containing growing points of length were collected. Adjust the length to 3-5cm, excluding extra leaves, so that the growing point can be easily removed.
70% ethanol was sprayed. The following operations were performed aseptically in a clean bench. Under a stereoscopic microscope, a micro-tissue piece including a growth point having a size of about 0.2 to 0.5 mm was excised using a sharp scalpel. The growth point was located at the base of the axillary bud, and the surrounding area was covered with a large amount of mushrooms, making it difficult to identify the growth points. Therefore, the growth points were removed after removing the nearby mushrooms.

Example 3 Induction of Shoot from Growth Point A micro-tissue piece containing a growth point extracted in Example 2 was placed on a medium and cultured for a certain period. The medium used is as shown in Table 1. The culture was performed in a culture room at 25 ° C. for 12 hours / day under illumination.

[0019]

[Table 1]

After 1-2 weeks from the implantation in the medium, the growth point
It became green and began to grow slightly. When a plant growth regulator was added, depending on the culture medium, callus formation of the nearby tissue was observed along with hypertrophy of the growth point and greening. When the growth point became clearly visible to the naked eye, transplantation to a rootstock in a test tube was performed. The enlarged tissue portion on the bottom surface of the growth point is cut with a scalpel, and the cut surface is placed on the floor so that the cut surfaces of the rootstock in the test tube prepared by the method described in Example 1 are in contact with each other, thereby growing the growth point. Was transplanted. When the growth point was too small to cut the bottom, it was placed on the cutting surface of the stock in a test tube without cutting. In the case of severe hypertrophy or callus, the excess was cut off and transplanted to a rootstock in a test tube.

Two weeks after transplantation of the growing point, the shoot elongated. When tomato was used as a rootstock in a test tube, shoots grew particularly vigorously. When the hypocotyl above the cotyledon was cut, the shoot extended from the growth point of the tomato itself from the base of the cotyledon and was removed, but when cut at the lower hypocotyl below the cotyledon, the growth point of the tomato itself was removed. Did not extend and did not need to be removed. When the eggplant was used as a rootstock in a test tube, it was necessary to remove all the adventitious buds because the rootstock itself appeared adventitiously from the cut surface of the hypocotyl.

Example 4 Induction of Plant Seedlings from Shoots When the shoots obtained by transplanting the growing points onto the cut surface of the hypocotyl of the growing stock are elongated to a length of 2 to 3 cm, the shoot base is The plant was further cut, transplanted to a rooting medium, and continued to be cultured to promote rooting to obtain a plant. The obtained plants were acclimated in a greenhouse. Wash the agar of the medium attached to the roots, transfer to a pot filled with culture soil, place in a mist room for the first week under light-shielded conditions, use only light-shielded conditions for the second week, and use normal Greenhouse cultivation. It was also possible to transfer the shoots elongated in the rootstock in a test tube to the rootstock without acclimation without cutting the shoots and transplanting them to the rooting medium. The acclimatization condition was performed by the method described above.

Example 5 Confirmation of Virus Freeness of Plant Seedlings Obtained by Growth Point Culture The 64 plants obtained in Example 4 were tested for virus free by ELISA. TMV-L, TMV-OM, TMV-P,
As a result of testing for 5 viruses, CMV-O and BBWV, 56 individuals showed a negative response to all the viruses (virus-free ratio: 87.5%).

Example 6 Propagation of Shoots in Test Tubes Shoots obtained by transplanting growth points to rootstocks in test tubes of tomatoes are continuously cultured, and split growth of shoots 4 weeks after transplantation Grew to the point where possible. The shoot was cut and divided, and grafted onto a rootstock in a tomato test tube again. In this case, the cut surface of the chute and the cut surface of the stock are cut at an angle of approximately 45 degrees to the horizontal so that the cut surfaces are adhered to each other. Fixing aids (Super Wiz, Super Idol (Nasnix Corp.) ). By this method, it was possible to grow at more than twice the growth rate in one month. Eggplant seedlings were obtained from the grown shoots in the same manner as in Example 4.

[0025]

Industrial Applicability According to the present invention, efficient eggplant growth point cultivation and culture expansion can be achieved. This enables virus-free eggplant plant seedling production.

Claims (6)

[Claims] 1. A method for producing eggplant seedlings, comprising the following steps (1) to (4): (1) Growing solanaceous plants as rootstocks (2) Extracting tissues containing growing points from eggplants and culturing them (3) Cutting stems of solanaceous plants as rootstocks, Transplant the tissue containing the growth point on the cut surface and extend the shoot from the growth point. (4) Grow a seedling from the elongated shoot 2. The method for producing eggplant seedlings according to claim 1, wherein the culture period of the tissue containing the growing point is 1 to 2 weeks. 3. The method for producing eggplant seedlings according to claim 1, wherein the solanaceous plant used as a rootstock belongs to the genus Solanum or Lycopersicon. 4. The method for producing eggplant seedlings according to claim 1, wherein the solanaceous plant used as the rootstock is eggplant, hylanus, or tomato. 5. The method for producing eggplant seedlings according to claim 1, wherein after growing shoots elongated from the growth point, the shoots are divided and seedlings are grown from each shoot. 6. The method includes the following steps (1) to (7):
A method for producing eggplant seedlings, wherein the steps (5) and (6) are performed once or more. (1) Growing solanaceous plants as rootstocks (2) Extracting tissues containing growing points from eggplants and culturing them (3) Cutting stems of solanaceous plants as rootstocks, A tissue containing a growing point cultured on a cut surface is transplanted, and a shoot is extended from the growing point. (4) A grown shoot is grown and then divided. (5) A solanaceous plant using the divided shoot as a rootstock (6) Growing and grafting the grafted shoots (7) Growing seedlings from the split shoots