JP2732184B2 - Culture method of plant tissue culture seedling - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for culturing plant tissue culture seedlings.
About the law. More particularly, the present invention relates to the culture how of seedlings using plant tissue culture techniques.

[0002]

2. Description of the Related Art Conventionally, the production of seeds and seedlings of plants has been multiplied by seeds, bulbs or cuttings. These methods are still important techniques for seed production in many plants. However, in these methods, as the production is repeated,
Problems have been pointed out, such as growth inhibition and death due to diseases and pests, and this is not a stable method.

[0003] Therefore, a technique for regenerating a plant from cells by biotechnology has been developed and has been used for breeding and seedling production. As an example of the method, the apical bud tissue is removed under aseptic conditions, placed on a medium such as a test tube, cultured, and regenerated into a plant. Many axillary buds are sprouted on the regenerated plant, divided into several, and the clone seedlings are proliferated in large quantities by repeating those steps. It is generally practiced to make the seedlings through a process of acclimatization that takes about one month to make it into an outdoor environment.

[0004] In such a conventional method of producing seedlings using plant tissue culture, plants with axillary buds are cut off one by one, and the process is repeated. And the labor required for the operation is large, and the propagation efficiency is not high. Furthermore, since the plants are placed one by one in a test tube or the like, the cost is high economically, and the cultivation time until seedling formation is long, and it is difficult to say that strong seedlings can be produced due to the empirical method of acclimation.

[0005] Further, in a conventional culture apparatus used for plant tissue culture or the like, only one pattern can be set for temperature, humidity, light intensity, carbon dioxide concentration and the like in one apparatus. As far as temperature is concerned, there are some devices that divide the room into multiple units with one device and set and control each at a different temperature, but it is possible to simultaneously control light intensity, humidity, and carbon dioxide concentration. There is nothing.

[0006] Recently, in plant tissue culturing technology, a technology has been developed to control the gas concentration, temperature, humidity, nutrient solution, and the like in a culture vessel by individually piping in order to promote the growth of the cultured plant body and restore healthy seedlings. Have been. However, this method is not practical because it requires a sterilization treatment to keep the inside of the culture container in an aseptic state, and requires time and labor for piping and the like. Such a conventional culture apparatus has the above-mentioned problems.When research or actual production of seeds and seedlings using plant tissue culture technology is to be performed, in the process of cultivation to acclimation, the plant is not cultivated. Different environmental controls are required depending on the type and culture / acclimation pattern, and for that purpose, many devices are required, which is not efficient. Also, controlling the environment for each culture vessel increases the size of the vessel, and is not efficient for research or production that requires many vessels.

Further, in the conventional plant tissue culturing apparatus, a fluorescent lamp is mainly used as a light source, and the distance between the light source and the culture vessel is 20 to 30 cm, which is often used for near illumination. Since the inside of the culture vessel is in a sealed state, the temperature inside the culture vessel becomes higher than that outside the vessel by 2 to 5 ° C. due to heat transfer from the light source, and phenomena such as inhibiting growth of the cultured plant are observed. .

[0008] In the conventional plant tissue culture, in the regeneration of plants from adventitious embryos or adventitious buds, test tubes or culture vessels of at most about 10 cm in diameter are used, and agar is added to the culture medium as a support. An agar medium gelled and gelled is often used. This method is suitable for small-scale breeding research. In some of these containers, the inside and outside of the container can be ventilated using a cotton plug or the like.

However, when attempting to produce seeds and seeds using these culture vessels, the size of the vessels is not suitable for producing large quantities of seeds and seedlings. In particular, it takes time and labor to culture,
The efficiency of the agar medium or the like used as the support is poor, for example, it is necessary to completely wash it off once in acclimation, and then re-inoculate it on another support.

[0010] Further, the ventilation inside and outside the container, which is performed in the culture container, is merely intended to keep the temperature and gas environment inside the container better than the sealed state, and aims at active control of the container environment. Not something. On the other hand, in consideration of these points, it is considered that the size of the culture vessel is increased, and piping is provided for each one to control the temperature, humidity and gas environment, but these are troublesome to handle. However, the cost is high and it is hard to say that it can be actually used at the seedling production site.

[0011]

Means for Solving the Problems The present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, in the plant tissue culture, control of the time of carbon dioxide fertilization, control of the humidity in the culture vessel, and control of the light intensity. The present inventors have found that the culturing days can be shortened and strong seedling production can be achieved by optimally performing control and the like, and the present invention has been completed .

That is, the gist of the present invention is to (1) perform fertilization of carbon dioxide in the culture vessel and reduce the humidity in the culture vessel from the time when the decrease amount of the medium sugar concentration in the culture vessel starts to decrease. And a method for culturing plant tissue culture seedlings, wherein the light intensity is gradually increased. (2) The plant tissue culture seedlings are adventitious buds, adventitious embryos or aseptic seeding.
The culture method according to the above (1), which is derived from a species or the like; (3) Temperature in the culture vessel, the humidity, the control <br/> culture container containing a support having Rousset Le molding having a opening control is via a sterile gas permeable membrane capable of atmosphere such as gas, the culture Container storage
Has multiple compartments where possible, with carbon dioxide to each compartment
Equipped with supply means and control means for temperature and humidity of each room
Was installed in a room of the culture apparatus of the plant tissue culture seedlings, timing culturing the culture vessel in a sealed state, the timing to perform aeration culture through the sterile gas permeable membrane, and conditioned by the culture vessel in an open state The present invention relates to a method for culturing plant tissue culture seedlings, which is performed sequentially.

[0013] The method for culturing plant tissue culture seedlings of the present invention is based on the finding that it is especially important to apply carbon dioxide fertilization, reduce the humidity and increase the light intensity. That is, a medium obtained by adding sucrose (3%), naphthalene acetic acid (1 mg / l) and kinetin (1 mg / l) to a culture medium under ordinary culture conditions, for example, a temperature of 25 ° C., a humidity of 70% RH, and light Strength 40μm
When plant tissue culture is performed at ol / m ² / sec under illumination for 24 hours, initially, nutrients in the medium are taken, heterotrophic growth takes place, and adventitious buds begin to form. Then, after a certain period of time, a transition from heterotrophic growth to mixed vegetative growth occurs. In this case, if the culture is continued without changing the culture conditions, the growth will be slowed down and the seedlings formed will be weak.

However, if carbon dioxide fertilization is performed at the time of transition from heterotrophic growth to mixed vegetative growth, photosynthesis is activated, the growth of adventitious shoots is promoted, and seedlings are advanced. In addition, when the humidity is controlled to about 90% RH during the mixed vegetative growth period, it becomes easy to make the adventitious buds healthy and acclimatized. Furthermore, when the light intensity is gradually increased during the mixed vegetative growth period, photosynthesis is further activated and plant growth is promoted. On the other hand, if carbon dioxide fertilization is performed during the heterotrophic growth period, the growth of adventitious buds is rather suppressed.

[0015] Therefore, it is indispensable for effective carbon dioxide fertilization and the like to determine the time of transition from heterotrophic growth to mixed vegetative growth. The timing is determined by tracking the concentration of carbon dioxide in the container.
That is, the time when the amount of decrease in the sugar concentration in the medium starts to decrease is determined as the transition period from heterotrophic growth to mixed vegetative growth. If carbon dioxide fertilization is performed before this transition period, the growth of adventitious buds is suppressed, and therefore, the judgment of the timing requires careful consideration.

The time at which the decrease in the sugar concentration of the culture medium in the culture vessel begins to decrease is not particularly limited, but is usually determined by measuring the sugar concentration in the culture medium using a refractometer. A transition period from heterotrophic growth to mixed vegetative growth is determined, and predetermined processing such as carbon dioxide fertilization is performed.

The fertilization of carbon dioxide into the culture vessel is performed by passing carbon dioxide gas through a sterile gas-permeable membrane into a culture apparatus containing the culture vessel. The concentration of carbon dioxide in the culture vessel is adjusted to be 500 to 1000 ppm.
The humidity in the culture vessel is also controlled through a sterile gas-permeable membrane, and the controlled humidity is usually 90 to 95% RH.

[0018] The longer the carbon dioxide fertilization period, the more the plant grows. However, in order to shorten the number of seedlings, the carbon dioxide fertilization period is limited to 10 to 15 days. The method of acclimatizing the step, which is performed for 5 to 10 days, has a high survival rate of the seedlings after the raising of the pots, which meets the object of the present invention.

Further, the present inventors have found that a method of gradually increasing light intensity in order to activate photosynthesis during mixed vegetative growth is effective for rapid growth and seedling of cultured plants. . As a method for increasing the light intensity, the heterotrophic growth period is 3
0μmol / m to about ² / sec, the mixing vegetative, twice that of 60~70μmol / m ² / sec, still twice that in the acclimatization period 120~140μmol / m ²
/ Sec step by step.

In the method for culturing plant tissue culture seedlings of the present invention, as described above, the transition period of the plant from heterotrophic growth to mixed vegetative growth is determined. By controlling the humidity and the light intensity of the illumination, it is possible to provide a means for promoting the growth of adventitious buds and achieving a healthy seedling as a synergistic effect of these operations. In addition, the main culture method that provides these effects can also reduce the number of days for adult seedlings.

The method for cultivating plant tissue culture seedlings of the present invention can be used not only for the culture of adventitious bud formation but also for the production of adventitious embryos, propagation by aseptic seeding such as orchids, and seedling production by scallop culture of lilies and the like. can do.

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

Therefore, in the present invention, a plant having a plurality of compartments capable of accommodating a culture vessel, a means for supplying carbon dioxide to each chamber, and a means for controlling the temperature and humidity of each chamber is provided. Preferably, a tissue culture seedling culture device is used. The carbon dioxide gas supply means is not particularly limited, and for example, a carbon dioxide gas cylinder is used. The means for controlling the temperature and humidity is not particularly limited, but for example, the humidity is controlled by the amount of air supplied from the ultrasonic humidifier, and the temperature is controlled by controlling the amount of cool air supplied from the cool air generator by opening and closing a damper. What is performed is preferably used. Further, by providing an infrared shielding filter that blocks heat from the artificial light source, it is possible to suppress a rise in temperature in the culture vessel 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. Reference numeral 1 denotes a divided room capable of storing a culture container. In this apparatus, three rooms (culture A room and culture B room) are provided.
An example is shown in FIG. Reference numeral 2 denotes a light source (daylight fluorescent lamp), which is a lighting facility capable of independently adjusting the intensity in each room. Reference numeral 3 denotes an infrared shielding filter, which can be mounted between, for example, the light source and each room to prevent a rise in temperature due to an increase in the intensity of light illumination from the artificial light source, thereby enabling temperature control. As the infrared shielding filter used here, Japanese Patent Publication No. 2-12
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 gas and the amount of ventilation. The carbon dioxide gas is supplied from a carbon dioxide gas supply source 6, and the ventilation is performed by a humidifier 7.
It is possible to supply 0% RH air. Reference numeral 5 denotes a temperature and humidity control damper, which controls the temperature and humidity of each room automatically by opening and closing the damper, and can independently control the temperature and humidity. Reference numeral 8 denotes a culture vessel, which is housed in each room. Reference numeral 9 denotes a support provided in the culture vessel, and reference numeral 10 denotes a plant to be cultured.

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

In each room of the culture apparatus of the present invention, a culture vessel for culturing a plant is disposed. Since the culture environment of the plant to be cultured is the environment in the culture vessel, it is necessary to maintain aseptic conditions, to supply aeration and supply of carbon dioxide gas, and to control the humidity. It is desirable to be able to produce. From such a viewpoint, the culture vessel of the present invention is a culture vessel capable of accommodating a cell-shaped support, and controls the temperature, humidity, gas, and the like in the culture vessel depending on the environment in the placed culture apparatus. A container having an opening through a sterile gas-permeable membrane capable of performing the above is used.

A preferred example of the culture vessel used in the present invention will be described with reference to FIG. FIG. 2 shows a schematic configuration diagram of the culture vessel of the present invention. Reference numeral 11 denotes a main body of the culture vessel, which is made of an autoclave-sterilizable container, for example, a plastic such as polycarbonate in order to create a sterile state and maintain the sterile state. It is preferable that the sterilization operation is easy. Reference numeral 12 denotes a lid, which is opened and closed to allow the support to be taken in and out. The lid is provided with a sealing gasket 13 in order to enhance the sealing property and prevent contamination during culture, and can be locked using a tablet or the like, preferably using a patchin tablet 14 or the like. A plurality of cell-shaped supports 17 can be stored 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 control of the humidity and carbon dioxide in the container is performed indirectly through an opening in which a sterile gas permeable membrane 15 is attached to the side surface of the container body 11. The degree of opening and closing of the opening is performed by moving a sterile gas permeable membrane cover 16 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 according to the degree of opening and closing of the sterile gas permeable membrane 15, and the desired humidity can be controlled by moving the sterile gas permeable membrane cover 16 to the desired scale. it can. As the sterile gas permeable membrane 15, for example, a fluorocarbon membrane filter is used.

The method for culturing plant tissue culture seedlings of the present invention can be effectively carried out using the culturing apparatus and the culturing container of the present invention as described above. Hereinafter, an embodiment of performing the method of the present invention using the culture apparatus and the culture container of the present invention will be described. That is, the method of the present invention comprises: (1) time for culturing the culture vessel in a sealed state, (2) time for aeration culture through a sterile gas-permeable membrane, and (3) acclimation with the culture vessel open. Depending on the time, all three of these steps can be performed with the apparatus of the present invention.

In the period (1), the temperature is kept constant, and it is not necessary to control the humidity and the carbon dioxide gas. 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. Illumination is performed continuously for 24 hours. This period varies depending on conditions, but is about 55 days.

In the period (2), the temperature is kept constant, but it is necessary to control the concentration of carbon dioxide and the humidity in the culture vessel.
Since the ventilation frequency of the culture vessel of the present invention is about three times per hour, when the humidity of each room of the culture apparatus is 70% RH and the carbon dioxide concentration is 1000 ppm, the humidity in the culture vessel under illumination is about 90% RH. Can be controlled. The intensity of the irradiation light is set to about 60 to 70 μmol / m ² / sec more strongly than in the case of (1) since the plant has started photosynthesis. The irradiation time is set to 12 to 16 hours, and a period during which no light is irradiated in one day is created, and a rhythm similar to a natural condition is created. Creating this rhythm is effective for producing strong seedlings. This period is 10 to 15 days.

(3) At the time of acclimatization, the humidity is 90%
It is particularly important to reduce the RH from high humidity to about 60% RH. The culture vessel is opened with the lid open, once the support is washed, the sugar is washed off, and the culture vessel is placed again in the culture vessel with the lid opened. The environment in each room of the culture apparatus is arbitrarily controlled under different conditions of temperature, humidity, and carbon dioxide concentration when the irradiation lamp is turned on and off. The humidity needs to be reduced gradually so that it is possible to move to raising seedlings in an outdoor environment. The mode of the reduction can be selected as appropriate, but preferably 90% R
H, 90-80% RH, 80-70% RH, 70-60
A method of gradually decreasing the ratio such as% RH is adopted to facilitate adaptation of the plant. The light intensity is 120 to 140 μmol / m ² / sec, which is even stronger than in the case of (2).
If it is made to the extent, the acclimatization efficiency is further improved. At this time,
5 to 10 days.

[0032]

The present invention will be described in more detail with reference to the following examples and test examples, but the present invention is not limited to these examples. Example 1 As a plant, an adventitious bud formation culture was carried out using a petiole (3 mm diameter, 2 mm thickness) of a sterile plant of Ligas begonia. The petiole pieces were placed on a cell-shaped support housed in the sterilized culture vessel of the present invention. The support was an 18 mm diameter cell forming tray packed with vermiculite. As a culture container, a molded container made of plastic such as polycarbonate was used for sterilization in an autoclave, and a lid was fitted with a patch tablet to keep 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 an MS (Murashige Skoog) medium supplemented with 3% sucrose, 1 mg / liter of naphthaleneacetic acid and 1 mg / liter of kinetin. The culture was performed using the culture apparatus of the present invention capable of controlling light intensity, temperature, humidity, and carbon dioxide concentration.

First, the cells were cultured 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). Sugar consumption began to decrease. This fact means that the petiole continued to grow heterotrophically until about the 55th day from the start of culture, and photosynthesis started around the 55th day. Thus, under the main culture conditions, 60 days after the start of the culture was determined as the transition period from heterotrophic growth to mixed vegetative growth.

Next, according to the culturing method of the present invention, the culture vessel was subjected to carbon dioxide fertilization, the humidity in the culture vessel was reduced, and the light intensity was gradually increased. The carbon dioxide fertilization was not performed forcibly, but the carbon dioxide concentration in the room of the culture apparatus was increased to about 1000 ppm, and the fermentation was performed indirectly into the culture vessel through a sterile gas-permeable membrane attached to the side of the culture vessel. As a result, the gas exchange is performed inside and outside the culture vessel, and at the same time, the humidity is also affected, and those that have been saturated up to that point are reduced to about 90% RH. These two effects promote the growth of adventitious buds and seedlings. Will progress. The light intensity was 60 to 70 μmol / m ² / sec.

The carbon dioxide fertilization was performed for 10 days. The longer the carbon dioxide fertilization was performed, the higher the growth of the plants and the higher the survival rate of seedlings after potting.However, since the number of seedlings was prolonged, the number of seedlings and the survival rate of seedlings after potting were increased. In consideration of the above, it was most efficient to set the carbon dioxide fertilization to about 10 days and the acclimatization period of the subsequent process to about 5 days.

For acclimatization, the support was washed with water to wash away the sugar, transferred again to the culture vessel, and the humidity was adjusted from 90% RH to 90%.
-80% RH, 80-70% RH, 70-60% RH. Illumination intensity is 120-140 μmol / m ²
/ Sec, and a rhythm is created by providing a lighting time and a lighting time during one day, thereby facilitating the transition to an outdoor environment. The thus-obtained cultured seedlings are about 1 day, which is the conventional number of days for seedling formation.
/ 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 conducted using a petiole piece of Saintpaulia in place of Legas Begonia. The transition 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 performed in Example 1,
Adventitious bud formation culture was performed using petiole pieces of a germ-free plant of Legas Begonia, and the changes over time in the sugar concentration and carbon dioxide concentration were tested. The sugar concentration was measured by a digital refractometer PR-1 (manufactured by Atago) every 10 days of culture, and the carbon dioxide concentration was measured by a detection tube method (Gastech No. 2LL) every 5 days of culture.

FIG. 3 shows the measurement results of the sugar concentration. Culture 55
On a daily basis, sugar consumption in the medium began to decrease. From this, around 55 days of culture was determined to be the transition 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 normal cultured seedlings (higher survival rate) than before. In addition, by using the culture apparatus of the present invention,
It is possible to perform everything from the start of cultivation to acclimatization in the stage of seedling production with one device, and it is also possible to reproduce various environmental conditions with one device in the scene of research, and it is possible to perform efficient comparative examination is there. The apparatus is particularly suitable for using the method for cultivating plant tissue culture seedlings of the present invention. Further, the use of the culture vessel of the present invention greatly improves the working efficiency, and thus enables a reduction in the production cost of cultured seedlings.

[Brief description of the drawings]

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

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

FIG. 3 shows a time-dependent change in a sugar concentration in a medium in a test example.

[Explanation of symbols]

 DESCRIPTION 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 container 9 Support body 10 Plant 11 Culture container main body 12 Lid 13 Seal packing 14 Patch tablet 15 Sterile air-permeable membrane 16 Sterile air-permeable membrane cover 17 Support 18 Plant

Claims (3)

(57) [Claims] 1. A method of fertilizing carbon dioxide in a culture vessel, reducing the humidity in the culture vessel, and gradually increasing the light intensity from the time when the decrease amount of the medium sugar concentration in the culture vessel begins to decrease. A method for culturing a plant tissue culture seedling, characterized by the following. 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. Atmosphere such as temperature, humidity and gas in the culture vessel.
A cell with an opening through a sterile gas-permeable membrane
The culture vessel containing the molded support is
It has several compartments that can be stored,
Acid gas supply means and temperature and humidity control means for each chamber
Installed in the room of the plant tissue culture seedling culture device equipped with
When the culture vessel is cultured in a sealed state, the aseptic gas permeable membrane
When aeration culture is performed, and open the culture vessel
Characterized in that acclimatization is performed sequentially in a state
Method of culturing a tissue culture seedling