CN103734017B - The method for tissue culture of a kind of water caltrop - Google Patents

The method for tissue culture of a kind of water caltrop Download PDF

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CN103734017B
CN103734017B CN201410029624.6A CN201410029624A CN103734017B CN 103734017 B CN103734017 B CN 103734017B CN 201410029624 A CN201410029624 A CN 201410029624A CN 103734017 B CN103734017 B CN 103734017B
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miii
water
sterilizing
water caltrop
cultivate
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CN103734017A (en
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徐润冰
常学秀
王小龙
王龙昌
刘磊
桂秘
鲍志豪
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Yunnan University YNU
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Abstract

A method for tissue culture for water caltrop, the method step is, clip stem-segment with node from submerged plant water caltrop, cuts off blade, with deionized water rinsing after soaking in running water; Sterilizing in superclean bench, is then cut into stem section the segment of about 1cm, and each section containing a stipes, must obtain the explant organizing training; Finally, be transferred to bottom to have in the vial of MIII solid culture medium and cultivate; Vial is placed in Sterile culture room, condition: temperature is 25 ± 1 DEG C, 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 DEG C, Light To Dark Ratio 12h:12h, cultivate the seedling becoming growing way health.Beneficial aspects of the present invention is, the water caltrop cultural method that the present invention gropes is very suitable, can overcome the defect of prior art, improves the survival rate of group training.

Description

The method for tissue culture of a kind of water caltrop
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 suppression is thereafter all high compared with other plant, tests each link and all likely occurs polluting.The kind of bactericidal agent is selected, the setting of concentration proportioning and operation sequence is all the key of sterilizing success or failure.Sterilization-intensity is excessive, overlong time, and plant can be all dead; Sterilizing is not enough, can all pollute.These two large technological difficulties often become the bottleneck of water plants tissue cultures.
Although more existing researchs for water caltrop tissue culture method in recent years, this plant specificity is comparatively strong, and condition of culture is difficult to unified.
Summary of the invention
The present invention is designed by kinds of experiments, and this experiment obtains 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, clip stem-segment with node from submerged plant water caltrop, cuts off blade, with deionized water rinsing after soaking in running water; Sterilizing in superclean bench, is then cut into stem section the segment of about 1cm, and each section containing a stipes, must obtain the explant organizing training; Finally, be transferred to bottom to have in the vial of MIII solid culture medium and cultivate; Vial is placed in Sterile culture room, condition: temperature is 25 ± 1 DEG C, 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 DEG C, Light To Dark Ratio 12h:12h, cultivate the seedling becoming growing way health.
The step that the inventive 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, cut off blade in running water, with deionized water rinsing 3 times; Sterilizing in superclean bench, is then cut into stem section the segment of about 1cm, and each section containing a stipes, must obtain the explant organizing training; Finally, be transferred to bottom to have in the vial of MIII solid culture medium and cultivate; Be placed in Sterile culture room, condition: 25 ± 1 DEG C, Light To Dark Ratio 12h:12h, cultivate 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 DEG C, Light To Dark Ratio 12h:12h, cultivate the seedling becoming 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, 0.0425gNaNO 3, 0.0068gKH 2pO 4, 0.086gCaSO 42H 2o, 0.00745gKCl, 0.0735gCaCl 22H 2o, 0.0615gMgSO 47H 2o;
2) add the hydrochloric acid solution of a little 1N, stir and make substance dissolves (being heated by microwave or ultrasonic process hydrotropy);
3) 100 times of micro-mother liquors of 1ml are added; The formula of trace element mother liquor is: get 30.8gH 3bO 4, 12.08gMnSO 4.4H 2o, 2.209gZnSO 4.7H 2o, 0.968gNa 2moO4.2H 2o, 1gCuSO 4.5H 2o, 3.792gAlK (SO 4) 2.12H 2o, 0.952gCoCl 2.6H 2o, 1.124gNiSO 4.7H 2o, 0.952gKBr, 0.664gKI, 0.774gH 2seO 3, be dissolved in water, be settled to 100ml;
4) add 10mlFe-EDTA solution and (FeCl need be prepared in advance 3stock solution: by 2.7gFeCl 3.6H2O be dissolved in distilled water with 10ml0.1NHCl, be finally settled to 100ml, obtain FeCl 3stock solution; Then Fe-EDTA solution is prepared: 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, takes out after being cooled to room temperature and is aseptically settled to 500ml with sterile distilled water);
5) be settled to 1L, adjustment pH is 6.0;
6) autoclave sterilization, takes out after being cooled to room temperature and uses, or be directly placed in refrigerator cold-storage, will return to room temperature before each use.
The formula of MIII solid culture medium of the present invention is: in cumulative volume 1L: after completing 1 ~ 4 step of MIII liquid nutrient medium preparation, add 6g agar, 20g sucrose; Heating, adds plant hormone 1mg6-BA, 0.1mgNAA, is settled to 1L after boiling, adjustment 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, and interval 10min, re-using concentration is 0.01% mercuric chloride sterilizing 4min.
Beneficial aspects of the present invention is, the present invention grope water caltrop cultural method very suitable, the defect of prior art can be overcome, avoid the bactericidal agent of High Fragmentation (NaClO and alcohol) and excessive concentrations, decrease the lethality of plant, adopt batch process simultaneously, extend the sterilization functions time in the mode of gentleness, while decreasing pollution rate, also improve plant survival rate.
Embodiment
With regard to embodiment, the present invention is further explained below.
First, applicant has done the preliminary experiment of some water caltrop tissue cultures.As follows.
1, different bactericidal agent is on the impact of water caltrop tissue cultures
Show in table 1 and use the mercuric chloride of variable concentrations, NaClO and alcohol to carry out the result of surface sterilizing to water caltrop explant.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 namely bactericidal agent concentration is higher, and the pollution rate that subsequent experimental occurs is lower, but explant is gone out, dead probability is then higher; Otherwise bactericidal agent concentration is lower, dead rate of going out is lower, but pollution rate is higher.Wherein, the susceptibility of water caltrop to alcohol is the highest, and the alcohol concentration (70%) being 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 ensure vitality under its effect; Comparatively speaking, water caltrop is just not so good as the above two to the susceptibility of mercuric chloride.Can to go out the breach obtaining 0.01% concentration processed group of dead rate and 0.075% and 0.1% concentration processed group of 0% pollution rate and solve sterilizing problem further from 0%.
Table 1 water caltrop is to the sensitivity of different bactericidal agent
0.1% concentration process easily produces higher dead rate of going out, and should not re-use.Consider low stain, object that height survives, the mercuric chloride that applicant have selected 0.01% and 0.075% does further experiment.
2,0.01% with the sterilization effect of the different using method of 0.075% mercuric chloride
The mercuric chloride of two concentration (0.01% and 0.075%) selected in being tested by previous step carries out 4min sterilizing and still can not reach desirable effect, so next have made some improvements this: to go out dead but that pollution rate is higher 0.01% mercuric chloride sterilizing to zero, tyndallization is adopted to extend its sterilization time, to reduce its pollution rate; To no pollution but the 0.075% mercuric chloride sterilizing of 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 using method of 0.075% mercuric chloride
As can be seen from Table 2, although 0.075% mercuric chloride of no pollution goes out after shortening sterilization time, dead rate declines to some extent, has occurred the pollution of 16%, has caused survival rate to reduce; And survival rate is not also improved because of the use of batch process.Although slightly going out extremely appears in zero 0.01% mercuric chloride going out dead rate after discontinuous sterilization, pollution rate declines greatly, and survival rate is to 85%.And sterilization effect does not improve because of the prolongation of sterilization time, produce higher dead rate of going out on the contrary.Therefore, the method for " 0.01% mercuric chloride sterilizing 4min+ interval 10min+0.01% mercuric chloride sterilizing 4min " is selected 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 comparatively early occurs, illustrate that liquid is more conducive to the Fast-propagation of water plants water caltrop tissue cultures, but in incubation, pollution rate is higher, this may be because liquid environment is more conducive to the cause of microbial reproduction.And the advantage of solid culture medium is that rooting rate is higher, and the possibility polluted in incubation is less.Sprout although liquid contamination is more conducive to explant, higher pollution probability of happening makes it be used for initial-stage culture.
Table 3 uses the group training effect of dissimilar medium
So after considering above result, the cultural method of " first with the solid culture medium with sucrose, be cultured to and take root, then be transferred to not with in the liquid nutrient medium of sucrose " will be adopted.
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, clip stem-segment with node from submerged plant water caltrop, cuts off blade, with deionized water rinsing after soaking in running water; Sterilizing in superclean bench, is then cut into stem section the segment of about 1cm, and each section containing a stipes, must obtain the explant organizing training; Finally, be transferred to bottom to have in the vial of MIII solid culture medium and cultivate; Vial is placed in Sterile culture room, condition: temperature is 25 ± 1 DEG C, 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 DEG C, Light To Dark Ratio 12h:12h, cultivate the seedling becoming growing way health.
The step that the inventive 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, cut off blade in running water, with deionized water rinsing 3 times; Sterilizing in superclean bench, is then cut into stem section the segment of about 1cm, and each section containing a stipes, must obtain the explant organizing training; Finally, be transferred to bottom to have in the vial of MIII solid culture medium and cultivate; Be placed in Sterile culture room, condition: 25 ± 1 DEG C, Light To Dark Ratio 12h:12h, cultivate 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 DEG C, Light To Dark Ratio 12h:12h, cultivate the seedling becoming 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, 0.0425gNaNO 3, 0.0068gKH 2pO 4, 0.086gCaSO 42H 2o, 0.00745gKCl, 0.0735gCaCl 22H 2o, 0.0615gMgSO 47H 2o;
2) add the hydrochloric acid solution of a little 1N, stir and make substance dissolves (being heated by microwave or ultrasonic process hydrotropy);
3) 100 times of micro-mother liquors of 1ml are added; The formula of trace element mother liquor is: get 30.8gH 3bO 4, 12.08gMnSO 4.4H 2o, 2.209gZnSO 4.7H 2o, 0.968gNa 2moO4.2H 2o, 1gCuSO 4.5H 2o, 3.792gAlK (SO 4) 2.12H 2o, 0.952gCoCl 2.6H 2o, 1.124gNiSO 4.7H 2o, 0.952gKBr, 0.664gKI, 0.774gH 2seO 3, be dissolved in water, be settled to 100ml;
4) add 10mlFe-EDTA solution and (FeCl need be prepared in advance 3stock solution: by 2.7gFeCl 3.6H2O be dissolved in distilled water with 10ml0.1NHCl, be finally settled to 100ml, obtain FeCl 3stock solution; Then Fe-EDTA solution is prepared: 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, takes out after being cooled to room temperature and is aseptically settled to 500ml with sterile distilled water);
5) be settled to 1L, adjustment pH is 6.0;
6) autoclave sterilization, takes out after being cooled to room temperature and uses, or be directly placed in refrigerator cold-storage, will return to room temperature before each use.
The formula of MIII solid culture medium of the present invention is: in cumulative volume 1L: after completing 1 ~ 4 step of MIII liquid nutrient medium preparation, add 6g agar, 20g sucrose; Heating, adds plant hormone 1mg6-BA, 0.1mgNAA, is settled to 1L after boiling, adjustment 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, and interval 10min, 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, clip stem-segment with node from submerged plant water caltrop, cuts off blade, with deionized water rinsing after soaking in running water; Sterilizing in superclean bench, is then cut into stem section the segment of about 1cm, and each section containing a stipes, must obtain the explant organizing training; Finally, have in the vial of MIII solid culture medium bottom being transferred to and cultivate, medium component is MIII liquid nutrient medium: containing 0.0848g/LNa 2siO 35H 2o, 0.0425g/LNaNO 3, 0.0068g/LKH 2pO 4, 0.086g/LCaSO 4, 2H 2o, 0.00745g/LKCl, 0.0735g/LCaCl 2, 2H 2o, 0.0615g/LMgSO 4, 7H 2o, 0.308g/LH 3bO 4, 0.1208g/LMnSO 44H 2o, 0.02296g/LZnSO 47H 2o, 0.00968g/LNa 2moO 42H 2o, 0.01g/LCuSO 45H 2o, 0.03792g/LAlK (SO 4) 212H 2o, 0.00952g/LCoCl 26H 2o, 0.01124g/LNiSO 47H 2o, 0.00952g/LKBr, 0.00664g/LKI, 0.00774g/LH 2seO 3+ agar+sucrose+hormone: 1mg/L6-BA+0.1mg/LNAA; Vial is placed in Sterile culture room, condition: temperature is 25 ± 1 DEG C, 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 MIII solid culture medium, continues to be placed in Sterile culture room, condition: 25 ± 1 DEG C, Light To Dark Ratio 12h:12h, cultivate the seedling becoming 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, with deionized water rinsing 3 times; Sterilizing in superclean bench, is then cut into stem section the segment of about 1cm, and each section containing a stipes, must obtain the explant organizing training; Finally, have in the vial of MIII solid culture medium bottom being transferred to and cultivate, medium component is MIII liquid nutrient medium: containing 0.0848g/LNa 2siO 35H 2o, 0.0425g/LNaNO 3, 0.0068g/LKH 2pO 4, 0.086g/LCaSO 4, 2H 2o, 0.00745g/LKCl, 0.0735g/LCaCl 2, 2H 2o, 0.0615g/LMgSO 4, 7H 2o, 0.308g/LH 3bO 4, 0.1208g/LMnSO 44H 2o, 0.02296g/LZnSO 47H 2o, 0.00968g/LNa 2moO42H 2o, 0.01g/LCuSO 45H 2o, 0.03792g/LAlK (SO 4) 212H 2o, 0.00952g/LCoCl 26H 2o, 0.01124g/LNiSO 47H 2o, 0.00952g/LKBr, 0.00664g/LKI, 0.00774g/LH 2seO 3+ 0.6% agar+20g/L sucrose+1mg/L6-BA+0.1mg/LNAA, pH6.0; Be placed in Sterile culture room, condition: 25 ± 1 DEG C, Light To Dark Ratio 12h:12h, cultivate until take root; In superclean bench, the explant of taking root is transferred in aseptic MIII liquid nutrient medium from MIII solid culture medium, continues to be placed in Sterile culture room, condition: 25 ± 1 DEG C, Light To Dark Ratio 12h:12h, cultivate the seedling becoming 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 35H 2o, 0.0425gNaNO 3, 0.0068gKH 2pO 4, 0.086gCaSO 42H 2o, 0.00745gKCl, 0.0735gCaCl 22H 2o, 0.0615gMgSO 47H 2o;
2) add the hydrochloric acid solution of a little 1M, stir and make substance dissolves, with heating using microwave or ultrasonic process hydrotropy;
3) 100 times of micro-mother liquors of 1ml are added; The compound method of trace element mother liquor is: get 30.8gH 3bO 4, 12.08gMnSO 44H 2o, 2.209gZnSO 47H 2o, 0.968gNa 2moO42H 2o, 1gCuSO 45H 2o, 3.792gAlK (SO 4) 212H 2o, 0.952gCoCl 26H 2o, 1.124gNiSO 47H 2o, 0.952gKBr, 0.664gKI, 0.774gH 2seO 3, be dissolved in water, be settled to 100ml;
4) 10mlFe-EDTA solution is added: FeCl need be prepared in advance 3stock solution: by 2.7gFeCl 36H 2o and 10ml0.1MHCl is dissolved in distilled water, is finally settled to 100ml, obtains FeCl 3stock solution; Then Fe-EDTA solution is prepared: by 0.372gNa 2eDTA2H 2o is dissolved in about 400ml distilled water, adds the above-mentioned FeCl of 5ml 3stock solution, uses high-pressure sterilizing pot sterilizing, takes out after being cooled to room temperature and is aseptically settled to 500ml with sterile distilled water;
5) be settled to 1L, adjustment pH is 6.0;
6) autoclave sterilization, takes out after being cooled to room temperature and uses, or be directly placed in refrigerator cold-storage, will return to room temperature before each use.
4. the method for tissue culture of a kind of water caltrop according to claim 3, is characterized in that, the compound method of MIII solid culture medium is: in cumulative volume 1L: after completing 1 ~ 4 step of MIII liquid nutrient medium preparation, add 6g agar, 20g sucrose; Heating, adds plant hormone 1mg6-BA, 0.1mgNAA, is settled to 1L after boiling, adjustment 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, it is characterized in that, the method for sterilizing in superclean bench is, first working concentration is 0.01% mercuric chloride sterilizing 4min, interval 10min, re-using concentration is 0.01% mercuric chloride sterilizing 4min.
CN201410029624.6A 2014-01-22 2014-01-22 The method for tissue culture of a kind of water caltrop Expired - Fee Related CN103734017B (en)

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