CN103734017A - Tissue culturing method for water caltrop - Google Patents
Tissue culturing method for water caltrop Download PDFInfo
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- CN103734017A CN103734017A CN201410029624.6A CN201410029624A CN103734017A CN 103734017 A CN103734017 A CN 103734017A CN 201410029624 A CN201410029624 A CN 201410029624A CN 103734017 A CN103734017 A CN 103734017A
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Abstract
The invention discloses a tissue culturing method for a water caltrop. The tissue culturing method comprises the following steps: clipping a stem with nodes from a submerged plant, namely water caltrop, soaking the stem in tap water, then cutting blades, and rinsing with deionized water; sterilizing in an ultraclean workbench, and then cutting the stem into small sections being about 1cm with each section including a node, thereby obtaining explants for tissue culture; finally, transferring the explants to a glass bottle with an MIII solid culture medium at the bottom for culture; placing the glass bottle in a sterile culture room at 25+/-1 DEG C with the light/darkness ratio being 12h:12h, and culturing till the explants root; and transferring the rooting explants from the solid culture medium in the ultraclean workbench to a sterile MIII liquid culture medium, continuing placing in the sterile culture room at 25+/-1 DEG C with the light/darkness ratio being 12h:12h, and culturing to obtain seedlings growing well. The tissue culturing method disclosed by the invention has the beneficial effects that the developed method for culturing the water caltrop is very appropriate, and can overcome the defects of the prior art and improve the survival rate in tissue culture.
Description
Technical field
The invention belongs to the method for tissue culture technical field of a kind of water caltrop.
Background technology
Compared with terrestrial plant, water plants very easily pollutes, and water caltrop is as a kind of water plants, and in its cultured in vitro process, the difficulty of Explant surface sterilizing and endophyte inhibition is thereafter all high compared with other plant, tests each link and all likely occurs polluting.The setting of kind selection, concentration proportioning and the operation sequence of bactericidal agent is all the key of sterilizing success or failure.Sterilization-intensity is excessive, overlong time, and plant can be all dead; Sterilizing deficiency, can all pollute.These two large technological difficulties often become the bottleneck that water plants tissue is cultivated.
Although more existing researchs for water caltrop tissue culture method in recent years, this plant specificity is stronger, and condition of culture is difficult to unified.
Summary of the invention
The present invention designs by kinds of experiments, and this experiment has obtained a set of suitable cultural method.The present invention adopts following technical scheme to realize.
A method for tissue culture for water caltrop, the method step is that clip stem-segment with node from submerged plant water caltrop, cuts off blade after soaking, with deionized water rinsing in running water; Sterilizing in superclean bench, is then cut into the segment about 1cm stem section, and the upper palpus of each section is containing a stipes, obtain organizing the explant of training; Finally, being transferred to bottom has in the vial of MIII solid culture medium and cultivates; Vial is placed in to Sterile culture room, condition: temperature is 25 ± 1 ℃, Light To Dark Ratio 12h:12h, cultivates until take root; In superclean bench, the explant of taking root is transferred in aseptic MIII liquid nutrient medium from solid culture medium, continues to be placed in Sterile culture room, condition: 25 ± 1 ℃, Light To Dark Ratio 12h:12h, cultivates the seedling that becomes growing way health.
The step that the inventive method is concrete is that from submerged plant water caltrop, the stem-segment with node of the tender nose part of clip, is about 10cm, soaks 1 hour in running water, cuts off blade, uses deionized water rinsing 3 times; Sterilizing in superclean bench, is then cut into the segment about 1cm stem section, and the upper palpus of each section is containing a stipes, obtain organizing the explant of training; Finally, being transferred to bottom has in the vial of MIII solid culture medium and cultivates; Be placed in Sterile culture room, condition: 25 ± 1 ℃, Light To Dark Ratio 12h:12h, cultivates until take root; In superclean bench, the explant of taking root is transferred in aseptic MIII liquid nutrient medium from solid culture medium, continues to be placed in Sterile culture room, condition: 25 ± 1 ℃, Light To Dark Ratio 12h:12h, cultivates the seedling that becomes growing way health.
The compound method step of MIII liquid nutrient medium of the present invention is: in cumulative volume 1L:
1) macroelement weighs and is successively dissolved in a certain amount of pure water according to order, and macroelement comprises: 0.0848gNa
2siO
3.5H
2o, 0.0425g NaNO
3, 0.0068g KH
2pO
4, 0.086g CaSO
42H
2o, 0.00745g KCl, 0.0735gCaCl
22H
2o, 0.0615g MgSO
47H
2o;
2) add the hydrochloric acid solution of a little 1N, stir and make substance dissolves (capable of being heated by microwave or ultrasonic are processed hydrotropy);
3) add 100 times of micro-mother liquors of 1ml; The formula of trace element mother liquor is: get 30.8g H
3bO
4, 12.08gMnSO
4.4H
2o, 2.209g ZnSO
4.7H
2o, 0.968g Na
2moO4.2H
2o, 1g CuSO
4.5H
2o, 3.792gAlK (SO
4)
2.12H
2o, 0.952g CoCl
2.6H
2o, 1.124g NiSO
4.7H
2o, 0.952g KBr, 0.664g KI, 0.774gH
2seO
3, be dissolved in water, be settled to 100ml;
4) add 10ml Fe-EDTA solution (need prepare in advance FeCl
3stock solution: by 2.7g FeCl
3.6H2O be dissolved in distilled water with 10ml0.1N HCl, be finally settled to 100ml, obtain FeCl
3stock solution; Then prepare Fe-EDTA solution: by 0.372gNa
2eDTA.2H2O is dissolved in about 400ml distilled water, adds the above-mentioned FeCl of 5ml
3stock solution, uses high-pressure sterilizing pot sterilizing, after taking-up is cooled to room temperature, under aseptic condition, with sterile distilled water, is settled to 500ml);
5) be settled to 1L, adjusting pH is 6.0;
6) autoclave sterilization, is used after taking-up is cooled to room temperature, or is directly placed in refrigerator cold-storage, before each use, will return to room temperature.
The formula of MIII solid culture medium of the present invention is: in cumulative volume 1L: complete after 1～4 step of MIII liquid nutrient medium preparation, add 6g agar, 20g sucrose; Heating, adds plant hormone 1mg6-BA, 0.1mg NAA after boiling, be settled to 1L, and adjusting pH is 6.0; Autoclave sterilization, takes out and solidifies rear use, or be directly placed in refrigerator cold-storage.
The method of the present invention's sterilizing in superclean bench is, first working concentration is 0.01% mercuric chloride sterilizing 4min, intermittently 10min, and re-using concentration is 0.01% mercuric chloride sterilizing 4min.
Effect intentionally of the present invention is, the present invention grope water caltrop cultural method very suitable, can overcome the defect of prior art, avoided the bactericidal agent of High Fragmentation (NaClO and alcohol) and excessive concentrations, reduced the lethality of plant, adopt batch process simultaneously, in gentle mode, extend the sterilization functions time, when reducing pollution rate, also improved plant survival rate.
Embodiment
With regard to embodiment, the present invention is further explained below.
First, applicant has done the preliminary experiment that some water caltrop tissues are cultivated.As follows.
1, the impact that different bactericidal agents are cultivated water caltrop tissue
In table 1, shown and used mercuric chloride, NaClO and the alcohol of variable concentrations water caltrop explant to be carried out to the result of surface sterilizing.Can see, the explant of water plants water caltrop is all very responsive to these three kinds of surface sterilants.No matter be mercuric chloride, NaClO, or alcohol, sterilization effect all presents identical rule, and bactericidal agent concentration is higher, and the pollution rate that subsequent experimental occurs is lower, and but explant is gone out, dead probability is higher; Otherwise bactericidal agent concentration is lower, the dead rate of going out is lower, but pollution rate is higher.Wherein, water caltrop is the highest to the susceptibility of alcohol, and the alcohol concentration (70%) that is usually used in Plant Tissue Breeding is also fatal concerning water caltrop; Water caltrop is also higher to the susceptibility of NaClO, and is that go out dead rate and high pollution rate of height exists simultaneously with NaClO sterilizing, and explant is difficult to guarantee vitality under its effect; Comparatively speaking, water caltrop is just not so good as the above two to the susceptibility of mercuric chloride.Can go out and obtain the breach that further solves sterilizing problem 0.01% concentration processed group of dead rate and 0.075% and 0.1% concentration processed group of 0% pollution rate from 0%.
The sensitivity of table 1 water caltrop to different bactericidal agents
0.1% concentration is processed the higher dead rate of going out that easily produces, and should not re-use.Consider the object that low pollution, height survive, applicant has selected 0.01% and 0.075% mercuric chloride to do further experiment.
2,0.01% with the sterilization effect of the different usings method of 0.075% mercuric chloride
The mercuric chloride of two concentration (0.01% and 0.075%) of selecting in being tested by previous step carries out 4min sterilizing and still can not reach desirable effect, so next this is had made some improvements: go out extremely but 0.01% higher mercuric chloride sterilizing of pollution rate to zero, adopt tyndallization to extend its sterilization time, to reduce its pollution rate; To zero pollution but the 0.075% mercuric chloride sterilizing of the dead plant of easily going out, attempt shortening sterilization time and discontinuous sterilization reduces its dead rate of going out.
The sterilization effect of table 20.01% and the different usings method of 0.075% mercuric chloride
As can be seen from Table 2, although zero 0.075% mercuric chloride polluting goes out after shortening sterilization time, dead rate declines to some extent, has occurred 16% pollution, causes survival rate to reduce; And survival rate is not also because the use of batch process is improved.Although zero 0.01% mercuric chloride that goes out dead rate occurs slightly going out extremely after discontinuous sterilization, pollution rate declines greatly, and survival rate reaches 85%.And sterilization effect does not improve because of the prolongation of sterilization time, produce on the contrary the higher dead rate of going out.Therefore, select the method for " 0.01% mercuric chloride sterilizing 4min+ is 10min+0.01% mercuric chloride sterilizing 4min intermittently " to carry out ensuing experiment.
The group training effect of dissimilar medium
As can be seen from Table 3, the liquid medium within that sprouts and take root early occurs, illustrate that liquid is more conducive to the Fast-propagation that water plants water caltrop tissue is cultivated, but in cultivation process, pollution rate is higher, this may be the cause that is more conducive to microbial reproduction due to liquid environment.And the advantage of solid culture medium is that rooting rate is higher, and the possibility of polluting in cultivation process is less.Although liquid contamination is more conducive to explant, sprout, higher pollution probability of happening makes it should not be used for initial-stage culture.
Table 3 is used the group training effect of dissimilar medium
So consider after above result, will adopt the cultural method of " first, with the solid culture medium with sucrose, be cultured to and take root, then be transferred in the liquid nutrient medium that does not add sucrose ".
According to above-mentioned preliminary experiment, obtain technical scheme of the present invention.
A method for tissue culture for water caltrop, the method step is that clip stem-segment with node from submerged plant water caltrop, cuts off blade after soaking, with deionized water rinsing in running water; Sterilizing in superclean bench, is then cut into the segment about 1cm stem section, and the upper palpus of each section is containing a stipes, obtain organizing the explant of training; Finally, being transferred to bottom has in the vial of MIII solid culture medium and cultivates; Vial is placed in to Sterile culture room, condition: temperature is 25 ± 1 ℃, Light To Dark Ratio 12h:12h, cultivates until take root; In superclean bench, the explant of taking root is transferred in aseptic MIII liquid nutrient medium from solid culture medium, continues to be placed in Sterile culture room, condition: 25 ± 1 ℃, Light To Dark Ratio 12h:12h, cultivates the seedling that becomes growing way health.
The step that the inventive method is concrete is that from submerged plant water caltrop, the stem-segment with node of the tender nose part of clip, is about 10cm, soaks 1 hour in running water, cuts off blade, uses deionized water rinsing 3 times; Sterilizing in superclean bench, is then cut into the segment about 1cm stem section, and the upper palpus of each section is containing a stipes, obtain organizing the explant of training; Finally, being transferred to bottom has in the vial of MIII solid culture medium and cultivates; Be placed in Sterile culture room, condition: 25 ± 1 ℃, Light To Dark Ratio 12h:12h, cultivates until take root; In superclean bench, the explant of taking root is transferred in aseptic MIII liquid nutrient medium from solid culture medium, continues to be placed in Sterile culture room, condition: 25 ± 1 ℃, Light To Dark Ratio 12h:12h, cultivates the seedling that becomes growing way health.
The compound method step of MIII liquid nutrient medium of the present invention is: in cumulative volume 1L:
1) macroelement weighs and is successively dissolved in a certain amount of pure water according to order, and macroelement comprises: 0.0848gNa
2siO
3.5H
2o, 0.0425g NaNO
3, 0.0068g KH
2pO
4, 0.086g CaSO
42H
2o, 0.00745g KCl, 0.0735gCaCl
22H
2o, 0.0615g MgSO
47H
2o;
2) add the hydrochloric acid solution of a little 1N, stir and make substance dissolves (capable of being heated by microwave or ultrasonic are processed hydrotropy);
3) add 100 times of micro-mother liquors of 1ml; The formula of trace element mother liquor is: get 30.8g H
3bO
4, 12.08gMnSO
4.4H
2o, 2.209g ZnSO
4.7H
2o, 0.968g Na
2moO4.2H
2o, 1g CuSO
4.5H
2o, 3.792gAlK (SO
4)
2.12H
2o, 0.952g CoCl
2.6H
2o, 1.124g NiSO
4.7H
2o, 0.952g KBr, 0.664g KI, 0.774gH
2seO
3, be dissolved in water, be settled to 100ml;
4) add 10ml Fe-EDTA solution (need prepare in advance FeCl
3stock solution: by 2.7g FeCl
3.6H2O be dissolved in distilled water with 10ml0.1N HCl, be finally settled to 100ml, obtain FeCl
3stock solution; Then prepare Fe-EDTA solution: by 0.372gNa
2eDTA.2H2O is dissolved in about 400ml distilled water, adds the above-mentioned FeCl of 5ml
3stock solution, uses high-pressure sterilizing pot sterilizing, after taking-up is cooled to room temperature, under aseptic condition, with sterile distilled water, is settled to 500ml);
5) be settled to 1L, adjusting pH is 6.0;
6) autoclave sterilization, is used after taking-up is cooled to room temperature, or is directly placed in refrigerator cold-storage, before each use, will return to room temperature.
The formula of MIII solid culture medium of the present invention is: in cumulative volume 1L: complete after 1～4 step of MIII liquid nutrient medium preparation, add 6g agar, 20g sucrose; Heating, adds plant hormone 1mg6-BA, 0.1mg NAA after boiling, be settled to 1L, and adjusting pH is 6.0; Autoclave sterilization, takes out and solidifies rear use, or be directly placed in refrigerator cold-storage.
The method of the present invention's sterilizing in superclean bench is, first working concentration is 0.01% mercuric chloride sterilizing 4min, intermittently 10min, and re-using concentration is 0.01% mercuric chloride sterilizing 4min.
Claims (5)
1. a method for tissue culture for water caltrop, is characterized in that, the method step is that clip stem-segment with node from submerged plant water caltrop, cuts off blade after soaking, with deionized water rinsing in running water; Sterilizing in superclean bench, is then cut into the segment about 1cm stem section, and the upper palpus of each section is containing a stipes, obtain organizing the explant of training; Finally, being transferred to bottom has in the vial of MIII solid culture medium and cultivates; Vial is placed in to Sterile culture room, condition: temperature is 25 ± 1 ℃, Light To Dark Ratio 12h:12h, cultivates until take root; In superclean bench, the explant of taking root is transferred in aseptic MIII liquid nutrient medium from solid culture medium, continues to be placed in Sterile culture room, condition: 25 ± 1 ℃, Light To Dark Ratio 12h:12h, cultivates the seedling that becomes growing way health.
2. the method for tissue culture of a kind of water caltrop according to claim 1, is characterized in that, the step that the method is concrete is, from submerged plant water caltrop, the stem-segment with node of the tender nose part of clip, is about 10cm, soaks 1 hour in running water, cut off blade, use deionized water rinsing 3 times; Sterilizing in superclean bench, is then cut into the segment about 1cm stem section, and the upper palpus of each section is containing a stipes, obtain organizing the explant of training; Finally, being transferred to bottom has in the vial of MIII solid culture medium and cultivates; Be placed in Sterile culture room, condition: 25 ± 1 ℃, Light To Dark Ratio 12h:12h, cultivates until take root; In superclean bench, the explant of taking root is transferred in aseptic MIII liquid nutrient medium from solid culture medium, continues to be placed in Sterile culture room, condition: 25 ± 1 ℃, Light To Dark Ratio 12h:12h, cultivates the seedling that becomes growing way health.
3. the method for tissue culture of a kind of water caltrop according to claim 1 and 2, is characterized in that, the compound method step of MIII liquid nutrient medium is: in cumulative volume 1L:
1) macroelement weighs and is successively dissolved in a certain amount of pure water according to order, and macroelement comprises: 0.0848gNa
2siO
3.5H
2o, 0.0425g NaNO
3, 0.0068g KH
2pO
4, 0.086g CaSO
42H
2o, 0.00745g KCl, 0.0735gCaCl
22H
2o, 0.0615g MgSO
47H
2o;
2) add the hydrochloric acid solution of a little 1N, stir and make substance dissolves (capable of being heated by microwave or ultrasonic are processed hydrotropy);
3) add 100 times of micro-mother liquors of 1ml; The formula of trace element mother liquor is: get 30.8g H
3bO
4, 12.08gMnSO
4.4H
2o, 2.209g ZnSO
4.7H
2o, 0.968g Na
2moO4.2H
2o, 1g CuSO
4.5H
2o, 3.792gAlK (SO
4)
2.12H
2o, 0.952g CoCl
2.6H
2o, 1.124g NiSO
4.7H
2o, 0.952g KBr, 0.664g KI, 0.774gH
2seO
3, be dissolved in water, be settled to 100ml;
4) add 10ml Fe-EDTA solution (need prepare in advance FeCl
3stock solution: by 2.7g FeCl
3.6H2O be dissolved in distilled water with 10ml0.1N HCl, be finally settled to 100ml, obtain FeCl
3stock solution; Then prepare Fe-EDTA solution: by 0.372gNa
2eDTA.2H2O is dissolved in about 400ml distilled water, adds the above-mentioned FeCl of 5ml
3stock solution, uses high-pressure sterilizing pot sterilizing, after taking-up is cooled to room temperature, under aseptic condition, with sterile distilled water, is settled to 500ml);
5) be settled to 1L, adjusting pH is 6.0;
6) autoclave sterilization, is used after taking-up is cooled to room temperature, or is directly placed in refrigerator cold-storage, before each use, will return to room temperature.
4. the method for tissue culture of a kind of water caltrop according to claim 1 and 2, is characterized in that, the formula of MIII solid culture medium is: in cumulative volume 1L: complete after 1～4 step of MIII liquid nutrient medium preparation, add 6g agar, 20g sucrose; Heating, adds plant hormone 1mg6-BA, 0.1mg NAA after boiling, be settled to 1L, and adjusting pH is 6.0; Autoclave sterilization, takes out and solidifies rear use, or be directly placed in refrigerator cold-storage.
5. the method for tissue culture of a kind of water caltrop according to claim 1 and 2, is characterized in that, in superclean bench, the method for sterilizing is, first working concentration is 0.01% mercuric chloride sterilizing 4min, intermittently 10min, and re-using concentration is 0.01% mercuric chloride sterilizing 4min.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106954549A (en) * | 2017-02-27 | 2017-07-18 | 水生藻安生物科技（武汉）有限公司 | A kind of comb tooth bog pondweed tissue culture and rapid proliferation method |
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| CN1742563A (en) * | 2005-10-13 | 2006-03-08 | 华中师范大学 | Method for fast breeding water caltrop seedlings |
| CN1943324A (en) * | 2005-10-13 | 2007-04-11 | 华中师范大学 | Method for fast breeding water caltrop seed and seedling |
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2014
- 2014-01-22 CN CN201410029624.6A patent/CN103734017B/en not_active Expired - Fee Related
Patent Citations (4)
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| CN1545861A (en) * | 2003-12-08 | 2004-11-17 | �Ϻ���ͨ��ѧ | Method for cultivating curly pondweed embryo and curly pondweed karren tissue |
| CN1701661A (en) * | 2003-12-08 | 2005-11-30 | 南京大学 | Curly pondweed rigid bud tissue culturing method |
| CN1742563A (en) * | 2005-10-13 | 2006-03-08 | 华中师范大学 | Method for fast breeding water caltrop seedlings |
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Non-Patent Citations (1)
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| CN106954549A (en) * | 2017-02-27 | 2017-07-18 | 水生藻安生物科技（武汉）有限公司 | A kind of comb tooth bog pondweed tissue culture and rapid proliferation method |
| CN106954549B (en) * | 2017-02-27 | 2019-05-28 | 水生藻安生物科技（武汉）有限公司 | A kind of comb tooth bog pondweed tissue culture and rapid proliferation method |
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