Efficient tissue culture and rapid propagation method for idesia polycarpa

Abstract

The invention relates to an efficient tissue culture and rapid propagation method for idesia polycarpa, and belongs to the technical field of idesia polycarpa culture, the method uses a tissue culture device, the device comprises a circular-ring-shaped flow channel and a supporting column, and the method comprises the following steps: obtaining a plurality of explants, and washing and disinfecting the explants; injecting the bud induction culture medium into the flow channel, sequentially putting a plurality of explants into the bud induction culture medium, driving the bud induction culture medium to drive the explants to flow in the flow channel, and starting to appear calluses and germinate new buds after 18 days; when the new buds grow to 1cm, replacing the bud induction culture medium with a planting increasing culture medium, driving the planting increasing culture medium to drive the explants to flow in the flow channel, and forming clustered plants after 22 days; putting a rooting substrate into a culture dish, then selecting 2cm single buds from clustered plants, treating the single buds with rooting powder, inserting the single buds into the rooting substrate with tweezers, rooting after 20 days, and after rooting, placing the culture dish and the single buds in the sun for 15 days to form idesia seedlings; and transplanting the idesia polycarpa seedlings outdoors.

Description

Technical Field

[0001] The invention belongs to the technical field of Castanopsis japonici culture, and particularly relates to an efficient Castanopsis japonici tissue culture rapid propagation method. Background Art

[0002] Castanopsis truncatum, also known as oil grape, is a tall deciduous tree belonging to the genus Castanopsis of the family Caryophyllaceae. It has a beautiful tree shape, deep red fruits in autumn, unisexual flowers, dioecious or polygamous, and conical drooping. It is an excellent horticultural ornamental tree species. Castanopsis truncatum has a high fruit yield per plant, the whole fruit contains oil, the oil is of high quality, and it has multiple health functions. It is an important woody oil species and is known as the "oil warehouse" on the tree.

[0003] With the advancement of science, tissue culture technology has become increasingly mature. Since Castanopsis japonici has a high economic value, many producers have used tissue culture technology to cultivate Castanopsis japonici plants and have achieved good results. Moreover, the Castanopsis japonici tissue culture method and culture medium type are no longer trade secrets in the industry and can be easily obtained by all producers.

[0004] However, when the existing tissue culture method is used to cultivate Castanopsis schrenkiana plants, the explants are usually placed in a fixed culture dish for culture. During this process, the explant culture process consumes nutrients, which leads to a decrease in the nutrient content near the explant. Although there are some technologies that shake the culture dish to evenly distribute the nutrients in the culture dish, the effect is not ideal, and the explants usually maintain a certain posture in the culture solution, making it difficult to ensure that all areas of the explant are evenly exposed to sunlight and absorb nutrients evenly. Summary of the invention

[0005] The invention provides an efficient rapid propagation method of Castanopsis japonici tissue culture, which is used to solve the technical problem raised in the background technology.

[0006] The present invention is achieved through the following technical scheme: an efficient method for rapid propagation of Castanopsis schrenkiana tissue culture, using a tissue culture device to culture Castanopsis schrenkiana seedlings, the tissue culture device comprising a ring-shaped flow channel and a support column for supporting the flow channel, the method comprising:

[0007] Step 1: Obtain a plurality of explants and rinse and sterilize the explants;

[0008] Step 2: injecting a bud induction medium into the flow channel, and placing a plurality of explants into the bud induction medium in sequence, and then driving the bud induction medium to drive the explants to flow in the flow channel, callus tissue begins to appear after 18 days, and new buds sprout;

[0009] Step 3: When the new buds grow to 1 cm, the bud induction medium is replaced with the growth medium, and the growth medium is driven to drive the explants to flow in the flow channel, and clustered plants are formed after 22 days;

[0010] Step 4: Place the rooting medium in a culture dish, then select a 2 cm single bud from the clump of plants and treat the single bud with rooting powder, then insert it into the rooting medium with tweezers, and take root after 20 days. After taking root, place the culture dish and the single bud in it in the sun for 15 days to form a Castanopsis truncatula seedling;

[0011] Step 5: Transplant the Castanopsis chinensis seedlings outdoors.

[0012] Further, in order to better implement the present invention, in step 1, the method for flushing and disinfecting the explant is specifically as follows:

[0013] First clean with detergent for 10 minutes, then rinse with running water for 30 minutes, then disinfect with 75% sodium sulfate for 30 seconds, then disinfect with 0.1% mercury solution for 10 minutes, and finally rinse with sterile water 6 times.

[0014] Further, in order to better realize the present invention, the cross-sectional shape of the flow channel is semicircular, the tissue culture device also includes a water pump installed below the flow channel, a water outlet hole is opened at the bottom of the flow channel, a first blocking net is arranged at the water outlet hole, the water inlet of the water pump is connected with the water outlet hole through a water inlet pipe, a curved convex block is convexly arranged at the bottom of the flow channel, a water channel is arranged in the curved convex block, the water outlet of the water pump is connected with the water channel through the water outlet pipe, and the water outlet direction of the water channel is the same as the flow direction of the liquid in the flow channel;

[0015] In step 2, the method for driving the bud induction medium to flow in the flow channel is specifically:

[0016] The water pump is started, and the bud induction medium in the flow channel is extracted from the water outlet by the water pump, and the bud induction medium is injected into the flow channel from the water channel again.

[0017] Furthermore, in order to better implement the present invention, in step 3, the method of driving the growth medium to flow in the flow channel is specifically:

[0018] The water pump is started, and the planting medium in the flow channel is extracted from the water outlet by the water pump, and the planting medium is injected into the flow channel from the water channel again.

[0019] Furthermore, in order to better realize the present invention, a drainage hole is provided at the bottom of the flow channel, a second barrier net is installed in the drainage hole, and a drainage pipe is provided in communication with the drainage hole, and a sealing plug is installed at the drainage pipe;

[0020] In step 3, the method of replacing the bud induction medium with the planting medium is specifically as follows:

[0021] Place the bucket under the drain pipe, then remove the sealing plug. The bud induction medium in the flow channel will flow from the drain hole and the drain pipe into the bucket. Then install the sealing plug, and finally inject the planting medium into the flow channel from above.

[0022] Furthermore, in order to better implement the present invention, during the process of step 2, it is observed whether the explant is browned. If browning occurs, the explant with browning is removed from the flow channel.

[0023] Furthermore, in order to better implement the present invention, a mounting frame is provided on the inner side of the flow channel, a fixed camera facing the flow channel is installed on the mounting frame, the fixed camera is connected to a computer, and an alarm light connected to the computer is also installed on the mounting frame;

[0024] The specific method for observing whether the explants are browning is:

[0025] Turn on the fixed camera and use it to collect image information of the explant in the flow channel in real time, and transmit the collected image information to the computer. Use image processing technology to process the image information. When brown appears near the explant, it is determined that the explant has browned, and the computer controls the alarm light to light up.

[0026] Furthermore, in order to better realize the present invention, a circular track is installed on the mounting frame, a sliding arm is installed on the circular track, an active camera electrically connected to the computer is installed on the sliding arm, and a motor is installed on the mounting frame, and the sliding arm is connected to the rotating shaft of the motor through a transmission mechanism, so that the motor drives the sliding arm to drive the active camera to move on the circular track;

[0027] When brown appears near the explant, the motor starts to drive the active camera to move to aim at the brown explant nearby, and the motor drives the active camera to rotate synchronously with the explant, so that the active camera can collect image information of the explant in real time.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] (1) The efficient rapid propagation method of Castanopsis chinensis tissue culture provided by the present invention is to culture Castanopsis chinensis plants with the help of a tissue culture device, the tissue culture device comprising a flow channel in the shape of a ring and a support column for supporting the flow channel, the method comprising obtaining a plurality of explants and washing and disinfecting the explants; injecting a bud induction culture medium into the flow channel, and placing a plurality of explants in turn into the bud induction culture medium, and then driving the bud induction culture medium to drive the explants to flow in the flow channel, callus tissue begins to appear after 18 days, and new buds germinate; when the new buds grow to 1 cm, the bud induction culture medium is replaced with a planting culture medium, and the planting culture medium is driven to drive the explants to flow in the flow channel, and clustered plants are formed after 22 days; a rooting matrix is ​​placed in a culture dish, and then a single bud of 2 cm is selected from the clustered plants and treated with rooting powder, and then inserted into the rooting matrix with tweezers, and roots are formed after 20 days. After rooting, the culture dish and the single bud therein are placed in the sun for 15 days to form Castanopsis chinensis seedlings; and the Castanopsis chinensis seedlings are transplanted outdoors.

[0030] Through the above method, the culture is allowed to flow with the culture solution during the bud induction stage and the root formation stage. In this way, not only can the nutrients in the culture solution be fully mixed, but also harmful substances can be prevented from accumulating in local areas, and the flow process is more convenient for gas exchange. In addition, by controlling the flow rate, the culture can sometimes roll in the culture solution, thereby changing the posture of the culture in the culture solution, so that each area of ​​the culture can absorb nutrients and receive light more evenly, and it can also avoid the situation where a certain area of ​​the culture is always immersed in the culture and causes rot. Another point is that during the flow process, the situation of the plant being blown by the wind when growing outdoors can be simulated, which is more conducive to the growth of the culture and the adaptation to the outdoor wind blowing situation in advance before planting. Through this method, the tissue culture of Castanopsis sylvestris can be carried out more efficiently. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0032] Figure 1 It is a general flow chart of the efficient rapid propagation method of Castanopsis affine tissue culture provided by the embodiment of the present invention;

[0033] Figure 2 is a schematic structural diagram of a tissue culture device in an embodiment of the present invention;

[0034] Figure 3 yes Figure 2 Another perspective view of the tissue culture device shown.

[0035] In the figure:

[0036] 1-flow channel, 2-support column, 3-water pump, 4-water inlet pipe, 5-arc-shaped protrusion, 6-water outlet pipe, 7-drain pipe, 8-water bucket, 9-mounting frame, 10-fixed camera, 11-annular track, 12-sliding arm, 13-movable camera. DETAILED DESCRIPTION

[0037] To make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other implementation methods obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0038] Embodiment 1:

[0039] like Figure 1-Figure 3 As shown, this embodiment provides an efficient method for rapid propagation of Castanopsis schrenkiana tissue culture, using a tissue culture device to culture Castanopsis schrenkiana seedlings, the tissue culture device comprising a ring-shaped flow channel 1 and a support column 2 for supporting the flow channel 1, the flow channel 1 is a structural member made of transparent glass, and the method comprises the following steps:

[0040] Step 1: Obtain a number of explants and rinse and disinfect the explants. Specifically, pick the newly grown young shoots of the Castanopsis truncatula plant in the morning of March, then hydroponically cultivate and disinfect the young shoots, cut the hydroponically cultivated and disinfected young shoots into a number of explants, and then rinse and disinfect the explants. The specific method of rinsing and disinfecting is as follows:

[0041] First clean with detergent for 10 minutes, then rinse with running water for 30 minutes, then disinfect with 75% sodium sulfate for 30 seconds, then disinfect with 0.1% mercury solution for 10 minutes, and finally rinse with sterile water 6 times.

[0042] Step 2: inject the bud induction medium into the flow channel 1, and place a plurality of explants in the bud induction medium in sequence, and then drive the bud induction medium to drive the explants to flow in the flow channel 1, and callus tissue begins to appear and new buds sprout after 18 days. Before the explants are placed in the bud induction medium in the flow channel 1, the explants can also be placed in the callus induction medium for 2 days, and the explants are kept in a dark environment during these two days.

[0043] Step 3: When the new buds grow to 1 cm, replace the bud induction medium with the proliferation medium, and drive the proliferation medium to drive the explants to flow in the flow channel 1, and form clustered plants after 22 days.

[0044] Step 4: Place the rooting medium in a culture dish, then select a 2 cm single bud from the clump of plants and treat the single bud with rooting powder, then insert it into the rooting medium with tweezers, and take root after 20 days. After taking root, place the culture dish and the single bud in it in the sun for 15 days to form a Castanopsis truncatula seedling;

[0045] Step 5: Transplant the Castanopsis chinensis seedlings outdoors.

[0046] Through the above method, the culture is allowed to flow with the culture solution during the bud induction stage and the root formation stage. In this way, not only can the nutrients in the culture solution be fully mixed, but also harmful substances can be prevented from accumulating in local areas, and the flow process is more convenient for gas exchange. In addition, by controlling the flow rate, the culture can sometimes roll in the culture solution, thereby changing the posture of the culture in the culture solution, so that each area of ​​the culture can absorb nutrients and receive light more evenly, and it can also avoid the situation where a certain area of ​​the culture is always immersed in the culture and causes rot. Another point is that during the flow process, the situation of the plant being blown by the wind when growing outdoors can be simulated, which is more conducive to the growth of the culture and the adaptation to the outdoor wind blowing situation in advance before planting. Through this method, the tissue culture of Castanopsis sylvestris can be carried out more efficiently.

[0047] In step 2 and step 3, the culture solution can be made to flow at a constant speed of 1 cm / s in the flow channel 1. Optimally, the culture solution is driven to flow at a variable speed between 1 cm / s and 5 cm / s in step 2 and step 3, so that the culture can tumble in the culture solution, and the slow variable speed flow will not cause damage to the culture.

[0048] Optionally, the cross-sectional shape of the flow channel 1 in this embodiment is semicircular, and the tissue culture device further includes a water pump 3 installed below the flow channel 1. A water outlet is provided at the bottom of the flow channel 1, and a first barrier net is provided at the water outlet. The water inlet of the water pump 3 is connected to the water outlet through a water inlet pipe 4. A curved convex block is also provided at the bottom of the flow channel 1, and a water channel is provided in the curved convex block. The water outlet of the water pump 3 is connected to the water channel through a water outlet pipe 6, and the water outlet direction of the water channel is the same as the liquid flow direction in the flow channel 1. In step 2, the method for driving the bud induction culture medium to flow in the flow channel 1 is specifically as follows:

[0049] Start the water pump 3, use the water pump 3 to extract the bud inducing medium in the flow channel 1 from the water outlet and inject the bud inducing medium from the water channel into the flow channel 1 again. The first barrier net can prevent the explant from entering the water outlet. The water pump 3 extracts the bud inducing medium in the flow channel 1 and injects it into the flow channel 1 again, and the direction in which the bud inducing medium is injected into the flow channel 1 is the same as the direction of liquid flow in the flow channel 1, so that the bud inducing medium in the flow channel 1 can flow. By accurately controlling the pumping speed of the water pump 3, the speed of the bud inducing medium flowing out of the water channel can be accurately controlled, and then the flow speed of the bud inducing medium in the flow channel 1 can be controlled.

[0050] Optionally, in step 3, the method for driving the growth medium to flow in the flow channel 1 is specifically:

[0051] Start the water pump 3, use the water pump 3 to extract the planting medium in the flow channel 1 from the water outlet and inject the planting medium from the water channel into the flow channel 1 again. The first barrier net can prevent the culture from entering the water outlet. The water pump 3 extracts the planting medium in the flow channel 1 and injects it into the flow channel 1 again, and the direction in which the planting medium is injected into the flow channel 1 is the same as the direction of liquid flow in the flow channel 1, so that the planting medium in the flow channel 1 can flow. By accurately controlling the pumping speed of the water pump 3, the speed at which the planting medium flows out of the water channel can be accurately controlled, thereby controlling the flow speed of the planting medium in the flow channel 1.

[0052] Optionally, a drainage hole is provided at the bottom of the flow channel 1, a second barrier net is installed in the drainage hole, and a drainage pipe 7 is provided in communication with the drainage hole, and a sealing plug is installed at the drainage pipe 7. In step 3, the method of replacing the bud induction medium with the planting medium is specifically as follows:

[0053] The bucket 8 is placed under the drain pipe 7, and then the plug is removed. The bud induction medium in the flow channel 1 flows into the bucket 8 from the drain hole and the drain pipe 7, and then the plug is installed, and finally the planting medium is injected into the flow channel 1 from above. The released bud induction medium is caught by the bucket 8, so that it can be recycled for later disinfection and further treatment before reuse, or collected and poured out. The second barrier net can prevent the culture from entering the drain hole.

[0054] In step 3, after the water pump 3 is restarted, some bud induction medium will remain in the outlet pipe 6 and the inlet pipe 4, and this part of the bud induction medium will be injected into the proliferation medium again, so as to be mixed with the proliferation medium. In this way, it can avoid the overreaction of the culture due to excessive changes in the culture medium composition, which is more conducive to the tissue culture of Castanopsis schrenkiana.

[0055] Optionally, during step 2, observe whether the explant is browned. If browning occurs, remove the browned explant from the flow channel 1. The removal method is to use a spoon to scoop out the explant. Specifically, a mounting frame 9 is set up on the inner side of the flow channel 1, and a fixed camera 10 facing the flow channel 1 is installed on the mounting frame 9. The fixed camera 10 is connected to a computer, and an alarm light connected to the computer is also installed on the mounting frame 9. The specific method for observing whether the explant is browned is:

[0056] Turn on the fixed camera 10, use the fixed camera 10 to collect image information of the explant in the flow channel 1 in real time, and transmit the collected image information to the computer, use image processing technology to process the image information, and when brown color appears near the explant, it is determined that the explant has browned, and the computer controls the alarm light to light up.

[0057] More preferably, a circular track 11 is also installed on the mounting frame 9, a slide arm 12 is installed on the circular track 11, a movable camera 13 electrically connected to the computer is installed on the slide arm 12, and a motor is also installed on the mounting frame 9, the slide arm 12 is connected to the rotating shaft of the motor through a transmission mechanism, so that the motor can drive the slide arm 12 to drive the movable camera 13 to move on the circular track 11.

[0058] When brown appears near the explant, the motor starts and drives the active camera 13 to move to the explant near which brown appears, and the motor drives the active camera 13 to rotate synchronously with the explant, so that the active camera 13 can collect image information of the explant in real time. Of course, the image information is also processed using image processing technology to further determine whether the explant has produced browning.

[0059] In this method, with the help of the fixed camera 10 and the movable camera 13, the browning explants can be found in time and the staff can be prompted so that the browning explants can be removed from the culture medium as soon as possible to avoid contamination of other normal explants.

[0060] It should be noted that the above-mentioned callus induction medium, bud induction medium, proliferation medium and rooting matrix are all existing technologies, so they will not be described in detail here. In addition, image processing technology is also existing technology.

[0061] By the above method, in the bud induction stage and the root generation stage, the culture is made to flow with the culture solution, so that not only the nutrients in the culture solution can be fully mixed, but also the harmful substances can be prevented from accumulating in the local area, and the flow process is more convenient for gas exchange. In addition, by controlling the flow rate, the culture can sometimes roll in the culture solution, thereby changing the posture of the culture in the culture solution, so that each area of ​​the culture can absorb nutrients and receive light more evenly, and it can also avoid the situation where a certain area of ​​the culture is always immersed in the culture and causes rot. Another point is that during the flow process, the situation of the plant being blown by the wind when growing outdoors can be simulated, so as to be more conducive to the growth of the culture and to adapt to the outdoor wind blowing in advance before planting. Through this method, the tissue culture of the mountain ash can be carried out more efficiently. Efficient culture is specifically reflected in: more explants can be cultured at one time; the bud induction time and the time required for rooting are shorter.

[0062] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope of the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. An efficient method for rapid propagation of Castanopsis affine tissue culture, characterized in that: A tissue culture device is used to culture Castanopsis schrenkiana seedlings, wherein the tissue culture device comprises a flow channel in a ring shape and a support column for supporting the flow channel. The method comprises: Step 1: Obtain a plurality of explants and rinse and sterilize the explants; Step 2: injecting a bud induction medium into the flow channel, and placing a plurality of explants into the bud induction medium in sequence, and then driving the bud induction medium to drive the explants to flow in the flow channel, callus tissue begins to appear after 18 days, and new buds sprout; Step 3: When the new buds grow to 1 cm, the bud induction medium is replaced with the growth medium, and the growth medium is driven to drive the explants to flow in the flow channel, and clustered plants are formed after 22 days; Step 4: Place the rooting medium in a culture dish, then select a 2 cm single bud from the clump of plants and treat the single bud with rooting powder, then insert it into the rooting medium with tweezers, and take root after 20 days. After taking root, place the culture dish and the single bud in it in the sun for 15 days to form a Castanopsis truncatula seedling; Step 5: Transplant the Castanopsis chinensis seedlings outdoors.

2. The efficient rapid propagation method of Castanopsis affine tissue culture according to claim 1, characterized in that: In step 1, the method for flushing and disinfecting the explant is specifically as follows: First clean with detergent for 10 minutes, then rinse with running water for 30 minutes, then disinfect with 75% sodium sulfate for 30 seconds, then disinfect with 0.1% mercury solution for 10 minutes, and finally rinse with sterile water 6 times.

3. The efficient rapid propagation method of Castanopsis affine tissue culture according to claim 1, characterized in that: The cross-sectional shape of the flow channel is semicircular, and the tissue culture device also includes a water pump installed below the flow channel. A water outlet is opened at the bottom of the flow channel, and a first blocking net is arranged at the water outlet. The water inlet of the water pump is connected with the water outlet through a water inlet pipe. A curved convex block is also convexly arranged at the bottom of the flow channel, and a water channel is arranged in the curved convex block. The water outlet of the water pump is connected with the water channel through the water outlet pipe, and the water outlet direction of the water channel is the same as the flow direction of the liquid in the flow channel. In step 2, the method for driving the bud induction medium to flow in the flow channel is specifically: The water pump is started, and the bud induction medium in the flow channel is extracted from the water outlet by the water pump, and the bud induction medium is injected into the flow channel from the water channel again.

4. The efficient rapid propagation method of Castanopsis affine tissue culture according to claim 2, characterized in that: In step 3, the method for driving the culture medium to flow in the flow channel is specifically: The water pump is started, and the planting medium in the flow channel is extracted from the water outlet by the water pump, and the planting medium is injected into the flow channel from the water channel again.

5. The efficient rapid propagation method of Castanopsis affine tissue culture according to claim 1, characterized in that: A drainage hole is also provided at the bottom of the flow channel, a second barrier net is installed in the drainage hole, and a drainage pipe is connected to the drainage hole, and a sealing plug is installed at the drainage pipe; In step 3, the method of replacing the bud induction medium with the planting medium is specifically as follows: Place the bucket under the drain pipe, then remove the sealing plug. The bud induction medium in the flow channel will flow from the drain hole and the drain pipe into the bucket. Then install the sealing plug, and finally inject the planting medium into the flow channel from above.

6. The efficient rapid propagation method of Castanopsis affine tissue culture according to any one of claims 1 to 5, characterized in that: During step 2, observe whether the explants are browned. If browned, remove the explants from the flow channel.

7. The efficient rapid propagation method of Castanopsis affine tissue culture according to claim 6, characterized in that: A mounting frame is arranged on the inner side of the flow channel, a fixed camera facing the flow channel is mounted on the mounting frame, the fixed camera is connected to a computer, and an alarm light connected to the computer is also mounted on the mounting frame; The specific method for observing whether the explants are browning is: Turn on the fixed camera and use it to collect image information of the explant in the flow channel in real time, and transmit the collected image information to the computer. Use image processing technology to process the image information. When brown appears near the explant, it is determined that the explant has browned, and the computer controls the alarm light to light up.

8. The efficient rapid propagation method of Castanopsis affine tissue culture according to claim 7, characterized in that: A circular track is also installed on the mounting frame, a sliding arm is installed on the circular track, a movable camera electrically connected to the computer is installed on the sliding arm, and a motor is also installed on the mounting frame, the sliding arm is connected to the rotating shaft of the motor through a transmission mechanism, so that the motor drives the sliding arm to drive the movable camera to move on the circular track; When brown appears near the explant, the motor starts to drive the active camera to move to aim at the brown explant nearby, and the motor drives the active camera to rotate synchronously with the explant, so that the active camera can collect image information of the explant in real time.

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