CN103333801B - Sterile culture device of plant root border cells and method - Google Patents
Sterile culture device of plant root border cells and method Download PDFInfo
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- CN103333801B CN103333801B CN201310272549.1A CN201310272549A CN103333801B CN 103333801 B CN103333801 B CN 103333801B CN 201310272549 A CN201310272549 A CN 201310272549A CN 103333801 B CN103333801 B CN 103333801B
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- 241000196324 Embryophyta Species 0.000 title claims abstract description 51
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- 238000011065 in-situ storage Methods 0.000 claims abstract description 7
- 238000005070 sampling Methods 0.000 claims abstract description 7
- 238000005507 spraying Methods 0.000 claims abstract description 4
- 239000000428 dust Substances 0.000 claims description 41
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- 239000012528 membrane Substances 0.000 claims description 9
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 claims description 6
- 238000002474 experimental method Methods 0.000 claims description 6
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- 239000012498 ultrapure water Substances 0.000 claims description 6
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 claims description 5
- 239000007844 bleaching agent Substances 0.000 claims description 5
- 239000000460 chlorine Substances 0.000 claims description 5
- 229910052801 chlorine Inorganic materials 0.000 claims description 5
- 239000000284 extract Substances 0.000 claims description 5
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 4
- 238000004140 cleaning Methods 0.000 claims description 3
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- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Cultivation Of Plants (AREA)
Abstract
The invention discloses a sterile culture device of plant root border cells and a method. The invention aims to overcome the problems of being high in border cell loss amount and long in experimental period in the culture methods, such as water culture, soil culture and the like, and solve the disadvantages of being incapable of ensuring a sterile environment and cell collection amount and the like in the existing culture device of plant root border cells. The sterile culture device of plant root border cells disclosed by the invention comprises a cubic cabinet, a pulley track, a pulley in the track, a drawer clapboard, a pulley on the drawer clapboard, a switchable door, a switchable door hinge, a magnetic bump, a handle, an ultraviolet sterilizing lamp and a universal wheel; the cubic cabinet is formed from six surfaces in an encircling manner; the pulley track, the drawer clapboard, the ultraviolet sterilizing lamp and the like are arranged in the cubic cabinet; a through hole for plant roots to pass through is arranged on the drawer clapboard. The method disclosed by the invention comprises the following steps of: 1, preparing the sterile culture device of plant root border cells; 2, sterilizing plant seeds; 3, transferring the seeds into the sterile culture device of plant root border cells after germinating; and 4, carrying out spraying culture by using a humidifier, and carrying out in-situ sampling after achieving experimental requirements.
Description
Technical field
The present invention relates to a kind of plant root edge cell sterile culture apparatus and method.
Background technology
Root border cell derives from root cap meristematic tissue, be present in the loose connected water-soluble adhesive-layer of root cap, in water, stir gently or mechanical friction under easily from the tip of a root, separate, be the viable cell colony that has the active and biological significance of special physiological that is different from root cap cell.They have built a bioelectric interface between Gen Biao and soil; can synthesize specifically, secrete a series of metabolite; as materials such as the peroxidase of anthocyanidin, microbiotic, root nodule bacterium induction generation, tilactase, low molecular weight protein (LMWP), phenols, flavonoids; affect rhizosphere microecological system, play suppress pathogenic bacteria intrusion, reduce the vital role such as root system mechanical resistance, the protection tip of a root.The low molecular weight protein discharging as root border cell provides stable carbon source to extracellular environment, promotes beneficial microorganism growth, suppresses the growth of pathogenic bacteria; When aluminium poison exists, border cell strengthens the resistibility to aluminium by secreting mucus, produces active oxygen ROS and guides the programmed death of cell, and aluminium is fixed in the pectin of cell walls.
Root border cell sticks to root outer rim by water-soluble saccharan matrix, once this middle layer contact wetting or intracellular Rohapect MPE, will be dissolved, thus border cell is come off.Border cell, come off or people is for removing in 1h, just can obtain new border cell from root cap, after 24h, one group of 4000 new cell can form.But root border cell is very easy to come off, touching or friction just very easily split away off from root cap surface easily.When utilizing nutrient solution or substratum to cultivate, border cell easily comes off and enters in nutrient solution; And utilizing soil incubation, experimental observation is front in order to remove root table soil, thereby also easily border cell is eluted to the loss that causes border cell when root is cleaned.Under the condition of lack of water, root border cell can be close to root cap and be difficult to distinguish observation around.In addition, many environment all likely affect border cell's generation as nematode, germ, fungi and temperature etc.
Conventional root border cell collection method is: will to inserting bottom after showing money or valuables one carries unintentionally, be coated with in the culturing bottle of thick 7 ‰ nutrient agars of 2cm through sterilization, the clean seed culture of sterilized water, show money or valuables one carries unintentionally place outwardly, again culturing bottle back-off on the disk of 1/3 volume distilled water, put into incubator and carry out agar and hang empty cultivation by the feet, carry out afterwards root border cell's collection.This method steps is loaded down with trivial details, and need to prepare a large amount of glassware etc. to obtain the border cell of considerable amount.Existing plant root edge cell culturing device does not relate to gnotobasis, and in the system of opening wide, root border cell's generation is likely subject to the infection of the microorganism such as germ and fungi in environment, thereby causes root border cell death and the decomposition to release secretory product; Meanwhile, when cutting the tip of a root and collect root border cell, need running gear and inner netted culturing rack thereof, the mechanical external force that produces in its process is as easy as rolling off a log makes root border cell shake off from the tip of a root, thereby causes border cell's loss, affects the collimation of experimental result.Based on utilizing now the shortcomings such as water planting, earth culture training method border cell loss amount is large, experimental period is long, and existing plant root edge cell culturing device cannot guarantee the deficiencies such as gnotobasis and cell harvesting amount, in conjunction with collect observing border cell, need the method for high humidity, need invent a kind of cultivate simple to operate, culture environment is aseptic, it is efficient and convenient to sample, a large amount of root border cells' of disposable effective acquisition plant root edge cell sterile culture apparatus and method are carried out border cell's cultivation and collection.
Summary of the invention
The object of the invention is the shortcomings such as border cell's loss amount of the training methods such as water planting, earth culture is large in order to overcome, experimental period is long, and solve existing plant root edge cell culturing device and cannot guarantee the deficiencies such as gnotobasis and cell harvesting amount, a kind of plant root edge cell sterile culture apparatus and method are provided.These apparatus and method provide suitable border cell's morphologic observation environment, in the situation that guaranteeing that growing environment is aseptic, easy, cultivate and original position is collected border cell quickly and efficiently.
The technical solution adopted for the present invention to solve the technical problems is as follows:
Plant root edge cell sterile culture device of the present invention comprises that cubes cabinet, pulley track, track place pulley, dust panel, dust panel top sheave, switch gate, switch gate hinge, magnetic touch, handle, ultraviolet sterilization lamp, universal wheel.
Described cubes cabinet is enclosed and is formed by six faces, inner hollow; An one side is provided with switch gate, and the lower edge of switch gate is apart from cubes cabinet bottom surface 5cm, upper edge and cubes cashier's office in a shop Surface Edge along overlapping;
Switch gate is flexibly connected with adjacent left and right side, on its outer wall, is provided with handle; In a side inner walls adjacent with switch gate, be provided with magnetic and touch, magnetic touches and overlaps near the sidewall inside edge of switch gate, and switch gate inwall is provided with this magnetic and touches corresponding iron block; Switch gate is connected by switch gate hinge with another adjacent side;
On the left and right side inwall adjacent with switch gate, apart from lower surface, 2/3rds places are provided with pulley track, and pulley track is provided with track place pulley near one end of switch gate; On dust panel, be provided with dust panel top sheave, and be connected with pulley track by dust panel top sheave; Ultraviolet sterilization lamp is arranged on the medial surface of cubes cabinet, and this medial surface is corresponding with switch gate, and ultraviolet sterilization lamp is at cubes cashier's office in a shop between surface and dust panel.
Cubes cashier's office in a shop surface has a plurality of through holes, and for gas flow, through hole top is provided with diameter, and to be greater than through-hole diameter, aperture be the filter membrane of 0.22 micron, for filtering microbes in air; Cubes cabinet lower surface has bottom discharge hole, and is provided with mating valve, and its four right angle ends are provided with universal wheel; In cubes cabinet, the side corresponding with switch gate is provided with supply lead hole.
Described ultraviolet sterilization lamp is with bucker; On described dust panel, be provided with a plurality of through holes, hole size is determined according to actual size of plant seed.
A kind of plant root edge cell Sterile culture method comprises the following steps:
Step (1): select the full plant seed of uniform particles, tap water is soaked seed 10min for seed-coat sterilization with 10% chlorine bleach liquor after cleaning, and ultrapure water fully cleans to be placed in ultrapure water and soaks 6 hours;
Step (2): the seed after soaking is transferred in the culture dish that is stamped moistening gauze, and the constant incubator that is placed in lower 25 ℃ of dark surrounds is sprouted;
Step (3): be ready to plant root edge cell sterile culture device used;
Preparation comprises: the cubes of plant root edge cell sterile culture device cashier's office in a shop stickup aperture, surperficial through hole is 0.22 micron of filter membrane, on dust panel, put the two-layer gauze that soaks through high-temperature sterilization, switch gate is opened, put into closing switch door after the humidifier of the 0.1mM calcium chloride solution that fills sterilized water configuration, utilize ultraviolet sterilization lamp to carry out ultraviolet sterilization;
Step (4): after seed shows money or valuables one carries unintentionally, its uniform spreading is placed on the dust panel gauze of plant root edge cell sterile culture device.Humidifier atomizing is cultivated 48～72h, every 5min spraying once, the 30s that at every turn sprays, diurnal temperature is 26～22 ℃, lucifuge.After cultivation finishes, opening unit switch gate, extracts dust panel out plant root edge cell is carried out in situ sampling.
In described plant root edge cell sterile culture device, seed needs vertically to place to prevent dropping of border cell; Device and container used in described experiment all need through ultraviolet sterilization, and all solution need adopt sterilized water preparation; The adjustable humidifier switch of described mist flow is controlled; The timer that the described spray intervals time can connect by the outer humidifier plug of plant root edge cell device is controlled.
Plant root edge cell sterile culture device of the present invention use procedure is as follows:
1, cubes is pasted filter membrane in surperficial through hole cashier's office in a shop, on dust panel, put the two-layer gauze that soaks through high-temperature sterilization, switch gate is opened, put into closing switch door after the humidifier of the 0.1mM calcium chloride solution that fills sterilized water configuration, utilize ultraviolet sterilization lamp to carry out ultraviolet sterilization;
2, open switch gate again, put with the seed after 10% chlorine bleach liquor's sterilization on gauze, door closes a switch;
3, device is pushed to darkroom, start humidifier, seed starts growth, and the root that seed stretches out is through gauze and dust panel hole;
4, after seed growth reaches requirement of experiment, switch gate to be opened, dust panel is extracted out can carry out in-situ sampling to root border cell.
Beneficial effect of the present invention is as follows:
In device of the present invention, be provided with ultraviolet sterilization lamp, can be fine kill the inner bacterium of device, for border cell's cultivation provides gnotobasis, improve experiment accuracy; Device top is provided with through hole and is beneficial to circulation of air, is stamped one deck filter membrane for filtrated air microorganism on through hole simultaneously, contributes to keep the gnotobasis of seed growth; For seed is grown under high humidity environment, on dust panel upper berth, warp layer cloth is conducive to keep moisture, improves the humidity in seed periphery subenvironment, is beneficial to seed and root border cell's growth; Device dust panel can advance and extract out along pulley track, is convenient to root border cell to carry out in-situ sampling, and pulley track also plays the effect of fixedly maintenance level to dust panel; Bottom of device hole and valve are used for the inner unnecessary ponding of remover.Circuit hole is left in device dust panel starboard quarter, for walking ultraviolet sterilization lamp electric wire; Ultraviolet sterilization lamp and humidifier electric wire all can be by device and supply lead hole, rear portion pass UNICOM's power supply; Device is furnished with universal wheel, is convenient to movement; Device materials be take polyethylene as main.
The present invention compares with existing technology, can provide almost aseptic growing environment for plant root edge cell, have advantages of easy, quick, effectively cultivate and original position is collected plant root edge cell.
Accompanying drawing explanation
Fig. 1 is the structural representation of plant root edge cell sterile culture device of the present invention.
In figure, through hole 1, pulley track 2, track place pulley 3, dust panel 4, dust panel top sheave 5, switch gate 6, switch gate hinge 7, magnetic touch 8, handle 9, supply lead hole 10, bottom discharge hole 11, ultraviolet sterilization lamp 12, universal wheel 13.
Embodiment
Below in conjunction with accompanying drawing and example, plant root edge cell sterile culture apparatus and method of the present invention are described in detail:
As shown in Figure 1, plant root edge cell sterile culture device, comprises that cubes cabinet, pulley track 2, track place pulley 3, dust panel 4, dust panel top sheave 5, switch gate 6, switch gate hinge 7, magnetic touch 8, handle 9, ultraviolet sterilization lamp 12, universal wheel 13.
Described cubes cabinet is enclosed and is formed by six faces, inner hollow; On it, bottom surface is that the length of side is the square of 50 cm * 50 cm, and height is 60cm; An one side is provided with switch gate 6, and the lower edge of switch gate 6 is apart from cubes cabinet bottom surface 5cm, upper edge and cubes cashier's office in a shop Surface Edge along overlapping;
Switch gate 6 is flexibly connected with adjacent left and right side, is provided with handle 9 on its outer wall; In a side inner walls adjacent with switch gate 6, be provided with magnetic and touch 8, magnetic touch 8 with near the sidewall inside edge of switch gate 6, overlap, and switch gate 6 inwalls are provided with this magnetic and touch 8 corresponding iron blocks; Switch gate 6 is connected by switch gate hinge 7 with another adjacent side;
On the left and right side inwall adjacent with switch gate, apart from lower surface, 2/3rds places are provided with pulley track 2, and pulley track 2 is provided with track place pulley 3 near one end of switch gate; On dust panel 4, be provided with dust panel top sheave 5, and be connected with pulley track 2 by dust panel top sheave 5; Ultraviolet sterilization lamp 12 is arranged on the medial surface of cubes cabinet, and this medial surface is corresponding with switch gate, and ultraviolet sterilization lamp 12 cubes cashier's office in a shop surface and dust panel 4 between.
The upper surface of cubes cabinet has the through hole 1 that a plurality of diameters are 6cm, and for gas flow, through hole 1 top is provided with diameter, and to be greater than through-hole diameter, aperture be the filter membrane of 0.22 micron, for the airborne microorganism of filtering; The lower surface of cubes cabinet has bottom discharge hole 11, and is provided with mating valve, and its four right angle ends are provided with universal wheel 13; In cubes cabinet, the side corresponding with switch gate is provided with supply lead hole 10.
Described ultraviolet sterilization lamp 12 is with bucker; On described dust panel 4, be provided with a plurality of through holes, hole size is according to actual size of plant seed setting.
A kind of plant root edge cell Sterile culture method comprises the following steps:
Step (1): select the full plant seed of uniform particles, tap water is soaked seed 10min for seed-coat sterilization with 10% chlorine bleach liquor after cleaning, and ultrapure water fully cleans to be placed in ultrapure water and soaks 6 hours;
Step (2): the seed after soaking is transferred in the culture dish that is stamped moistening gauze, and the constant incubator that is placed in lower 25 ℃ of dark surrounds is sprouted;
Step (3): be ready to plant root edge cell sterile culture device used;
Preparation comprises: the cubes of plant root edge cell sterile culture device cashier's office in a shop surperficial through hole is pasted filter membrane, on dust panel, put the two-layer gauze that soaks through high-temperature sterilization, switch gate is opened, put into closing switch door after the humidifier of the 0.1mM calcium chloride solution that fills sterilized water configuration, utilize ultraviolet sterilization lamp to carry out ultraviolet sterilization;
Step (4): after seed shows money or valuables one carries unintentionally, its uniform spreading is placed on the dust panel gauze of plant root edge cell sterile culture device.Humidifier atomizing is cultivated 48～72h, every 5min spraying once, the 30s that at every turn sprays, diurnal temperature is 26～22 ℃, lucifuge.After cultivation finishes, opening unit switch gate, extracts dust panel out plant root edge cell is carried out in situ sampling.
In described plant root edge cell sterile culture device, seed needs vertically to place to prevent dropping of border cell; Device and container used in described experiment all need through ultraviolet sterilization, and all solution need adopt sterilized water preparation; The adjustable humidifier switch of described mist flow is controlled; The timer that the described spray intervals time can connect by the outer humidifier plug of plant root edge cell device is controlled.
Plant root edge cell sterile culture device of the present invention use procedure is as follows:
1, cubes surperficial through hole is pasted filter membrane cashier's office in a shop, extracts dust panel out and puts one deck thereon through the gauze that soaks of high-temperature sterilization, and switch gate is opened, and puts into closing switch door after humidifier, utilizes ultraviolet sterilization lamp to carry out ultraviolet sterilization;
2, open switch gate again, put with the seed after 10% chlorine bleach liquor's sterilization on gauze, door closes a switch;
3, device is pushed to darkroom, start humidifier, seed starts growth, and the root that seed germination goes out is through gauze and dust panel hole;
4, after seed growth reaches requirement of experiment, switch gate to be opened, dust panel is extracted out just can carry out in-situ sampling to root border cell.
Claims (2)
1. a plant root edge cell sterile culture device, is characterized in that comprising that cubes cabinet, pulley track, track place pulley, dust panel, switch gate, switch gate hinge, magnetic touch, handle, ultraviolet sterilization lamp, universal wheel;
Described cubes cabinet is enclosed and is formed by six faces, inner hollow; An one side is provided with switch gate, and the lower edge of switch gate is apart from cubes cabinet bottom surface 5cm, upper edge and cubes cashier's office in a shop Surface Edge along overlapping;
Described switch gate is flexibly connected with adjacent left and right side, on its outer wall, is provided with handle; In a side inner walls adjacent with switch gate, be provided with magnetic and touch, magnetic touches and overlaps near the sidewall inside edge of switch gate, and switch gate inwall is provided with this magnetic and touches corresponding iron block; Switch gate is connected by switch gate hinge with another adjacent side;
On the described left and right side inwall adjacent with switch gate, apart from lower surface, 2/3rds places are provided with pulley track, and pulley track is provided with track place pulley near one end of switch gate; On dust panel, be provided with dust panel top sheave, and be connected with pulley track by dust panel top sheave; Ultraviolet sterilization lamp is arranged on the medial surface of cubes cabinet, and this medial surface is corresponding with switch gate, and ultraviolet sterilization lamp is at cubes cashier's office in a shop between surface and dust panel;
The upper surface of described cubes cabinet has a plurality of through holes, and for gas flow, through hole top is provided with diameter, and to be greater than through-hole diameter, aperture be the filter membrane of 0.22 micron, for filtering microbes in air;
The lower surface of described cubes cabinet has bottom discharge hole, and is provided with mating valve, and its four right angle ends are provided with universal wheel, and in cubes cabinet, the side corresponding with switch gate is provided with supply lead hole;
Described ultraviolet sterilization lamp is with bucker; On described dust panel, be provided with a plurality of through holes, on it, be covered with the two-layer gauze that soaks.
2. utilize the plant root edge cell sterile culture device sterile culture plant root edge cell method described in claim 1, it is characterized in that comprising the following steps:
Step (1): select the full plant seed of uniform particles, tap water is soaked seed 10min for seed-coat sterilization with 10% chlorine bleach liquor after cleaning, and ultrapure water fully cleans to be placed in ultrapure water and soaks 6 hours;
Step (2): the seed after soaking is transferred in the culture dish that is stamped moistening gauze, and the constant incubator that is placed in lower 25 ℃ of dark surrounds is sprouted;
Step (3): be ready to plant root edge cell sterile culture device used
Preparation comprises: the cubes of plant root edge cell sterile culture device cashier's office in a shop stickup aperture, surperficial through hole is the filter membrane of 0.22 micron, on dust panel, put the two-layer gauze that soaks through high-temperature sterilization, switch gate is opened, put into closing switch door after the humidifier of the 0.1mM calcium chloride solution that fills sterilized water configuration, utilize ultraviolet sterilization lamp to carry out ultraviolet sterilization;
Step (4): after seed shows money or valuables one carries unintentionally, its uniform spreading is placed on the dust panel gauze of plant root edge cell sterile culture device; Humidifier atomizing is cultivated 48～72h, every 5min spraying once, the 30s that at every turn sprays, diurnal temperature is 26～22 ℃, lucifuge; After cultivation finishes, opening unit switch gate, extracts dust panel out plant root edge cell is carried out in situ sampling;
In described plant root edge cell sterile culture device, seed needs vertically to place to prevent dropping of border cell; Device and container used in described experiment all need through ultraviolet sterilization, and all solution need adopt sterilized water preparation; The adjustable humidifier switch of described mist flow is controlled; The timer that the described spray intervals time can connect by the outer humidifier plug of plant root edge cell sterile culture device is controlled.
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| CN2692156Y (en) * | 2004-03-16 | 2005-04-13 | 喻敏 | plant root edge cell culturing device |
| CN101384717A (en) * | 2006-02-14 | 2009-03-11 | 瑞克斯旺种苗集团公司 | Non-destructive procedure for the isolation of DNA from plants |
| CN101534633A (en) * | 2006-09-14 | 2009-09-16 | 欧·福斯博格 | Method and device for micropropagation of plants |
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2013
- 2013-06-28 CN CN201310272549.1A patent/CN103333801B/en active Active
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|---|---|---|---|---|
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