JPS61242522A - Method for converting plant tissue to seedling - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a method for culturing plant tissue into seedlings and producing seedlings that can be transplanted into soil or the like and cultivated.

Traditionally, seedling culture is used to obtain young seedlings by redifferentiating plant tissues such as callus and meclones into root, stem, and leaf tissues.
It is carried out exclusively using an agar medium. Other methods have been proposed, including a method in which the paper is suspended in a liquid medium and cultured to form seedlings, and a paper wick method in which the edges of paper are immersed in a liquid medium and cultured to form seedlings on the paper.

■I'm trying to do it, I@ By the way, in the seedling culture of plant tissues, generally,
The optimal medium composition differs at each stage of differentiation and growth. In particular, it is necessary to change the type and concentration of plant hormones between the differentiated growth of a plant tissue into stems and leaves and the differentiated growth into roots.

Therefore, in the conventional method using an agar medium described above, it is necessary to transplant a plant tissue undergoing single differentiation growth from an agar medium with the initial composition to an agar medium with the next composition, which requires detailed manual work that requires skill. However, it has been extremely difficult to automate and save labor in mass culture.

Furthermore, when transplanting and cultivating seedlings after seedling culture into soil, etc., if the agar medium attached to the roots is not completely removed, bacteria will proliferate, and fine roots will easily break during the removal operation, causing roots to take root. There were problems such as the occurrence of many defective products.

On the other hand, in the seedling cultivation method using a liquid medium and the paper wick method, there is a problem that the grass leaves and roots often grow bent, which significantly reduces the commercial value of the seedlings.

This invention was made in order to solve the above problem, and the purpose of this invention is to produce seedlings of plant tissues that can omit detailed manual work that requires skill and provide seedlings with high commercial value. The purpose is to propose a culture method.

4. Means for Solving the Problems As a result of intensive research to solve the above-mentioned problems, the present inventor surprisingly found that Avicel, waste paper powder,
Even in a medium in which a powdery or flaky carrier such as perlite, vermiculite, rock wool, akadama soil, black soil, etc. is immersed in a culture solution, the plant tissues grow differentiated, and the above-mentioned culture changes according to the differentiated growth of the plant tissues. It has been discovered that by changing the composition of the liquid, seedling cultivation can be continued as it is, and seedlings with straight stems, leaves and roots and high commercial value can be obtained.

The present invention has been made based on this knowledge, and is a method for producing plant tissue by growing the plant tissue into seedlings in a medium prepared by impregnating a water-insoluble powder or flake carrier with a culture solution. This is a seedling method.

The present invention can be applied to plant tissues in general, which are tissue pieces or organs of various plants, which have not yet been completed into plants fully equipped with root and foliage tissues obtained by tissue culture. Calli grown in
The present invention is suitable for use in somatic embryos, which correspond to embryos in natural seeds derived from calli, and protocorm-like bodies, which correspond to protocorms in plants such as orchids.

There is no particular problem with a carrier made of water-insoluble powder or flakes, as long as the roots can grow in the carrier and the roots can support the differentiated and grown seedlings themselves. As this carrier, for example, soil, mineral particles, vegetable fiber powder, etc. can be used. The particle size of these particles is appropriately selected depending on the type of plant, but from the viewpoint of normal root elongation, it is preferably 2 mm or less, and from the viewpoint of easy replacement of the culture solution, it is preferably 5 μ or more. ·stomach.

Avicel, waste paper powder, rock wool, perlite, vermiculite, red clay, black clay, and the like are suitable.

This carrier may be used by being spread in a culture container in the form of powder or pieces, or may be processed into a sheet and spread. In this case, the thickness of the carrier needs to be such that the elongated roots can support the seedlings themselves. Although this thickness cannot be determined because it varies depending on the plant species, it is preferably at least 10 mm.

For seedling cultivation, the carrier is placed in a culture container, a culture solution of a predetermined composition is added, and the carrier is immersed. In this case, as long as the carrier has hygroscopic properties and absorbs the culture solution by capillary action, it goes without saying that it is sufficient to immerse a portion of the carrier in the culture solution. In addition, if it is necessary to carry out seedling culture aseptically, it is preferable to sterilize the culture container, carrier, and culture solution in advance or after adding the culture solution as described above.

The composition of the culture solution varies depending on the plant species, so it cannot be determined generally, but examples include Murashige-Skoog medium, Gamborg B5 medium, and White medium ("Plant Cell Tissue Culture" Drift, edited by Komamine, 1983 Science and Engineering) (Refer to p. 390, published by the company) In addition, it is possible to use various hydroponic culture solutions to which plant hormones such as auxins, cytokinins, and gibberellins, and sugars such as sucrose and glucose are added in desired amounts as necessary. .

Next, a part of the plant tissue is transplanted to a medium in which this carrier is soaked with a culture solution, and cultured to form seedlings. By the way, since the carrier is water-insoluble and in the form of powder or strips, if a drainage port is provided at the bottom of the culture container, it is extremely easy to draw out only the culture solution by opening the drainage port. Therefore, at each desired stage of seedling culture, part or all of the culture solution is extracted from the lower drainage port of the culture container, and a newly prepared predetermined culture solution is added. Thereby, seedling cultivation can be continued without re-transplanting the plant tissue. Of course, the extraction and addition of the culture solution can also be performed continuously, and the extracted culture solution can be circulated and reused by adjusting the solution composition.

The seedlings after seedling cultivation are taken out from the culture container, transplanted into soil, etc., and used for cultivation.

In this case, it is possible to take out only the young seedlings and transplant them, but it is also possible to transplant them together with the above-mentioned carrier into soil etc. and culture them. Furthermore, if the above-mentioned carrier is designed as a seedling growing mat for a transplanting machine such as a rice transplanter, the seedlings can be transplanted directly into soil or the like using the transplanting machine.

Of course, it is also possible to directly perform hydroponic cultivation by replacing the culture solution with a hydroponic culture solution without transplanting the seedlings.

Examples (Example 1) As a plant tissue, Happolopappus gr
The KH-1 (2n=4) shoot primordium of S. acillis was transplanted and the 1st month old strain was used.

The seedling medium used as a carrier was Avicel for column chromatography (particle size 100-120μ, manufactured by Asahi Kasei Industries) 10
Using g, this carrier was placed in a wire mesh tray with an opening diameter of 0.5 mm (the thickness of the carrier was 10 mm), and the tray was placed in a circular glass container with an inner diameter of 6 cmφ. After impregnating 24ml of modified culture solution (hereinafter abbreviated as 1/2MS7), it was sterilized in an autoclave at 120°C for 15 minutes and used.

Next, the above-mentioned shoot primordium aggregate was divided into 3-5 mm diameter pieces and 10 pieces were implanted in each medium, about half buried in the carrier. I prepared 10 of these.

For seedling cultivation, seal the gap between the lid of the glass container of the above-mentioned culture medium and the container with vinyl tape (do not seal only about 10 mm width) to prevent the medium from drying, and heat at 20℃ and 4000 lux. The test was carried out by standing under the following conditions (16 hours light period, 8 hours dark period).

As a result, two weeks after seedling cultivation, the development of minute shoots was observed from 81 shoot primordium agglomerates, and after one month, 96
From each shoot primordium cluster, 1 to 5 shoots of approximately 1 cm in length were produced, and 4 shoot primordium clusters died.

In the first month of seedling cultivation, the liquid portion of the seedling cultivation medium was replaced. The composition of this new culture solution is the same as the 1/2 MS7 culture solution shown in Table 1 except for 6-benzylaminopurine, the concentrations of naphthalene acetic acid and sucrose are the same, and the other components are doubled in concentration (hereinafter referred to as MS2). ), 120
It was used after being sterilized in an autoclave at a temperature of .degree. C. for 15 minutes.

For liquid replacement, take out the seedling medium with the tray and place it in MS
Completely submerge the carrier part in 500 ml of the second solution, and soak for about 3 hours, taking care not to topple the stems. Next, remove the medium tray from the solution, shake it up and down 2-3 times, drain it, and add it again to 500 ml of fresh MS2 replacement solution.
After soaking for about 3 hours in the same manner as described above, the water was drained and placed in a glass container again, and seedling culture was continued under the same conditions as above.

As a result, vigorous rooting was observed in the vicinity of the base of the shoots within one week after the liquid replacement, and within one month, the roots were elongated in the seedling medium, and seedlings with shoots and leaves measuring 5 to 6 cm were formed. , 1 to 5 shoots per shoot primordium aggregate (this was taken as one plant),
A total of 96 stocks were obtained.

Two months after the start of seedling culture, the seedlings were taken out from the glass container along with the tray, and the medium was soaked in running water for about 6 hours, then the medium was divided into individual seedlings and transplanted directly into vermiculite pots. After about two weeks of acclimatization and cultivation in a greenhouse, all 96 plants grew firmly and became nucleating bodies.

(Left below) (Example 2) A culture medium using carriers prepared according to the method described in Table 2, and adding the carrier and culture solution with the same composition as in Example 1 at the ratio shown in the right column of Table 2. Two of each were prepared, and the shoot primordia of Happolopappus gracilli S used in Example 1 were planted and cultured to seedlings in the same manner as in Example 1, and the resulting seedlings were transplanted into vermulite pots. I did this.

As a result, the results shown in Table 3 were obtained.

Table 2 (blank below) Tsukuda Reimori 1 (Example 3) As a plant tissue, Vasabia japanic
20 thin pieces obtained by sub-planting Dharma type callus and dividing the 3rd month callus into pieces of about 5 mm each were used.

For the seedling medium, add 1 part coconut milk to the basic medium shown in Table 4.
It was prepared in the same manner as described in Example 1, except that Avicel was impregnated with a culture solution containing 50 g/l of kinetin and 1.0 mg/l of kinetin.

The method for implanting the callus pieces was the same as in Example 1.

Seedling cultivation was carried out at a temperature of 10°C under conditions of 4000 lux (16 hours light period, 8 hours dark period) without replacing the liquid during the cultivation.

As a result, after about 4 months, adventitious bud formation was observed in 8 callus strips, and at the same time, rooting was observed in the lower part of the callus strips. Twelve callus pieces were withered and dead. By further culturing for about one month, the roots elongate into the seedling medium, and the seedlings with stems and leaves of 3 to 4 cm in length are produced, with 1 to 3 seedlings per callus strip (this is counted as one plant). I got a total of 8 shares.

In the 5th month of seedling cultivation, they were transplanted to vermiculite and cultivated using the same method as described in Example 1. As a result, 7 plants took root and became nucleating bodies, and 1 plant died.

Table 4 (Example 4) As the plant tissue, somatic embryos of Dacus carota Kuroda five-sun line 10 days after the start of induction were used.

The density of somatic embryos in the induction medium was 317 cells/m 1. Seedlings were grown in the same manner as described in Example 1, except that the White basic medium shown in Table 5 was used as the culture medium. The medium for this purpose was prepared.

The somatic embryos were implanted by dropping the above-mentioned somatic embryo fluid onto the entire surface of the medium at a rate of 1 ml per seedling medium.

Seedling cultivation was carried out at a temperature of 26°C, and other conditions were the same as those described in Example 3.

As a result, after 2 weeks of seedling culture, 2-5 sprouts of 20 m in length were observed per medium, 28 in total, in 7 seedling media, and after 1 month, 6-7 cm sprouts with leaves were observed.
It became a young seedling.

After one month of seedling culture, the seedlings were transplanted to the field and cultivated. As a result, all of the above seedlings took root and became nucleating bodies. Because it is carried out using a medium made of an insoluble powder-like or flaky carrier impregnated with a culture solution,
It produces the following special effects.

■There is no need to transplant the plant tissue to another medium at each stage of differentiation and growth, and detailed manual work that requires skill is no longer necessary.

(2) The composition of the culture solution can be arbitrarily adjusted at each stage of seedling cultivation, and root growth can be maintained in good condition by adding oxygen to the culture solution.

■Especially in the later stages of seedling culture, it is easy to use measures such as blowing air into the seedling culture vessel to acclimate the seedlings to dry conditions, and gradually increase the light intensity to acclimatize them to strong light conditions. Therefore, when seedlings are transplanted into soil or the like, it is possible to obtain strong seedlings that can grow sufficiently even if the steps of acclimatizing them to dryness and strong light are omitted.

■Since the roots are spread in the carrier, it is possible to obtain seedlings with high commercial value with straight stems, leaves and roots.

■When transplanting the produced seedlings to soil, etc., automation is possible, and there is no possibility of poor root establishment due to proliferation of various bacteria or damage to hairy roots.

■Seedlings can be directly used for hydroponic cultivation without transplanting, making it possible to have an integrated process from seedling culture to final plant cultivation.

Claims (1)

[Claims] 1. A method for growing plant tissue into seedlings, which comprises growing plant tissue into seedlings in a medium prepared by impregnating a water-insoluble powder-like or strip-like carrier with a culture solution.