JPH0297341A - Method for propagating plants and seeds - Google Patents

Abstract

PURPOSE:To simply contrive the improvement of the photosynthesis of a plant body and the multiplication thereof by treating the plant body cultured in a tissue-culturing device and having a photosynthesis ability with a quaternary ammonium salt (excluding choline and salts thereof) and culturing the treated plant body in the tissue-culturing device. CONSTITUTION:A plant body prepared by culturing a plant body (cell, tissue, etc.), in a tissue-culturing liquid or solid medium such as Murashige.Squuge culture medium (containing approximately 1-100g/l of sucrose, etc., as a carbon source) is subjected to the spreading, coating, etc., of an aqueous solution containing a quaternary ammonium salt (e.g., benzyltrimethylammonium hydroxide) preferably in an amount of 10-1000ppm and, if necessary, further containing an auxiliary such as ethanol and subsequently cultured under the irradiation of light of 2000-20000 luxes with aeration, etc., to carry out the multiplication of the objective plant body or seedling.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a method for propagating plants and seeds and seedlings, and particularly relates to a method for propagating plants and seeds that can increase their photosynthetic activity. be.

[Conventional technology]

Conventionally, it has been thought that the photosynthetic ability of plants undergoing tissue culture is considerably reduced, and therefore, in order to grow the plants undergoing tissue culture, they are used in the medium for the growth of the plants. It is common practice to add sugars such as sucrose, glucose, and fructose as carbon sources to achieve mixotrophic growth with a strong aspect of heterotrophic growth.

However, in the conventional propagation method as mentioned above,
Because a large amount of sugar is added to the medium, the medium itself is susceptible to contamination from external bacteria during the culture period, often causing problems such as plants being cultured to become sluggish. Therefore, in order to prevent this, culture environments such as culture vessels and culture chambers must be maintained in a substantially sterile state, and particularly strict care must be taken in handling them.

Moreover, in conventional methods, usually 3000
Since the culture was carried out without supplying carbon dioxide gas into the culture container while irradiating the light at a level of approximately 1000 mL, the carbon dioxide concentration in the culture container increased to several + ppm within a short period of time after the start of light irradiation.
It had declined to a certain extent. In this state, photosynthesis of the plant was suppressed, and the plant grew and multiplied heterotrophically using sugars added to the medium. However, in the process in which the plants grown as described above are then transplanted to a conditioned medium and acclimatized, the growth process is different from that in which they are grown in a sterile culture system using only sugars as a carbon source without photosynthesis. , suddenly faced with the need to carry out photosynthesis in a natural environment exposed to sterilization, this change in the external environment, the prosperity of the lifestyle,
This resulted in a large stress, which ultimately led to a decrease in the survival rate during the acclimatization process and a delay in growth.

On the other hand, attempts have also been made to supply carbon dioxide to the plants in the culture container by storing the culture container in a box that transmits light and controlling the carbon dioxide concentration within the box to 950 to 11,000 pp. However, this method supplies carbon dioxide gas,
Special equipment is required to control the amount, leading to an increase in the size and cost of the culture device.

[Problem to be solved by the invention]

An object of the present invention is to provide a novel method for propagating plants and seedlings, which solves the problems of the conventional techniques described above.

[Means for Solving the Problems] As a result of conducting research to achieve the above object, the present inventor unexpectedly found that, contrary to conventional technical common sense, plants in tissue culture were able to achieve sufficient autotrophic growth. However, it was discovered that this potential could not be fully demonstrated under conventional culture conditions based on heterotrophy.

In addition, the present inventor noticed that choline and quaternary ammonium salts other than choline salts have carbon dioxide absorption ability, and
We investigated whether it is possible to improve the photosynthetic ability of plants in tissue culture using grade ammonium salts, and confirmed that good results could be obtained.

Plants to be subjected to the plant and seedling propagation method of the present invention may be cells, tissues, organs, individuals, etc. that have photosynthetic horns, and are not particularly limited by the type of plant, tissue culture method, etc. .

For example, if the plant tissue culture method is the shoot tip culture method,
If the formed shoot or protocone has photosynthetic ability, a plant obtained by cutting the shoot or protocone into small pieces, or a plant obtained by germinating this cut plant in a germination medium, etc. can be mentioned.

In addition, if the plant tissue culture method is a callus culture method,
If the formed callus has photosynthetic ability,
Examples include this callus, a plant obtained by germinating the callus in a germination medium, and a plant obtained by propagating and dividing this germinated plant. Furthermore, even if fibers such as ceramic fibers are used as a medium material and plant tissue culture is performed consistently until just before acclimatization by exchanging the liquid medium, the plants targeted for tissue culture will develop their photosynthetic ability. The method for propagating a plant of the present invention can be applied from the stage when the plant has a plant.

Furthermore, the present invention can also be applied to plants that have been propagated by conventional methods known as plant tissue culture methods, such as in vitro cutting, adventitious bud formation, and asexual embryo formation, and have reached the point where they have photosynthetic ability. Can be done. It can also be applied as a method for propagating seeds and seedlings obtained by acclimatizing and rooting the plants.

Furthermore, the medium used in the present invention is usually composed of a basic medium and saccharides added thereto, and examples of the basic medium include Murashige-Skoog medium, White medium, Linsmeyer-Skoog medium, Niche and Niche medium, etc. Known plant tissue culture media and modified media thereof may be mentioned. Specifically, the inorganic components of the medium include nitrogen,
Examples include inorganic salts containing elements such as phosphorus, potassium, sodium, calcium, magnesium, sulfur, iron, manganese, zinc, boron, molybdenum, chlorine, iodine, and cobalt, specifically potassium nitrate, sodium nitrate, and ammonium nitrate. , ammonium chloride, potassium chloride, calcium chloride, potassium monohydrogen phosphate, diphosphoric acid
Potassium hydrogen, magnesium sulfate, magnesium chloride,
Examples include compounds such as sodium sulfate, ferrous sulfate, ferric sulfate, manganese sulfate, copper sulfate, sodium molybdate, molybdenum trioxide, potassium iodide, zinc sulfate, boric acid, and cobalt chloride.

Examples of carbon sources for the medium include carbohydrates such as sucrose and their derivatives, organic acids such as fatty acids, and primary alcohols such as ethanol.

Examples of plant hormones in the medium include naphthaleneacetic acid (NAA), indoleacetic acid (IAA), p-chlorophenoxyacetic acid, 2,4-dichlorophenoxyacetic acid (2,4-D), indolebutyric acid (IBA), and these. Examples include auxins such as derivatives of , and cylinics such as hensyl adenine (BA), kinetin, and zeatin.

Vitamins in the medium include biotin, thiamine (vitamin B1), pyridoxine (vitamin B6), pyridoxal, pyridoxamine, calcium pantothenate, ascorbic acid (vitamin C), inositol, nicotinic acid, nicotinamide, and riboflavin (vitamin B
2) etc. can be exemplified.

Examples of amino acids in the medium include glycine, alanine, glutamic acid, and lysine.

The culture medium of the present invention usually contains an inorganic component of about 0.1 μH to about 100 mM and a carbon source of about 1 g/p to about 1
00g/l, plant hormones from about 0.01μ to about 100μi, vitamins from about 0.1mg/ff to about 150mg/j2, and amino acids from 0 to about 1
00mg/l.

The medium that can be used in the present invention is a liquid medium or a solid medium containing usually 0.2 to 1% of agar, gellan gum, gelatin, or the like.

Further, as the quaternary ammonium salt used in the present invention, those of the following formula can be exemplified.

RR HO-(CHt2)--N"-R3X-R2-N"-R
'XR2R3 Specifically, hensyltrimethylammonium hydroxide (a), tetramethylammonium iodide (b)
, diethylhexylmethylammonium chloride (C)
Examples include: Moreover, these structural formulas are listed below.

(a) Benzyltrimethylammonium-hydroxide [C6H3-CIl□-N (CHt) 3 ] 00
w1b) Tetramethylammonium-iosito[N(C
H3)4 ] I (C) Diethylhexylmethylammonium-chloride CI□-Cl5 CH+-CHz-CHz-CHz-CHz-Cllz-
N''-CtL+ ClCH2-C11° The quaternary ammonium salt can be supplied by spraying or coating on the plant body during tissue culture.

Further, it can be used in an appropriate form such as an aqueous solution, powder, emulsion, granule or paste. In addition, the concentration is preferably 10 to 11000 pp as an aqueous solution.

In the present invention, as a method for treating plants that have acquired photosynthetic ability with quaternary ammonium salts, in addition to the above-mentioned methods of spraying and coating, choline or choline salts are usually used at an appropriate concentration of 10 to 110,007 pp. A method of culturing a plant body using a dissolved solution or solid medium is also included in the treatment method of the present invention.

Further, in order to enhance the carbon dioxide absorption capacity of the quaternary ammonium salt, various adjuvants commonly used in agricultural preparations can be used.

Examples of such adjuvants include solid carriers such as talc, bentonite, clay, kaolin, diatomaceous earth, white carbon, and vermiculite, ethanol, toluene, xylene, acetone, dioxane, cyclohexanone, methylnaphthalene, and dimethylformamide. Solvents, alkyl sulfates, alkyl sulfonates, polyoxyethylene glycol ethers, polyoxyethylene alkylaryl ethers, surfactants such as polyoxyethylene sorbicun monoalkyle-1, carboxymethyl cellulose, gum arabic, etc. Mention may be made of condensing agents.

In the present invention, when growing plants in a tissue culture vessel, examples include a method of aerating air into the container, or a method of bringing the plant into contact with outside air in an open system without actively aerating the container. In the case of aeration, examples include a method of supplying the medium to the gas phase section in the tissue culture vessel, or a method of bubbling the medium by aerating it to the leaflet section when the medium is liquid. Normally, air is sufficient for ventilation, but the effect will be further enhanced if air with a high carbon dioxide concentration is aerated.

In addition, lighting conditions vary depending on the type of plant, but basically they range from the light compensation point unique to each plant to the light saturation point, but from an economical point of view,
Good looks are preferred. Note that known methods can be applied to culture conditions such as light/dark cycle and culture temperature.

[For production]

According to the present invention, a quaternary ammonium salt (excluding choline and choline salts) that has excellent carbon dioxide absorption ability is treated with a method such as spraying or coating a plant body during tissue culture, thereby increasing the amount of water during the culture period. Above all, photosynthesis can be carried out efficiently in the culture vessel, enabling autotrophic growth and promoting the proliferation of these plants and seedlings.

In this case, the present invention does not use sugar as a carbon source, or
Even if it is used, the sugar concentration in the culture medium may be lower than the concentration normally used, for example, culture may be performed using a culture medium with a concentration range of 0 to 20 g/j2. Even when the sugar concentration is zero or low, the method of the present invention allows efficient photosynthesis and autotrophic growth, promoting the proliferation of these plants and seedlings, and eliminating the risk of exposing the culture system to contaminants. It also reduces

In addition, the plants obtained by the present invention are grown through photoautotrophic growth, and the transition from the propagation process to the acclimatization process is difficult. The survival rate during the acclimatization process and the subsequent growth condition are also improved.

Furthermore, in the method for propagating plants and seedlings of the present invention, there is no need to supply carbon dioxide gas into the culture container, so a device for supplying carbon dioxide gas and a control device for controlling the amount of supply thereof are provided. This eliminates the need for this, making it possible to downsize the culture device and reduce costs.

〔Effect of the invention〕

As described above, according to the present invention, carbon dioxide gas can be efficiently supplied to plants without using special equipment, so the photosynthetic ability of plants can be significantly improved. As a result, the growth of plants and seedlings is promoted. Moreover, since the stress that the plant body receives during acclimatization can be reduced, the survival rate during the acclimatization process and the subsequent growth condition can be improved. Moreover, the sugar concentration of the culture medium can be reduced if necessary.

Applicant: Mitsui Petrochemical Industries, Ltd. Agent Patent Attorney Yusuke Hiraki Same patent attorney Ishii page next

Claims (2)

[Claims] (1) When growing a plant that has been cultured in a tissue culture vessel and has the ability to photosynthesize, the plant is treated with quaternary ammonium salts (excluding choline and choline salts). A method for propagating a plant body, characterized by: (2) When growing a plant that has been cultured in a tissue culture vessel and has acquired photosynthetic ability in a tissue culture vessel, the plant is treated with quaternary ammonium salts (excluding choline and choline salts). A method for propagating seeds and seedlings, which is characterized in that the plants are acclimatized and rooted to obtain seedlings.