CN112753576A - Plant tissue culture method and plant tissue culture container thereof - Google Patents
Plant tissue culture method and plant tissue culture container thereof Download PDFInfo
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- CN112753576A CN112753576A CN202011632846.9A CN202011632846A CN112753576A CN 112753576 A CN112753576 A CN 112753576A CN 202011632846 A CN202011632846 A CN 202011632846A CN 112753576 A CN112753576 A CN 112753576A
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- 238000004161 plant tissue culture Methods 0.000 title claims abstract description 54
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The utility model provides a plant tissue culture container, is in including vessel (1), container lid (2) and setting breather (30) on the container lid (2), its characterized in that, it has side door (6) to articulate on the lateral wall of vessel (1), be provided with flourishing magazine (4) that hold the culture medium in the plant tissue culture container for hold layer board (5) of group's tissue culture plant, layer board (5) are shelved on flourishing magazine (4). Compared with the traditional plant tissue culture method, the invention places the plant tissues on the supporting plate, and only the nutrient solution is replaced during the subculture, and the tissue culture container is not required to be replaced, thereby greatly reducing the labor cost and the material cost. Because the plant tissues do not need to be cut from a solid culture medium and then transferred into a new tissue culture container, the cutting times of the tissue culture plants are greatly reduced, the risk of secondary pollution is reduced, the tissue culture period is shortened, and the survival rate is improved.
Description
Technical Field
The invention relates to the technical field of plant cultivation, in particular to a plant tissue culture method and a plant tissue culture container thereof.
Background
Plant tissue culture, plant tissue culture for short, refers to a process of separating plant materials (tissues or organs) as explants under aseptic conditions by utilizing the totipotency of plant cells, inoculating the explants onto a culture medium, and culturing under artificial conditions to grow, differentiate and regenerate the explants into complete plants. At present, the plant tissue culture technology is widely developed and applied in the aspects of plant in-vitro rapid propagation, detoxified seedling cultivation, genetic breeding, transgenosis, germplasm resource preservation and the like.
The traditional plant tissue culture method generally comprises four steps: inducing callus, namely sterilizing and inoculating the explant and the culture medium to obtain callus or organ. Multiplication and subculture, continuous differentiation to generate new plants, or direct generation of adventitious buds and embryoids, or repeated subculture as required to achieve mass propagation. And thirdly, rooting. And fourthly, domestication, namely after the test-tube plantlet is taken out of the bottle, performing an adaptation process to the external environment for a certain time, and after the four steps, taking the test-tube plantlet as a production plantlet. The main reasons are that the traditional tissue culture mode using agar as a coagulant and the tissue culture container space thereof are too small, which causes frequent subculture, difficult rooting and low survival rate of transitional seedlings, which are common problems in the tissue culture of plants, and the main reasons are that the traditional tissue culture mode using agar as a coagulant is not air-permeable and the structure of the tissue culture container is unreasonable, and the whole process of the tissue culture of plants needs frequent subculture operation to replace the nutrition and the tissue culture container, which increases the workload and labor cost in the tissue culture operation to a great extent, and also easily brings bacteria into the container, which causes secondary pollution and failure of the tissue culture, while various tissue culture containers and using methods thereof on the market can not be suitable for the whole-process culture of plant tissues from induction, callus proliferation and subculture to rooting and domestication.
Disclosure of Invention
Therefore, a plant tissue culture method and a plant tissue culture container thereof are needed, which reduce the subculture operation, reduce the labor cost, replace the traditional agar culture medium and are suitable for the whole-process culture of the plant tissue culture.
The technical scheme for solving the technical problems is as follows:
a method for tissue culture of plants, comprising the steps of: in the tissue culture process, plant tissues are placed on a bearing object in a tissue culture container, the bearing object can be contacted with or separated from nutrient substances, when the nutrient substances in a culture medium are exhausted or other nutrient substances are needed, only the nutrient medium is replaced, and the tissue culture plants are still kept on the bearing object.
Plant tissue carrier and nutrient substance can mutual separation, and when plant growth enters different stages, required environment and nutrient substance change, the tissue culture utensil does not need to be changed, and nutrient substance only needs to be changed, just can realize that a container carries out the operation of multiple stage step, has reduced the subculture operation, the cost is reduced.
Another object of the present invention is to provide a plant tissue culture container for implementing the above plant tissue culture method, wherein the technical scheme is as follows:
the utility model provides a plant tissue culture container, includes vessel, container lid and sets up breather on the container lid, it has the side door to articulate on the lateral wall of vessel, be provided with the flourishing magazine that holds the culture medium in the plant tissue culture container for hold the layer board of group's tissue culture plant, the layer board is shelved on flourishing magazine.
The containing box that holds the culture medium sets up separately with the layer board of placing plant tissue, when carrying out the successive transfer nutrition and changing, only need take out containing box, and the layer board need not to take out plant tissue culture container with the plant tissue that is in the cultivation in-process, has reduced the risk that the plant tissue of cultivating is by bacterial contamination, also the cost is reduced.
The inside flourishing magazine that is equipped with the culture medium of holding of container and places group culture plant alone both can contact the matrix and can break away from the layer board of matrix again, gets into different stages when vegetation, and when required environment and nutrient change, need not to change group and cultivate the utensil, only relies on the flourishing magazine that open and shut side door can take out to realize that a container carries out the operation of a plurality of stages step.
Adopt the side door design, operating personnel gets from the side and puts the flourishing magazine, when having avoided operating from the top, wind on the superclean bench easily blows in the tissue culture container with the germ of operator on hand, has reduced the pollution risk.
Further, be provided with seal part on the side door, when making the side door close, can with container body is sealed.
When the side door is closed, the side door and the container body form sealing, so that the pollution risk is reduced.
Further, be provided with the layer board suspension device that is used for placing the layer board in the plant tissue culture container.
When the material containing box is taken out, the supporting plate can be placed on the supporting plate suspension device, and the supporting plate, the material containing box, the supporting plate and the side door form a step to complete a plurality of successive transfer steps, so that the operation is simplified, the efficiency is improved, the traditional tissue culture method is changed, and the target of the tissue culture level is improved.
Further, the ventilation device comprises a natural ventilation assembly and a forced ventilation assembly.
Further, the natural ventilation assembly includes an intake air amount adjusting part capable of manually adjusting ventilation in the container in the absence of bacteria.
According to the needs, the air input of the natural ventilation assembly can be adjusted, the air exchange capacity of the culture container is completed, and the requirements of plant growth ventilation and light transmission are met.
Furthermore, the forced ventilation assembly is provided with an interface which can isolate bacteria and connect the air pump to fill air.
The forced ventilation assembly can be connected with the pump, when the container is required, the humidity, the carbon dioxide content and the temperature in the container can be rapidly adjusted through the pump, the defects of the natural air inlet assembly are overcome, and the purpose of arbitrarily, conveniently and adjustably adjusting the small air temperature in the container is achieved together with the natural ventilation assembly. Meanwhile, an antibacterial filtering membrane is also sealed in the forced air inlet device, so that the risk of bacteria entering the forced air inlet device is prevented.
Furthermore, a nutrient solution inlet channel and a liquid discharge channel are arranged on the container body.
When the liquid nutrient solution is independently adopted for tissue culture, the nutrient solution can be directly contained in the tissue culture container without using a material containing box. When the nutrient solution is supplemented, the nutrient solution is added into the container through the nutrient solution inlet channel. When the nutrient solution is replaced, the original nutrient solution is discharged from the liquid discharge channel, and the new nutrient solution is added from the liquid inlet channel, so that the nutrient solution is more convenient to supplement and replace. The liquid inlet pipeline and the liquid discharge pipeline can also be connected with a control system to realize automatic tissue culture.
Further, the container lid is a non-planar structure.
The non-planar structure of the container cover can prevent the water vapor in the container body from condensing on the container cover, and ensures that the light is transparent and does not influence the growth of plants. Furthermore, the inner wall of the container cover is sprayed with a material for preventing the adhesion of condensed water.
The condensation-preventing material can further enhance the effect of preventing condensation of water vapor.
Drawings
FIG. 1 is an assembly view of a plant tissue container;
FIG. 2 is a view showing the internal structure of the plant tissue container;
FIG. 3 is an assembly view of the material holding box and the support plate;
FIG. 4 is a perspective view of the vent;
fig. 5 is a cross-sectional view of the vent in an assembled state.
In the drawings, the components represented by the respective reference numerals are listed below:
1 container body, 2 container cover, 21 fourth sealing ring, 30 breather device, 3 natural ventilation component, 31 breather pipe, 311 first protrusion, 312 convex rib, 313 positioning rotation-stopping port, 32 second sealing ring, 33 anti-bacterial membrane cover, 331 first upper cover surface, 3311 first opening, 332 first side wall, 3321 positioning rotation-stopping sheet, 34 ventilating cover, 341 second upper cover surface, 3411 second opening, 342 second side wall, 3412 groove, 4 material containing box, 5 supporting plate, 51 supporting plate suspension device, 52 hanging hole, 6 side door, 61 first sealing ring, 7 forced ventilation component, 71 ventilating hole, 72 lower cover, 721 third side wall, 722 fourth side wall, 723 third cover surface, 724 fourth cover surface, 7231 first cover surface, 73 gasket, 74 third sealing ring, 75 upper cover, 751 fifth side wall, 7511 second protrusion, 752 fifth cover surface, 7521 second ventilating pipe, 81 short pipe, 82, 83 plug, 9 liquid discharge pipe.
Detailed Description
The principles and features of this invention are described below in conjunction with the following drawings, which are set forth by way of illustration only and are not intended to limit the scope of the invention.
As shown in fig. 1, the plant tissue culture container of the present invention comprises a container body 1, a container cover 2 and an air vent device 30 arranged on the container cover 2, wherein a side door 6 is hinged on the side wall of the container body 1, a material containing box 4 for containing culture medium is arranged in the plant tissue culture container, a support plate 5 for containing tissue culture plants is arranged in the plant tissue culture container, and the support plate 5 is arranged on the material containing box 4.
The material containing box 4 can contain a water absorption substrate bag and nutrient solution, can also be poured into a traditional agar culture medium or can contain a culture medium mixed with the nutrient solution, and has no strict limitation on the shape and the size. In order to enable the tissue culture plants to better absorb nutrition, the support plate 5 is preferably of a hollow structure, the support plate 5 is placed on the material containing box 4, and the size and the shape of the support plate 5 are matched with those of the material containing box 4. Preferably, a pallet suspension 51 is provided inside the container, said pallet suspension 51 being used for placing the pallets 5 when the magazine 4 is taken out. The pallet suspension device 51 is only required to temporarily place the pallet 5. In one embodiment, the tray suspension device is composed of two side-by-side hooks 51 disposed near the container wall, and correspondingly, the tray 5 is provided with a hanging hole 52 matched with the hooks.
Fig. 3 shows an embodiment of a cartridge 4 and a pallet 5. The material containing box 4 is a box matched with the bottom of the tissue culture container in shape and size, the supporting plate 5 comprises a bottom plate and a fence, and a clamping block is arranged on the fence and clamped on the material containing box 4. The bottom plate of the supporting plate 5 is of a frame structure, and two hanging holes 52 matched with the hooks are arranged on the bottom plate.
The side wall of the container is hinged with a side door 6, and in a preferred embodiment, a sealing component is arranged on the side door, and the sealing component is preferably a first sealing ring 61.
In a preferred embodiment, a liquid inlet channel and a liquid outlet channel are provided in the container body. When pure liquid nutrient solution is used for tissue culture, the material containing box 4 is not required to be placed, but the nutrient solution is directly filled in the container, and the nutrient solution is input or output through the liquid inlet channel and the liquid discharge channel. A seedbed for placing plant tissues is arranged in the container, and the seedbed structure adopts the prior art and is not shown in the figure. Fig. 1 shows a structure of a liquid inlet passage and a liquid discharge passage, the liquid inlet passage includes a through hole formed in a side wall of a container body, a short pipe 81 extending from a periphery of the through hole, a plug 82 for plugging the short pipe 81, and a pressing cover 83 for pressing the plug 82 against the short pipe 81; the liquid discharge channel comprises a liquid discharge pipe 9 arranged at the bottom of the container, and a valve is arranged on the liquid discharge pipe.
For regulating the microclimate in the plant tissue culture container, an aeration device 30 is provided on the container lid. The aeration device 30 preferably includes a natural aeration assembly 3 and a forced aeration assembly 7. The natural draft assembly 3 enables the size of the air inlet to be manually adjusted, thereby adjusting the amount of ventilation. When the natural ventilation component 3 is not enough to adjust the ventilation volume, a forced ventilation component 7 can be used, the forced ventilation component 7 comprises an interface connected with an air pump, the air in the plant tissue culture container can be rapidly pumped out through the air pump, and fresh air can also be rapidly introduced into the container.
In one embodiment, the natural-venting assembly 3 includes a vent tube 31 disposed on the container lid 2, a second sealing ring 32, an anti-microbial membrane cover 33, and a gas-permeable cover 34. The internal structure of the vent pipe 31 is shown in fig. 4. A circle of first protrusions 311 are arranged on the inner wall of the vent pipe 31 in the circumferential direction, and a circle of convex ribs 312 are arranged on the outer wall in the circumferential direction; the anti-bacterial membrane cover 33 comprises a first upper cover surface 331 and a first side wall 332, the first side wall 332 presses the second sealing ring 32 on the first protrusion 311, and the first upper cover surface 331 is provided with a first opening 3311; the breathable cover 34 includes a second upper cover 341 and a second sidewall 342, the second upper cover 341 has a second opening 3411, the inner wall of the second sidewall 342 has a groove 3421 along the circumferential direction, the groove 3421 is matched with the rib 312, and the groove 3421 is rotatably connected with the rib 312. When the air-permeable cover 34 is rotated, the repetition area of the second opening 3411 with the first opening 3311 can be adjusted, thereby adjusting the ventilation amount. In order to prevent bacteria in the air from entering the container from the natural ventilation assembly 3, a bacteria-proof membrane, which is not shown, may be press-fitted on the first protrusion 311 using the second sealing ring 32.
A plurality of positioning rotation stopping openings 313 are uniformly distributed on the inner wall of the vent pipe 31 along the circumferential direction, and positioning rotation stopping sheets 3321 matched with the positioning rotation stopping openings 313 are arranged on the outer wall of the first side wall 332 along the circumferential direction. The number and shape of the positioning rotation-stopping openings 313 and the positioning rotation-stopping pieces 3321 are not strictly limited, and can be set according to actual requirements.
The number of natural ventilation assemblies 3 is not strictly limited and may be set according to actual requirements.
The form of the natural ventilation assembly 3 is not limited strictly, and can be round square ball type or sliding push rotation, and preferably round rotation opening and closing.
In order to further improve the air permeability of the tissue culture container, a forced ventilation assembly 7 is further arranged on the container cover 2, the forced ventilation assembly 7 comprises a ventilation hole 71, a lower cover 72, an upper cover 75, a gasket 73 and a third sealing ring 74 which are arranged on the container cover 2, the lower cover 72 comprises a third side wall 721 and a fourth side wall 722, the end surface of the third side wall is provided with a third cover surface 723, the third cover surface 723 is provided with a first ventilation pipe 7231, the end surface of the fourth side wall 722 is provided with a fourth cover surface 724, the fourth cover surface 724 is provided with a plurality of through holes, and the outer surface of the fourth side wall 722 is provided with threads; the upper cover 75 comprises a fifth side wall 751 and a fifth cover surface 752, wherein the inner wall of the fifth side wall 751 is provided with threads, the end surface of the fifth side wall 751 extends outwards along the radial direction to form a circle of second protrusions 7511, and the fifth cover surface 752 is provided with a second vent pipe 7521; the lower cover 72 is clamped in the vent hole 71, the gasket 73 is sleeved on the fourth side wall 722, the third sealing ring 74 presses the antibacterial film on the fourth cover surface 724, and the lower cover 72 is in threaded connection with the upper cover 75.
The structure of each part of the natural air intake assembly 3 and the forced draft assembly 7 is shown in fig. 4, and the assembly mode of each part is shown in fig. 5.
The number of the forced ventilation assemblies 7 is not critical and can be set according to the actual requirements, and preferably one forced ventilation assembly 7 is arranged on the container cover 2.
The distribution of the natural-ventilation assembly 3 and the forced-ventilation assembly 7 on the container lid 2 is not critical, and it is preferable that the natural-ventilation assembly 3 is disposed at the center of the container lid 2 and the forced-ventilation assembly 7 is disposed adjacent to the natural-ventilation assembly 3.
In a preferred embodiment, the container lid 2 is dome shaped.
In a preferred embodiment, the container cover 2 is detachably connected to the container body 1, and a fourth sealing ring 21 is disposed between the container cover 2 and the container body 1.
The size of the plant tissue culture container is not strictly limited, and can be set according to actual requirements, and the length of the plant tissue culture container is preferably 20mm-500mm, the width of the plant tissue culture container is preferably 10mm-300mm, and the height of the plant tissue culture container is preferably 100mm-300 mm.
The working principle is as follows: the first protrusion 311 is pressed with a second packing 32 having the same size as the inner diameter of the vent pipe 31, and the vent pipe 31 is covered with the anti-bacterial film cover 33 and the ventilation cover 34. An anti-bacterial membrane is also provided in the forced air assembly 7.
The working modes of the plant tissue culture container comprise the following four working modes:
in the first working mode, water absorbing materials such as a substrate bag, a water retaining cloth bag or water absorbing cotton are placed in a material containing box 4, a layer of water absorbing cotton is sleeved on a supporting plate 5, and the plant tissue culture container is placed in a super-clean workbench after being sterilized integrally;
opening a container side door 6, and pouring the sterilized nutrient solution into the material containing box 4;
cutting the sterilized plant material or sterile plant tissue, inoculating the cut plant material or sterile plant tissue on a supporting plate 5 sleeved with absorbent cotton, and placing the plant material or sterile plant tissue on a material containing box 4 for tissue culture;
changing the culture medium according to the nutrient condition of the tissue culture plant, lifting a supporting plate 5 for culturing plant materials to be hung on a supporting plate suspension device 51 during the change, taking out the material containing box 4 to be changed, putting the newly sterilized material containing box 4, pouring new sterile nutrient solution, putting the supporting plate 5 down, and closing a side door 6;
and (3) placing the plant tissue culture container in a proper environment for culture.
The method for tissue culture can complete the secondary nutrition replacement only by replacing the material containing box 4 and the integrated matrix bag or water absorbing material, thereby greatly reducing the labor amount during the secondary tissue culture and reducing the secondary pollution in the operation process. The tissue culture plant by the method has faster growth speed than the traditional solid culture medium, and the production period is shortened.
In the second working mode, a layer of absorbent cotton material is placed on the seedbed and is placed in the container; opening the side door 6 of the container, and pouring the sterilized nutrient solution to the height of the seedbed;
cutting the sterilized plant material or aseptic plant tissue, inoculating to a seedbed covered with absorbent cotton material, and performing tissue culture;
the liquid discharge pipe 9 and the short pipe 81 are connected with a transfusion port and a liquid outlet, and the input and the output of the nutrient solution and the waste liquid are controlled by an automatic control system. The culture medium is replaced according to the nutrition condition of the tissue culture plant, and the nutrient solution is automatically replaced according to a freely set program, so that the automatic tissue culture is realized.
In the third working mode, the whole culture container is sterilized and then placed in a super clean workbench, a container side door 6 is opened, the sterilized agar culture medium is poured into a material containing box 4, and after the culture medium is solidified, plant tissues are inoculated for tissue culture; taking out the old material containing box according to the nutrition condition of the tissue culture plant, transferring the graft material to the material containing box 4 containing new culture medium, then putting the new material containing box into the culture container, and continuing culturing.
And in the fourth working mode, the whole culture container is sterilized and then placed in an ultra-clean workbench, a container side door 6 is opened, a culture medium mixed with nutrient solution after sterilization is poured into a material containing box 4, and the plant material to be rooted is transferred into the medium, so that the one-step culture of rooting and domestication of the plant material is realized.
The plant tissue culture method of the present invention will be further described with reference to the following specific examples.
The embodiment provides a method for carrying out the subculture proliferation of plant tissues by using the plant tissue culture container. The tissue culture vessels used in the examples and comparative examples had a length of 500mm, a width of 300mm and a height of 300 mm.
Example 1
The tissue culture material adopted in the embodiment is tobacco leaves, and is derived from aseptically sown tobacco bottle seedlings.
The embodiment provides a method for performing tobacco tissue culture by adopting the plant tissue culture container, which comprises the following steps:
s1, callus induction culture: the callus induction liquid culture medium formula is that MS +6-BA 1.5mg/L + NAA 0.1mg/L + sucrose 30g/L is sterilized at 121 ℃ for 30min under high pressure and cooled, then poured into a material containing box 4 in an aseptic operation platform, thick and dark green leaves of well-grown tobacco cultured for about 50 days by aseptic seeding are selected as explants, the edges of the leaves are cut off by aseptic operation, the leaves are cut to about 1cm and 1cm, and then inoculated on absorbent cotton on a supporting plate 5, callus is induced, and the inductivity is 100% about 7 days after the leaves are inoculated.
S2, shoot induction culture: the formula of the bud induction liquid culture medium is (1.5 mg/L of MS +6-BA +0.1mg/L of NAA + 30g/L of cane sugar), is consistent with the formula of callus induction, does not need to be replaced, and can differentiate buds about 13 days after the leaves are inoculated.
S3, subculture bud multiplication culture: clamping the supporting plate 5 and placing the supporting plate on a bracket suspension device 51, clamping out the material containing box 4 after nutrition consumption, placing the sterilized material containing box 4, pouring a sterilized cooled liquid culture medium into the material containing box 4, taking the supporting plate 5 off the bracket suspension device 51, placing the supporting plate on the material containing box 4 for subculture proliferation, continuously culturing for 25 days, wherein the proliferation of subculture buds is not limited, a large number of small buds are differentiated, and the proliferation coefficient is 16.5.
S4, rooting culture: clamping the supporting plate 5 and placing on a second bracket 51, clamping out the material containing box 4 after nutrition consumption, then placing the sterilized material containing box 4, pouring sterilized cooled rooting liquid culture medium (MS +0.1 mg/LIBA), taking out the differentiated 1-2cm small bud groups, cutting into small blocks, spreading the small blocks on the supporting plate 5 without overlapping, taking the supporting plate 5 off from the second bracket 51, placing on the material containing box 4 for rooting culture, and continuously culturing for 20d to obtain well-grown rooted seedlings.
The culture conditions were: dark culture is carried out at 24-25 ℃ in the first 2 days after inoculation, and light culture is carried out at 28 ℃, 1500-2000 LX from the 3 rd day after inoculation and is carried out for 12-13 h every day.
The culture period was 58 days.
Comparative example 1
The comparative example provides a method for tissue culture of tobacco by using a tissue culture container in the prior art, which comprises the following steps:
s1, callus induction culture: the callus induction solid culture medium formula is MS +6-BA 1.5mg/L + NAA 0.1mg/L + sucrose 30g/L + agar 9.4g/L, thick and dark green leaves of NC89 with good growth and cultured for about 50 days in aseptic seeding are selected as explants, the edges of the leaves are cut off by aseptic operation, the leaves are cut to about 1cm x 1cm in size and then inoculated on a culture dish, callus is induced, and the induction rate is 100 percent about 7 days after the leaves are inoculated.
S2, shoot induction culture: the formula of the bud induction solid culture medium is (1.5 mg/L of MS +6-BA +0.1mg/L of NAA + 30g/L of sucrose + 9.4g/L of agar, the bud induction solid culture medium is consistent with the formula of callus induction and does not need to be replaced, and buds are differentiated about 20 days after the leaves are inoculated.
S3, subculture bud multiplication culture: the subculture medium is a formula I, cutting callus blocks with buds, transferring the callus blocks into a new culture dish for subculture multiplication, wherein the multiplication coefficient is 8.5, and buds with the length of 1-1.5cm are differentiated after 25 days.
S4, rooting culture: a rooting solid culture medium, namely MS, 0.1mg/LIBA and 9.4g/L of agar, cutting the differentiated plantlet of 1-1.5cm, transferring the plantlet into a rooting bottle for rooting culture, and continuously culturing for 25d to obtain a rooted seedling.
The culture conditions were: dark culture is carried out at 24-25 ℃ in the front 2d after inoculation, and light culture is carried out at 26-27 ℃ and 1500-2000 LX from the 3 rd after inoculation for 12-13 h every day.
The culture period was 70 days.
As can be seen from the comparison between example 1 and comparative example 1, when the plant tissue culture container of the invention is used for tobacco tissue culture, the multiplication coefficient can reach 16.5, the culture period can be shortened to 58 days, the steps of cutting and healing in successive transfer operation are reduced, and the operation pollution risk is reduced.
The tissue culture material is rice callus.
Example 2
The embodiment provides a tissue culture method for rice callus differentiation by adopting the plant tissue culture container, which comprises the following steps:
s1, rice callus differentiation culture: the formula of the liquid culture medium for the callus differentiation of the rice is MS (improved) +6-BA2mg/L + KT2mg/L + NAA0.2mg/L + IAA0.2mg/L + CH 1g/L + sucrose 30g/L, the sterilized and cooled culture solution for the differentiation of the rice is poured into a material containing box 4, the sterilized and cooled culture solution for the differentiation of the rice is transferred to a cotton material of a supporting plate 5 for differentiation culture, differentiation and seedling emergence are started in 15 days, and the differentiation rate is 90% after 30 days.
The culture conditions were: after the inoculation, the culture is carried out by adopting light with the temperature of 28 ℃ and the light of 1500-2000 LX for 12-13 h every day.
The emergence time was 15 days.
Comparative example 2
The comparative example provides a method for performing rice callus differentiation tissue culture by using a tissue culture container in the prior art, which comprises the following steps:
s1, rice callus differentiation culture: the rice callus differentiation solid medium formula MS (modified) +6-BA2mg/L + KT2mg/L + NAA0.2mg/L + IAA0.2mg/L + CH 1g/L + sucrose 30g/L + agar 9.4g/L, the Nipponbare callus is transferred to a culture dish for differentiation culture, differentiation and seedling emergence are started in 28 days, and after 30 days, the differentiation rate is 60%.
The culture conditions were: after the inoculation, the culture is carried out by adopting light with the temperature of 28 ℃ and the light of 1500-2000 LX for 12-13 h every day.
The emergence time was 28 days.
As can be seen from the comparison between example 2 and comparative example 2, the plant tissue culture container of the present invention, when used for callus differentiation tissue culture of rice, can improve the differentiation rate by 30% and shorten the seedling emergence time by 12 days.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (10)
1. A method for plant tissue culture is characterized by comprising the following steps: in the tissue culture process, plant tissues are placed on a bearing object in a tissue culture container, the bearing object can be in contact with or separated from nutrient substances, and when the nutrient substances are exhausted or other nutrient substances are needed, only the nutrient substances are replaced, and the tissue culture plants are still kept on the bearing object.
2. A plant tissue culture container for realizing the plant tissue culture method according to claim 1, which comprises a container body (1), a container cover (2) and an air breather (30) arranged on the container cover (2), and is characterized in that a side door (6) is hinged on the side wall of the container body (1), a material containing box (4) for containing a culture medium is arranged in the plant tissue culture container, a supporting plate (5) for containing tissue culture plants is arranged in the plant tissue culture container, and the supporting plate (5) is arranged on the material containing box (4).
3. The plant tissue culture container according to claim 2, wherein the side door (6) is provided with a sealing component, so that the side door (6) can be sealed with the container body (1) when closed.
4. The plant tissue culture container according to claim 2, wherein a supporting plate suspension device (51) for placing the supporting plate (5) is arranged in the plant tissue culture container.
5. A plant tissue culture container according to claim 2, characterized in that the aeration means (30) comprise a natural aeration assembly (3) and a forced aeration assembly (7).
6. The plant tissue culture container according to claim 5, wherein the natural ventilation assembly (3) comprises an air inflow adjusting component, and the ventilation in the container can be manually adjusted under the condition of isolating bacteria.
7. The plant tissue culture container according to claim 5, wherein the forced ventilation component (7) is provided with an interface which can be connected with an air pump for air filling under the condition of isolating bacteria.
8. The plant tissue culture container according to claim 2, wherein the container body (1) is provided with a nutrient solution inlet channel and a nutrient solution outlet channel.
9. The plant tissue culture container according to claim 2, wherein the container lid (2) is a non-planar structure.
10. The plant tissue culture container according to claim 9, characterized in that the inner wall of the container cover (2) is sprayed with a material for preventing the adhesion of condensed water. And is sprayed with a material for preventing water from being attached.
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|---|---|---|---|---|
| CN102835310A (en) * | 2012-05-12 | 2012-12-26 | 姜伟 | Double-tray combined type plant tissue culture container |
| CN204482615U (en) * | 2015-03-16 | 2015-07-22 | 武汉叶动力生物科技有限公司 | One Plants aseptic culture device |
| CN205596816U (en) * | 2016-03-26 | 2016-09-28 | 山西大同大学 | Plant tissue culture case |
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| CN209201779U (en) * | 2018-12-18 | 2019-08-06 | 北京工商大学 | Plant callus subculture increment culture apparatus |
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| CN102835310A (en) * | 2012-05-12 | 2012-12-26 | 姜伟 | Double-tray combined type plant tissue culture container |
| CN204482615U (en) * | 2015-03-16 | 2015-07-22 | 武汉叶动力生物科技有限公司 | One Plants aseptic culture device |
| CN205596816U (en) * | 2016-03-26 | 2016-09-28 | 山西大同大学 | Plant tissue culture case |
| CN107484660A (en) * | 2017-09-16 | 2017-12-19 | 武汉叶动力生物科技有限公司 | A kind of plant tissue culture method using incubator |
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