JP2720049B2 - Cultured plant growth promoter - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to an agent for efficiently and rapidly growing a tissue of a plant cultured by an in vitro sterile culture method.

[Related Art] (Background) In recent years, as a method of mass-producing vegetative bodies of the best variety,
In vitro plant tissue aseptic culture techniques have been widely adopted. 2. Description of the Related Art A technique for aseptically removing and culturing a plant tissue to produce an embryoid body, a protocorm, a rhizome, or a callus has attracted attention. For example, at present, flower gardening crops, mainly orchids, woody plants such as fruit trees, and vegetables, the target plant tissue is aseptically extracted and cultured by applying tissue culture technology, and these are grown. Later, mass production of seedlings through a step of promoting plant regeneration has been attempted. However, current culture techniques believe that the addition of plant hormones and other growth regulators to the culture medium is essential for most of these cultures as a means of rapidly regenerating plants from these tissues. .

(Problems of the prior art) However, when the culture is continued in a medium containing plant hormones for a long period of time, mutation of the plant is easily induced, and during the culture, the organ formation ability of the tissue is reduced and the plant is not regenerated.

 Important traits of the variety are missing.

And the like, which are frequently caused by the addition of the plant hormone. This is one of the serious problems faced by the industry, as these mutations can severely impede the economic production of seedlings.

[Problems to be Solved by the Invention] Accordingly, the problem to be solved by the present invention is to minimize the application period of a growth regulator as a mutagen at the growth stage of a protocorm-like body of an orchid, such as a lysosome. Another object of the present invention is to provide a technique for producing a plant rapidly and efficiently by suppressing a decrease in organ formation ability such as regeneration of a plant and mutagenesis.

[Means for Solving the Problems] (Progress) Accordingly, the present inventor has set out the following in order to solve the above-mentioned problems.
As a result of intensive studies on a method of rapidly promoting plant regeneration without using known plant hormones in the process of tissue regeneration, various orchids in the growth stage of protocorm-like bodies and lysosomes in orchids in culture were found. By immersing the tissue in an aqueous solution of silver nitrate, it was found that plant regeneration was performed quickly and in synchrony. The present invention is based on the above findings.

(Summary) That is, the plant culture growth promoter according to the present invention contains silver nitrate as an active ingredient based on the above findings. To add,
The present invention promotes shoot and root differentiation promptly and efficiently by treating these tissues at the growth stage of protocorm-like bodies or lysosomes of orchids,
The gist of the present invention is an agent mainly composed of a water-soluble silver salt to enable the production of a large amount of seedlings that are genetically uniform within a short period of time.

 Hereinafter, the main items related to the invention will be described.

(Plant tissue) The protocorm-like body or lysosome which is the object of the agent of the present invention is a plant body containing a tissue obtained by culturing various organs (for example, gingiva, vase, etc.) of a floral horticultural crop centering on orchids. Means

Silver nitrate, which is an active ingredient of the present invention, is easy to handle in that it is easily soluble in water, easily available, and excellent in stability against heat. In addition to silver nitrate, silver salts such as silver sulfate, oxalic acid and tartaric acid can be sufficiently used.

(Medium) Examples of the above-mentioned medium include Murashige & Skoog medium, Hyponex medium, Nadson C medium, niche medium, Lindsmeyer-Squeeze medium, White medium, Virgin and bent medium, B-5 medium, Gumborg medium and the like. Is used.

As the carbon source, sucrose, glucose, fructose and the like are used alone or as a mixture of two or more.
The carbon source may be present in the medium at a concentration of about 1 to 50 g /, preferably about 5 to 30 g /.

It is appropriate to adjust the pH of the medium to 4.0 to 7.0, especially
A range from 4.8 to 5.8 is preferred.

These culture media may be either liquid culture media or fixed culture media, and the cultivation temperature is preferably in the range of 15 to 30 ° C, particularly preferably 18 to 25 ° C. And, more preferably, the culturing is performed under a constant temperature condition under light irradiation of about 500 to 2,000 lux.

Depending on the type, coconut milk (about 1-20
%), Banana juice (about 1 to 10% by weight), tomato juice (about 1 to 10% by weight), potato juice (about 1 to 10% by weight), etc., alone or as a mixture. In some cases. Further, if necessary, zeatin, kinetin, benzylaminopurine, 2i
P or 4PU or other natural or synthetic cytokinins or 2,4-
The use of natural or synthetic plant hormones such as D, auxin, α-indoleacetic acid, α-indolebutyric acid, α-naphthaleneacetic acid, abscisic acid, gibberellin may be preferred.

(Culture method) In tissue culture using the agent of the present invention, as before, orchid tissue that has been aseptically extracted is placed on a medium containing inorganic and organic salts and sucrose, and then activated. Is transplanted into a medium, and protocomb-like bodies or lysosomes are propagated.

Next, these grown tissues are immersed in a sterilized silver salt aqueous solution, washed with sterilized water, transplanted into a medium, and cultured.
These tissues are differentiated into complete seedlings having foliage and roots. In this case, the concentration of silver nitrate or other suitable water-soluble silver salt varies depending on culture conditions and the like, but is usually 0.01% in the immersion liquid.
-100 ppm, more preferably 0.1-10 ppm.

[Effect] The agent of the present invention promotes the differentiation of shoots and roots by rapidly and efficiently removing these hormones at the growth stage of protocorm-like bodies and lysosomes during the culture of orchids in place of plant hormones. It acts to differentiate large numbers of seedlings that are genetically uniform without mutation.

The nature of the above action is currently under investigation, but for the time being, silver salts have some effect on the metabolic system of methionine in plant tissues, for example, S-adenosylmethionine (SAM).
By inhibiting the production of ACC synthase from E. coli to 1-aminopropane-1-carboxylic acid.

[Examples] Production examples and usage examples will be described below, and the effects of the invention will be described in comparison with comparative examples. However, the examples are, of course, illustrative and not intended to limit the inventive idea.

Production Example (Preparation Example of Inventive Agent Aqueous Solution) Reagent grade silver nitrate was added to distilled water at concentrations of 0.1, 1.0 and 1.0, respectively.
Prepare 500 ml of each aqueous solution dissolved to 10 ppm, adjust the pH of each aqueous solution to 5.5 with hydrochloric acid, dispense 100 ml at a time into a 200 ml wide-necked Erlenmeyer flask, cover with an aluminum cap, and then pressurize in an autoclave. The solution was sterilized by applying pressure and steam at 1.2 kg / cm ² and 121 ° C. for 15 minutes and allowed to cool to prepare a silver nitrate solution.

Usage Example 1 Dendrobium shoots (3 to 6 cm) were collected and sterilized on the surface with a 2% aqueous sodium hypochlorite solution (or Wilson's solution), washed three times with sterile water, and the shoot shoots and axillary shoots were aseptically sterilized. Cut into coconut milk (150g /)
On a Murashige & Skoog agar medium containing, and cultured at a constant temperature of 25 ° C.
LB) formation was induced and propagated.

This PLB was divided and immersed in sterile silver nitrate aqueous solutions of the above concentrations for 0.5, 2, 6, 12 and 24 hours, respectively, and then placed on Murashige and Skoog medium containing no plant growth hormone. The culture was performed at a constant temperature of 25 ° C. for 2 months. As a control, in addition to the silver nitrate-treated sample, PLB not subjected to silver nitrate treatment was placed on Murashige and Skoog medium containing 10 ppm of 6-benzylaminopurine and cultured similarly.

In the silver nitrate-treated group, compared to the hormone-treated group (control group), good results were obtained in all of the shoot and root formation rates and elongation. In particular, silver nitrate concentrations of 1 and 10 ppm, immersion time of 2 In the 12-hour treatment, shoot and root formation rate: 50-70% increase, shoot and root elongation: 30-40% increase, and mutation occurrence rate of important traits: about 1 / The result was reduced to 3.

Usage Example 2 Shunran sprout (20-30 mm in length) was collected, 3 to 4 of its outer leaves were removed, the back surface was sterilized with Wilson's solution for 15 minutes, and then washed three times with sterile water. Buds and axillary buds were aseptically cut out, placed on Murashige and Skoog medium, and cultured at 25 ° C. under constant temperature conditions to form lysosomes and proliferate.

The tip of the proliferated lysosome was cut off to a length of about 5 mm, immersed in the same aqueous solution of silver nitrate as in the example, and cultured for 2 months at a constant temperature of 25 ° C.

From the lysosome placed in the medium containing 6-benzylaminopurine (1 ppm), shoot formation was observed, but almost no root formation was observed, and the plant did not differentiate into plants.

On the other hand, in the silver nitrate treatment group, the same good results as in the previous example were obtained. In particular, in the treatment group with the concentration of 1 to 10 ppm and the immersion time of 2 to 6 hours, the formation rate of shoots and roots was higher than in the control group. : 2-3 fold increase, shoot and root elongation: 1.5 to 2 fold increase, mutation rate of important trait: reduced to about 1/5, good results were obtained.

[Effects of the Invention] As described above, the present invention provides rapid and efficient differentiation of shoots and roots by treating these orchids during the growth stage of protocorm-like bodies or lysosomes during tissue culture. And to provide a practical means for enabling the production of a large number of seedlings that are genetically uniform in a short period of time, thereby contributing to the rationalization and development of the tissue culture industry.

 ──────────────────────────────────────────────────続 き Continued on the front page (56) References Laszlo Purnhauser, Plant Cell Reports, Vol. 6, p. 1-4 (1987) p. Gavinlertvatan a, J.A. Amer. Hort. Sci. Vol. 105, No. 3, p. 304-307 (1980)

Claims (1)

(57) [Claims] 1. An agent for promoting the growth of a cultured plant which contains silver nitrate as an active ingredient and promotes the formation of roots and shoots from a protocorm-like body or lysosome of orchids to differentiate them into seedlings.