JPH06217660A - Method for culturing seedling of tissue culture of plant and culture device therefor - Google Patents

Abstract

PURPOSE:To inexpensively obtain a strong, normal seedling of tissue culture of plant in reduced days of growth of seedling by supplying a carbon dioxide gas from a time of starting reduction in a reduced amount of medium concentration in a culture container, lowering humidity in the container, gradually increasing light intensity. CONSTITUTION:Plant materials 10 such as petiole pieces of leagous begonia as plant tissue are laid on substrates 9 shaped into cells put in culture containers 8, the substrates 9 are packed with vermiculite containing a medium and the plant materials are cultured in a culture chamber 1 at 40mumol/m<2>/second light intensity from a light source 2 such as fluorescent light of day color light at 25 deg.C at 70% humidity for 24 hours. At a point of time when the reduced amount of a saccharide concentration in the medium in the culture containers 8 starts to decrease, a carbon dioxide gas is supplied from a carbon dioxide feed source 6 through a solenoid valve 4 to the culture containers 8 and the humidity in the culture containers 8 is lowered by adjusting a humidifier 7. The culture is further carried out by gradually increasing the light intensity of the light source 2 so that the number of days for growing seedlings is reduced and strong, normal seedlings of tissue culture of plant are obtained at a reduced manufacturing cost.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method and apparatus for cultivating plant tissue culture seedlings. More specifically, the present invention relates to a method for cultivating seedlings using a plant tissue culturing technique, and a culturing device and a culturing container that can be used for mass production of seedlings used therein.

[0002]

2. Description of the Related Art Conventionally, seedling production of plants has been carried out by using seeds, bulbs or cuttings. These methods are still important seed production techniques for many plants today. However, with these methods, during repeated production,
Problems such as growth inhibition and death due to illness or pests have been pointed out, and it cannot be said to be a stable method.

Therefore, a technique for regenerating plants from cells has been developed by biotechnology and has come to be used for breed improvement and seedling production. As an example of such a method, the apical bud tissue is removed under aseptic condition, placed in a medium such as a test tube, cultured, and regenerated into a plant body. A large number of axillary buds are germinated in the regenerated plant, divided into several pieces, and these steps are repeated to multiply clone seedlings. It is generally practiced to make seedlings through the process of acclimatizing it to the outdoor environment for about one month.

[0004] In such a conventional seedling production method using plant tissue culture, individual axillary bud plants are cut off one by one and repeated, so a large number of seedlings can be obtained unless the number of repetitions is large. It is not possible to do so, the work for it is troublesome, and the proliferation efficiency is not high. Furthermore, since each strain is placed in a test tube or the like, it is economically expensive, and it takes a long time to culture the seedlings, and it is difficult to say that strong seedlings can be produced because of an empirical method for acclimatization.

Further, in the conventional culture device used for plant tissue culture and the like, one device can set only one pattern of temperature, humidity, light intensity, carbon dioxide concentration and the like. In terms of temperature only, there are some devices that divide the room into multiple parts with one device and set and control different temperatures, but it is possible to control light intensity, humidity, and carbon dioxide concentration at the same time. There is nothing.

Recently, in plant tissue culture technology, a technology has been developed for controlling gas concentration, temperature, humidity, nutrient solution, etc. in a culture container by individually piping to promote the growth of cultured plants and to make seedlings healthy. Has been done. However, this method is not practical because it requires a sterilization process to keep the inside of the culture container in a sterile state and requires time and effort such as piping. Such conventional culture devices have the above problems, and when conducting seedling production research or actual production using plant tissue culture technology, in the process of acclimation to culturing, the plant Different environmental controls are required depending on the type and culture / acclimation pattern, which requires many devices and is not efficient. In addition, controlling the environment for each culture container increases the size of the container, which is not efficient for research and production that require many containers.

Further, in the conventional plant tissue culture apparatus, a fluorescent lamp is mainly used as a light source, and the distance between the light source and the culture container is 20 to 30 cm, and in many cases close-up illumination is involved, and Since the inside of the culture container is hermetically sealed, the temperature inside the culture container becomes 2 to 5 ° C. higher than that outside the container due to heat transfer from the light source, and phenomena such as inhibition of growth of cultured plants are observed. .

[0008] In conventional plant tissue culture, when regenerating a plant from an adventitious embryo or adventitious bud system, a test tube or a culture container having a diameter of at most about 10 cm is used, and agar is added to the medium as a support. In many cases, an agar medium that has been gelled by the above method is used. This method is suitable for studies such as small-scale breeding purposes. In addition, some of these containers use a cotton plug or the like to allow ventilation inside and outside the container.

[0009] However, when trying to produce seedlings using these culture vessels, since the vessels are small, they are not suitable for mass production of seedlings. Especially, it takes time and effort for culturing,
The agar medium used as the support is inefficient because it needs to be completely washed off once during acclimation and then re-planted on another support.

Further, the ventilation inside and outside the container, which is carried out in the culture container, merely aims to keep the temperature and gas environment inside the container better than the sealed state, and aims at the active control of the inside environment of the container. Not a thing. On the other hand, in consideration of these points, an apparatus for controlling the temperature, humidity, and gas environment by enlarging the culture vessel and providing a pipe or the like for each one has been considered, but these are cumbersome to handle. However, it is difficult to say that it can be actually used at the seedling production site due to the high cost.

[0011]

Means for Solving the Problems As a result of intensive studies for solving the above problems, the present inventors have found that in plant tissue culture, control of the time of carbon dioxide fertilization, control of humidity in the culture vessel, and control of light intensity. The inventors have completed the present invention by finding that it is possible to shorten the number of culturing days and produce strong seedlings by optimally performing control and the like. Further, a novel plant tissue culture device and a culture container used for the culture device have been devised to facilitate such culture.

That is, the gist of the present invention is (1) carbon dioxide fertilization is performed in the culture container and the humidity in the culture container is reduced from the time when the decrease amount of the sugar concentration in the culture medium in the culture container starts to decrease. A method for cultivating plant tissue culture seedlings, characterized by gradually increasing the light intensity, (2) having a plurality of compartments capable of accommodating culture vessels, and supplying carbon dioxide gas to each compartment, And a plant tissue culture seedling culturing device, characterized by comprising temperature and humidity control means for each room,
(3) A culture container capable of accommodating a cell-molded support, with a sterile aeration membrane capable of controlling the temperature, humidity, gas, etc. in the culture container depending on the environment of the room of the culture device in which it is placed. The culture container having the above-mentioned opening, and (4) the culture container according to (3) containing the cell-molded support is installed in the chamber of the culture device according to (2) to perform culture. The present invention relates to a method for cultivating plant tissue culture seedlings, which comprises sequentially performing a period of culturing in a container in a sealed state, aeration culture through a sterile aeration membrane, and a period of acclimation with the culture container in an open state.

The method for cultivating plant tissue culture seedlings of the present invention is based on the finding that the time when carbon dioxide fertilization, the decrease in humidity and the increase in light intensity are particularly important. That is, a normal culture condition, for example, MS medium supplemented with sucrose (3%), naphthalene acetic acid (1 mg / liter) and kinetin (1 mg / liter) was used as a medium at a temperature of 25 ° C., a humidity of 70% RH, and a light. Strength 40 μm
When plant tissue culture is performed under illumination of ol / m ² / sec for 24 hours, the nutrients in the medium are initially ingested to cause heterotrophic growth and start adventitious bud formation. Then, after a certain period of time, the heterotrophic growth is changed to the mixed vegetative growth. In this case, if the culture is continued without changing the culture conditions, the growth becomes slow and the seedlings formed become weak.

However, when carbon dioxide fertilization is carried out at the time of transition from heterotrophic growth to mixed vegetative growth, photosynthesis is activated, adventitious bud growth is promoted, and seedling formation proceeds. Further, if the humidity is controlled to about 90% RH during the mixed vegetative growth period, the adventitious shoots can be easily transformed into healthy seedlings and acclimatized. Furthermore, when the light intensity was gradually increased during the mixed vegetative growth period, photosynthesis was further activated and the growth of plants was promoted. On the other hand, when carbon dioxide is applied during the heterotrophic growth period, the growth of adventitious shoots is rather suppressed.

Therefore, it is indispensable to determine the time of transition from heterotrophic growth to mixed vegetative growth for effective carbon dioxide fertilization and the like. In the present invention, however, the sugar concentration of the medium during the culture and the culture The time is determined by tracking the carbon dioxide concentration in the container.
That is, the time when the decrease in the sugar concentration in the medium begins to decrease is determined as the transitional period from heterotrophic growth to mixed vegetative growth. If carbon dioxide fertilization is performed before this transition period, the growth of adventitious buds will be suppressed, and thus the determination of the period is required carefully.

Although there is no particular limitation for determining the time when the decrease amount of the medium sugar concentration in the culture container begins to decrease, usually, the sugar concentration in the medium is measured by a saccharimeter to make a determination. The transitional period from heterotrophic growth to mixed vegetative growth is determined, and a predetermined treatment such as carbon dioxide fertilization is performed.

Fertilization of carbon dioxide gas into the culture vessel is carried out by aerating carbon dioxide gas through a sterile gas permeable membrane into the culture device accommodating the culture vessel. The carbon dioxide concentration in the culture vessel is adjusted to be 500 to 1000 ppm.
Further, the humidity control in the culture vessel is also carried out via a sterile air permeable membrane, and the controlled humidity is usually 90 to 95% RH.

The longer the period of carbon dioxide fertilization, the more the plants grow. However, in order to shorten the number of days for growing seedlings, the period of carbon dioxide fertilization is limited to 10 to 15 days. A method in which acclimation, which is a step, is performed for 5 to 10 days has a high survival rate of seedlings after being potted and meets the purpose of the present invention.

Further, the present inventors have discovered that a method of gradually increasing the light intensity in order to activate photosynthesis during the mixed nutritional growth period is effective for the rapid growth and seedling formation of cultured plants. . As a method of enhancing the light intensity, the heterotrophic growth period is 3
It is about 0 μmol / m ² / sec, which is doubled to 60 to 70 μmol / m ² / sec during the mixed nutritional growth period, and doubled to 120 to 140 μmol / m ² during the acclimation period.
/ Sec is gradually increased.

In the method for cultivating plant tissue culture seedlings of the present invention, as described above, the plant determines the transitional period from heterotrophic growth to mixed vegetative growth, and after this transitional period, carbon dioxide fertilization and culture container By controlling humidity and controlling the light intensity of illumination, a means for achieving growth promotion of adventitious shoots and establishment of healthy seedlings as a synergistic effect of these operations is provided. In addition, the number of days for adult seedling formation can be shortened by the main culture method that brings about these effects.

The method for cultivating plant tissue culture seedlings of the present invention is also used for adventitious embryo formation, growth by aseptic seeding of orchids, and seedling production by flake culture of lilies and the like, in addition to the above-mentioned culture of adventitious bud formation. can do.

In order to carry out the culture method of the present invention, not only temperature control in the culture container but also carbon dioxide fertilization and humidity control are required, and when the light intensity is increased, It is not appropriate to use the conventional plant tissue culture device because it is necessary to suppress the temperature rise.

Therefore, in the present invention, a plant having a plurality of compartments capable of accommodating a culture vessel, provided with a carbon dioxide gas supply means for each compartment and a temperature and humidity control means for each compartment. It is preferable to use a tissue culture seedling culture device. The carbon dioxide gas supply means is not particularly limited and, for example, a carbon dioxide gas cylinder is used. The temperature and humidity control means is not particularly limited, for example, the humidity is controlled by the amount of air supplied from the ultrasonic humidifier, and the temperature is adjusted by controlling the amount of cold air supplied from the cool air generator by opening and closing the damper. What is done is preferably used. Furthermore, by providing an infrared shielding filter that shields heat from the artificial light source, it is possible to suppress the temperature rise in the culture container when the light intensity is increased.

A preferred example of the culture device used in the present invention will be described with reference to FIG. FIG. 1 shows a schematic configuration diagram of the culture apparatus of the present invention. Numeral 1 is a divided chamber capable of accommodating a culture container. In this device, three chambers (culture A chamber, culture B)
Chamber, culture C chamber). Reference numeral 2 denotes a light source (daylight fluorescent lamp), which is an illumination facility whose intensity can be adjusted independently in each room. Reference numeral 3 denotes an infrared shielding filter, which can be mounted between the light source and each room, for example, to prevent a temperature rise due to an increase in the intensity of the light illumination from the artificial light source, and to control the temperature. Japanese Patent Publication No. 2-12
Japanese Patent Publication No. 535 or Japanese Patent Publication No. 4-48404 can be used. Reference numeral 4 denotes an electromagnetic on-off valve, which controls the supply amount of carbon dioxide and the ventilation amount. The carbon dioxide gas is supplied from the carbon dioxide gas supply source 6, and the aeration is performed by the humidifier 10
It is possible to supply 0% RH air. A temperature and humidity control damper 5 controls the temperature and humidity of each room by automatically opening and closing the damper to independently control the temperature and humidity. Reference numeral 8 denotes a culture container, which is housed in each room. 9 is a support provided in the culture container, and 10 is a plant to be cultured.

As described above, the culture apparatus of the present invention has a culture chamber divided into a plurality of chambers, and each of these chambers is a walk-in type, which is convenient in terms of work. By using the main culturing apparatus, it is possible to cultivate plant tissue culture seedlings with adjustment of illumination intensity, supply and control of carbon dioxide, aeration and control thereof, temperature control in a certain range, and the like.

A culture vessel for culturing a plant is arranged in each chamber of the culture apparatus of the present invention. Since the culture environment of the plant to be cultured is the environment inside the culture vessel, it is necessary to maintain the sterility, supply aeration and supply of carbon dioxide gas, and control the humidity. It is desirable that it can be produced. From such a viewpoint, the culture container of the present invention is a culture container capable of accommodating a cell-molded support, and controls the temperature, humidity, gas and the like in the culture container depending on the environment in the culture device in which it is placed. A container having an opening through a sterile gas-permeable membrane is used.

A preferred example of the culture container used in the present invention will be described with reference to FIG. FIG. 2 shows a schematic configuration diagram of the culture container of the present invention. Reference numeral 11 denotes a main body of a culture container, which is made of a container that can be sterilized by autoclave, for example, made of plastic such as polycarbonate in order to maintain an aseptic condition, and the capacity is not particularly limited, but in a clean bench. It is preferably such that the aseptic operation in 1. becomes easy. Reference numeral 12 is a lid, which is opened and closed to insert and remove the support. The lid is provided with a sealing packing 13 in order to enhance the hermeticity and prevent contamination during culture, and can be locked by using a lock or the like, preferably a patchon tablet 14 or the like. A plurality of cell-molded supports 17 can be housed in the container body. As the support 17, for example, a urethane cube, a rock wool cube, a plug tray, or the like is used. Further, the humidity and carbon dioxide gas in the container are controlled indirectly by the opening portion provided with the sterile gas permeable membrane 15 on the side surface of the container body 11. The degree of opening / closing of the opening is performed by moving the sterile gas-permeable membrane cover 16 that is movably installed so as to cover the sterile gas-permeable membrane 15. This controls the humidity and carbon dioxide in the container. That is, for example, a humidity scale is provided so that the humidity is adjusted in accordance with the degree of opening / closing of the sterile gas-permeable membrane 15, and the humidity can be controlled to a desired humidity by moving the sterile gas-permeable membrane cover 16 to a desired scale. it can. As the sterile gas permeable membrane 15, for example, a membrane filter made of fluorocarbon is used.

The method for cultivating plant tissue culture seedlings of the present invention can be effectively carried out using the above-described culture apparatus and culture container of the present invention. Below, the aspect which implements the method of this invention using the culture apparatus and culture container of this invention is demonstrated. That is, in the method of the present invention, (1) the time of culturing in a sealed state in the culture container, (2) the time of performing aeration culture through a sterile aeration membrane, and (3) the acclimation with the culture container in an open state. It is possible to perform all of these three steps with the apparatus of the present invention, depending on the time.

At the time of (1), the temperature is kept constant and the control of humidity and carbon dioxide gas is unnecessary. The light intensity is 30 μmol because the plant tissue has not yet reached photosynthesis.
A weak light of about / m ² / sec is sufficient. Lighting shall be performed continuously for 24 hours. This period is about 55 days although it depends on the conditions.

At the time of (2), the temperature is kept constant, but it is necessary to control the carbon dioxide concentration and humidity in the culture vessel.
Since the culture container of the present invention has a ventilation frequency of about 3 times per hour, assuming that the humidity of each chamber of the culture device is 70% RH and the carbon dioxide concentration is 1000 ppm, the humidity in the culture container under illumination is about 90% RH. Can be controlled. The intensity of the irradiation light is stronger than in the case of (1) because the plant has started photosynthesis, and is set to about 60 to 70 μmol / m ² / sec. The irradiation time is 12 to 16 hours, and the time during which the light is not irradiated is made during the day to create a rhythm similar to the natural condition. This rhythm creation is effective for the production of strong seedlings. This period is 10 to 15 days.

(3) Humidity is 90% at the time of acclimation.
It is particularly important to reduce the high humidity of RH to about 60% RH. With respect to the culture container, the lid is opened, the support is once washed, the sugar is washed away, and the culture container is installed again in the culture container with the lid opened. The environment in each room of the incubator is arbitrarily controlled under different conditions of temperature, humidity and carbon dioxide concentration when the irradiation lamp is turned on and off. Humidity needs to be gradually reduced to allow for transfer to seedlings in an outdoor environment. The mode of the reduction can be appropriately selected, but preferably 90% R
H, 90-80% RH, 80-70% RH, 70-60
The method of gradually lowering such as% RH is adopted to facilitate the adaptation of plants. The light intensity is 120 to 140 μmol / m ² / sec, which is stronger than in the case of (2).
The degree of acclimation further improves the acclimation efficiency. At this time,
5 to 10 days.

[0032]

EXAMPLES The present invention will be described in more detail with reference to Examples and Test Examples, but the present invention is not limited to these Examples. Example 1 As a plant body, adventitious bud formation culture was performed using a petiole piece (3 mm diameter, 2 mm thickness) of a sterile plant of Regus Begonia. The petiole pieces were placed on a cell-molded support housed in a sterilized culture container of the present invention. The support was an 18 mm diameter cell molding tray packed with vermiculite. As the culture container, a plastic molded container such as polycarbonate was used for autoclave sterilization, and a lid was fitted with a patch tablet to maintain hermeticity. The size of the culture vessel is 210 mm x 250 mm (height 100 m to facilitate aseptic operation in a clean bench).
m) was used.

The medium used was MS (Murashige Skoog) medium supplemented with 3% sucrose, 1 mg / liter naphthalene acetic acid and 1 mg / liter kinetin. The culturing was carried out using the culturing apparatus of the present invention capable of controlling the light intensity, temperature, humidity and carbon dioxide concentration.

First, when the culture was carried out at a temperature of 25 ° C., a humidity of 70% RH, and a light intensity of 40 μmol / m ² / sec for 24 hours under illumination (daylight fluorescent lamp), about 60 days after the start of the culture, Sugar consumption began to decline. This fact means that the petiole pieces continued to grow heterotrophically from the start of the culture until about the 55th day, and started the photosynthesis from about the 55th day. Therefore, under the main culture conditions, 60 days after the start of culture was determined as the transitional period from heterotrophic growth to mixed vegetative growth.

Next, according to the culture method of the present invention, carbon dioxide fertilization was performed in the culture container, the humidity in the culture container was lowered, and the light intensity was gradually increased. The carbon dioxide fertilization was not forcibly performed, but the concentration of carbon dioxide gas in the chamber of the culture device was increased to about 1000 ppm, and the fertilization was indirectly performed into the culture container through a sterile aeration membrane attached to the side surface of the culture container. As a result, gas exchange is carried out both inside and outside the culture vessel, and at the same time, humidity is affected, and what was previously saturated is reduced to about 90% RH. These two effects promote the growth of adventitious shoots Will be promoted. The light intensity was set to 60 to 70 μmol / m ² / sec.

Carbon dioxide fertilization was performed for 10 days. The longer the carbon dioxide fertilization was, the more the plants grew and the survival rate of seedlings after pot raising was high.However, since the number of days for seedling formation is prolonged, the number of days for seedling formation and seedling survival rate after potting are increased. In consideration of the above, it was most efficient to apply the carbon dioxide gas for about 10 days and then acclimate the process for about 5 days.

The acclimation was carried out by washing the support with water to wash away the sugar, transferring it again into the culture vessel, and changing the humidity from 90% RH to 90%.
-80% RH, 80-70% RH, 70-60% RH. Illumination intensity is 120 to 140 μmol / m ²
/ Sec to increase the lighting time and extinguish time during the day to create a rhythm and facilitate the transition to the outdoor environment. The culture seedlings obtained in this way are about 1
It took only 1/2 (75 days), but the survival rate after potting was 95% or more, which proved to be an excellent method.

Example 2 A similar experiment was carried out by using petiole pieces of Saintpaulia instead of Ligas begonia. The transitional period from heterotrophic growth to mixed vegetative growth was about 60 days after the start of culture.

Test Example In the same manner as the adventitious bud formation culture carried out in Example 1,
Adventitious bud-forming culture was performed using petiole pieces of a sterile plant of Regus begonia, and the changes in sugar concentration and carbon dioxide concentration with time were examined. The sugar concentration was measured by a digital sugar content meter PR-1 (manufactured by Atago Co.) every 10 days of culture, and the carbon dioxide concentration was measured by a detector tube method (Gastech No. 2LL) every 5 days of culture.

The results of measuring the sugar concentration are shown in FIG. Culture 55
The daily consumption of sugar in the medium began to decrease. From this, it was determined that about 55 days of culture was the transitional period from heterotrophic growth to mixed vegetative growth.

[0041]

By using the culture method of the present invention,
It is possible to shorten the number of days for forming seedlings and obtain a normal cultured seedling that is stronger (higher survival rate) than before. In addition, by using the culture device of the present invention,
It is possible to perform everything from the start of culturing to acclimation in the production of seeds with one device, and also to reproduce various environmental conditions with one device in the situation of research, which enables efficient comparison and examination. is there. The apparatus is particularly suitable for using the method for cultivating plant tissue culture seedlings of the present invention. Further, since the work efficiency is greatly improved by using the culture container of the present invention, the production cost of the cultured seedlings can be reduced.

[Brief description of drawings]

FIG. 1 shows a schematic configuration diagram of a culture device of the present invention.

FIG. 2 shows a schematic configuration diagram of a culture container of the present invention.

FIG. 3 shows the time-dependent changes in the sugar concentration in the medium in Test Examples.

[Explanation of symbols]

 1 Culture A room 2 Light source (daylight fluorescent lamp) 3 Infrared shielding filter 4 Electromagnetic on-off valve 5 Temperature and humidity control damper 6 Carbon dioxide gas supply source 7 Humidifier 8 Culture vessel 9 Support body 10 Plant body 11 Culture vessel body 12 Lid 13 Seal packing 14 Patchon tablet 15 Aseptic breathable membrane 16 Aseptic breathable membrane cover 17 Support 18 Plant

Claims (6)

[Claims] 1. From the time when the decrease amount of the medium sugar concentration in the culture container begins to decrease, carbon dioxide fertilization is performed in the culture container, the humidity in the culture container is decreased, and the light intensity is gradually increased. A method for cultivating plant tissue culture seedlings, which comprises: 2. The culture method according to claim 1, wherein the plant tissue culture seedling is derived from an adventitious bud, an adventitious embryo, or aseptic seeding. 3. A plant characterized by having a plurality of compartments capable of accommodating a culture vessel, comprising carbon dioxide supply means for each compartment, and temperature and humidity control means for each compartment. Tissue culture seedling culture device. 4. The culture device according to claim 3, further comprising an infrared shielding filter that shields heat from the artificial light source. 5. A culture vessel capable of accommodating a cell-molded support, and a sterile aeration membrane capable of controlling temperature, humidity, gas and the like in the culture vessel according to the environment of the room of the culture apparatus in which it is placed. A culture container having an opening through the culture container. 6. The cell-shaped support member is housed therein.
The culture container according to claim 3 is installed in the chamber of the culture device according to claim 3, the culture is performed in a sealed state in the culture container, the aeration culture is performed through a sterile aeration membrane, and the culture container is opened to acclimatize. A method for cultivating plant tissue culture seedlings, which comprises sequentially performing the steps.