CN110959330A - Application of agapanthus cystatin in improving survival rate of plant cells after ultralow-temperature preservation - Google Patents
Application of agapanthus cystatin in improving survival rate of plant cells after ultralow-temperature preservation Download PDFInfo
- Publication number
- CN110959330A CN110959330A CN201911108062.3A CN201911108062A CN110959330A CN 110959330 A CN110959330 A CN 110959330A CN 201911108062 A CN201911108062 A CN 201911108062A CN 110959330 A CN110959330 A CN 110959330A
- Authority
- CN
- China
- Prior art keywords
- agapanthus
- cystatin
- survival rate
- cryoprotectant
- liquid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01C—PLANTING; SOWING; FERTILISING
- A01C1/00—Apparatus, or methods of use thereof, for testing or treating seed, roots, or the like, prior to sowing or planting
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N3/00—Preservation of plants or parts thereof, e.g. inhibiting evaporation, improvement of the appearance of leaves or protection against physical influences such as UV radiation using chemical compositions; Grafting wax
Abstract
The invention relates to an application of agapanthus cystatin in improving the survival rate of plant materials after ultralow-temperature preservation, wherein the agapanthus cystatin is added into at least one solution of a loading liquid, a cryoprotectant and a washing liquid as an additive, so that the survival rate of the plant materials after vitrification ultralow-temperature preservation can be improved, the survival rate of agapanthus embryonic callus is improved from 33% to 80%, and the survival rate of arabidopsis thaliana seeds germinating for 60h seedlings is improved from 30% to 50%.
Description
Technical Field
The invention relates to the technical field of low-temperature or ultralow-temperature preservation of biological samples, in particular to application of agapanthus cystatin in improving the survival rate of plant cells after ultralow-temperature preservation.
Background
The development of Cryopreservation (Cryopreservation) started in the last 60 th century, meaning the preservation of biological materials at temperatures of-80 ℃ and below, also known as liquid nitrogen preservation, since it is generally preserved in liquid nitrogen (-196 ℃). The metabolism and growth of the materials in the cells are almost completely stopped in liquid nitrogen, and the materials are in a relatively stable biological state, so that the purpose of preserving germplasm for a long time is achieved, and the ultra-low temperature preservation is the only medium-long term preservation mode which does not need continuous subculture at present. The vitrification cryopreservation is a preferred method for long-term storage of excellent germplasm resources in the last decade, because of the advantages of simple and rapid operation, low cost, wide suitable storage variety, stable hereditary property of the stored material, good storage effect and the like.
The plant vitrification cryopreservation comprises 7 main steps: preculture (Preculture), Osmoprotection (Osmoprotection), Dehydration of vitrification solution (Dehydration), liquid nitrogen freezing (Rapid cooling), thawing (Rapid warming), washing (Dilution) and recovery culture (Plating for re-growth). The material is required to be subjected to pre-culture, permeation protection and vitrification solution dehydration treatment before being frozen and stored in liquid nitrogen. Sufficient penetration of the plant material by the vitrification solution and a sufficiently rapid rate of temperature decrease to increase the viscosity of the cells, thereby inhibiting the formation and growth of ice crystals. The pre-freezing treatment can also reduce the free water content of cells as much as possible, increase the protective substances such as soluble sugar and soluble protein, and improve the stability of cell membranes under severe dehydration conditions. During thawing, a quick thawing method is generally used, and a high-concentration sucrose solution is used for removing cryoprotectant components such as dimethyl sulfoxide and the like in plant materials, so that the inhibition and toxic effects of residual cryoprotectants on cells are avoided, and the restoration damage of the cell parenchyma wall is also reduced. The recovery culture is to place the plant material in an environment suitable for recovery and growth for recovery growth, and the final survival rate of the plant material can be influenced by factors such as illumination, temperature, humidity, culture medium nutrition and the like at the stage. In the application of plant cryopreservation, a plurality of key technical problems still exist, so that the plant material is low in cryopreservation rate and cannot be applied in production, and more plants are limited to be preserved and applied.
Cystatins, also known as cystatins, are the most abundant reversible natural inhibitors of cysteine proteases, and are widely found in animals, plants and microorganisms, and are closely related to defense mechanisms, protection of cells from endogenous or exogenous abnormal proteolytic interference, involvement in stress regulation, and other processes.
Disclosure of Invention
In view of the above disadvantages of the prior art, the present invention aims to provide an application of agapanthus cystatin in improving the survival rate of plant cells after cryopreservation, so as to overcome the disadvantage of low survival rate of biological samples after cryopreservation in the prior art.
The purpose of the invention is realized by the following technical scheme:
the invention provides application of agapanthus cystatin in improving the survival rate of plant materials after ultralow-temperature preservation.
Preferably, the agapanthus cystatin is added into at least one solution of a loading liquid, a cryoprotectant and a washing liquid as an additive.
The nucleotide sequence (SEQ ID NO.1) of the agapanthus cystatin is as follows:
ATGCGATCTCAACCGGCGATTTGTTCTCTCGCTCTCGCTCTATTGTTATTTTCAGCGACTTTAGGGTTTCATTCGATCATCGCCATGGCCACGCTCGGAGGCGTTCACGAGAAGGAGGGAACCGAGAACAGCGTCGAGGTCGAGGAGCTCGCTCGTTTCGCTGTCGACGAACACAACAAAAAGGAGAATGCGCTCCTGGAATTTGGACGACTGGTGAAGGCGAAGGAGCAAGTGGTTGCAGGCACCATGTACCATCTGACTGTGGAGGCAATCGAAGCTGGGAAGAAGAAGATCTATGAGGCGAAGGTGTGGGTTAAGCCATGGCTCAACTTCAAGGAGCTTCAGGAGTTTAGGCACGCTGGCGATGCCGACTCTTCTTCCAACATCACCCCTGCGGACCTCGGCGCTAAGCGAGGTGATTGA。
the protein coding sequence (SEQ ID NO.2) of the agapanthus cystatin is as follows:
MRSQPAICSLALALLLFSATLGFHSIIAMATLGGVHEKEGTENSVEVEELARFAVDEHNKKENALLEFGRLVKAKEQVVAGTMYHLTVEAIEAGKKKIYEAKVWVKPWLNFKELQEFRHAGDADSSSNITPADLGAKRGD。
preferably, the concentration of the agapanthus cystatin in the loading solution, the cryoprotectant or the washing solution is 0.4-1.6 mg/L.
Preferably, the agapanthus cystatin is mixed with a phosphate buffer solution and then added to each solution.
Preferably, the phosphate buffer comprises the following components in percentage by weight: 137mM NaCl, 2.7mM KCl, 10mM Na2HPO4、2mM KH2PO4(ii) a The phosphoric acidThe pH of the buffer solution is 7.0 to 7.8, and more preferably 7.4.
Preferably, the agapanthus cystatin is obtained by performing prokaryotic expression on an escherichia coli Transetta defective strain, wherein the prokaryotic expression conditions are that the temperature is 16-25 ℃, the IPTG concentration is 0.1-1 mM, and the protein expression induction time is 18-24 h; and (3) carrying out GST-tag purification after protein induction expression.
The invention provides a method for improving the survival rate of plant materials after ultralow-temperature preservation, which comprises the following steps:
A. pretreating plant materials, and soaking in a loading solution;
B. transferring the soaked plant material into a cryoprotectant, dehydrating, and then placing in liquid nitrogen for ultralow temperature preservation;
C. taking out the plant material from liquid nitrogen, thawing in water bath, removing cryoprotectant, and treating with washing solution;
D. removing the washing liquid, and transferring to a recovery culture medium for recovery culture;
at least one solution of the loading liquid, the freezing protective agent and the washing liquid contains agapanthus cystatin; the concentration of the agapanthus cystatin in the loading liquid, the cryoprotectant or the washing liquid is 0.4-1.6mg/L respectively.
Preferably, the loading liquid comprises the following components: 4.3g/L MS powder, 12mol/L glycerol, 0.4mol/L sucrose and 10mmol/L KNO3(ii) a The pH value of the loading liquid is 5.6-6.2, and the pH value is more preferably 5.8.
Preferably, the cryoprotectant comprises the following components: 2.15g/L MS powder, 136.92g/L sucrose, 300g/L glycerol, 150g/L ethylene glycol and 136.4mL/L dimethyl sulfoxide; the pH value of the cryoprotectant is 5.6-6.2, and the pH value is more preferably 5.8.
Preferably, the washing solution comprises the following components: 4.3g/L MS powder, 1.2mol/L sucrose and 10mmol/LKNO3(ii) a The pH of the washing liquid is 5.6-6.2, and the pH is more preferably 5.8.
Preferably, the MS powder is prepared by adopting the following components in percentage by weight: 1900mg/L KNO3，1650mg/L NH4NO3，170mg/L KH2PO4，370mg/L MgSO4·7H2O，440mg/L CaCl2·2H2O，37.3mg/L Na2-EDTA，27.8mg/L FeSO4·7H2O, 100mg/L inositol, 0.5mg/L nicotinic acid, 0.5mg/L pyridoxine hydrochloride, 0.1mg/L thiamine hydrochloride, 2mg/L glycine, 0.83mg/L KI, 6.2mg/L H3BO3，22.3mg/L MnSO4·4H2O，8.6mg/L ZnSO4·7H2O，0.25mg/L Na2MoO4·2H2O，0.025mg/L CuSO4·5H2O，0.025mg/L CoCl2·6H2O and the balance of water.
Preferably, the recovery medium is 4.3g/L MS powder, 30g/L sucrose and 10g/L agar, and the pH value is 5.6-6.2.
Preferably, in step a, the soaking conditions are as follows: soaking at room temperature for 30-60 min.
More preferably, the soaking is carried out at room temperature for 60 minutes.
Preferably, in step B, the conditions of the dehydration treatment are: dehydrating at 0-4 deg.C for 40-60 min.
More preferably, the treatment is carried out at 4 ℃ for 40 minutes.
Preferably, in step C, the conditions for thawing the water bath are as follows: thawing at 37-40 deg.C for 60-90 s.
More preferably, the thawing is carried out at 40 ℃ for 90 seconds.
Preferably, in step D, the washing treatment conditions are: soaking at room temperature for 10-40 min.
More preferably, the soaking is carried out at room temperature for 30 minutes, with a new wash being replaced every 10 minutes.
Preferably, the plant material is at least one of agapanthus embryonic callus and arabidopsis seed germination 60h seedling.
Compared with the prior art, the invention has the following beneficial effects:
according to the invention, only the agapanthus cystatin is added into the loading liquid, the cryopreservation rate of agapanthus embryonic callus is improved from 33% to 55%, and the cryopreservation rate of the seedling of arabidopsis thaliana seed germination 60h is improved from 30% to 40%. The survival rate of the agapanthus embryonic callus is improved from 33% to 75% and the survival rate of the arabidopsis seed germinating seedling is improved from 30% to 37% when the agapanthus cystatin is only added into the cryoprotectant. Only adding the agapanthus cystatin into the washing solution, the survival rate of the agapanthus embryonic callus is improved from 33% to 70%, and the survival rate of the arabidopsis seed germination 60h seedling is improved from 30% to 45%. The agapanthus cystatin is added into the loading liquid, the cryoprotectant and the washing liquid at the same time, the survival rate of the agapanthus embryonic callus is improved from 33% to 80%, and the survival rate of the arabidopsis seed germination 60h seedling is improved from 30% to 50%. The method disclosed by the invention has the advantages that the preservation rate of the agapanthus embryonic callus and the arabidopsis seedlings germinating for 60h is obviously optimized, and the survival rate of the plant material after vitrification ultralow temperature preservation is promoted by adding the agapanthus cystatin ultralow temperature preservation solution.
Detailed Description
The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that it would be obvious to those skilled in the art that various changes and modifications can be made without departing from the spirit of the invention. All falling within the scope of the present invention.
Example 1
The formula of the experimental reagent in this example is as follows:
1) the MS culture solution is as follows: MS culture solution containing 1900mg/L KNO3，1650mg/L NH4NO3，170mg/L KH2PO4，370mg/L MgSO4·7H2O，440mg/L CaCl2·2H2O，37.3mg/L Na2-EDTA，27.8mg/L FeSO4·7H2O, 100mg/L inositol, 0.5mg/L nicotinic acid, 0.5mg/L pyridoxine hydrochloride, 0.1mg/L thiamine hydrochloride, 2mg/L glycine, 0.83mg/L KI, 6.2mg/L H3BO3，22.3mg/L MnSO4·4H2O，8.6mg/L ZnSO4·7H2O，0.25mg/LNa2MoO4·2H2O，0.025mg/L CuSO4·5H2O，0.025mg/L CoCl2·6H2O and the balance of water. The pH of the MS culture solution is 5.8.
2) The MS solid culture medium is: MS solid culture medium containing 1900mg/L KNO3，1650mg/L NH4NO3，170mg/LKH2PO4，370mg/L MgSO4·7H2O，440mg/L CaCl2·2H2O，37.3mg/L Na2-EDTA，27.8mg/LFeSO4·7H2O, 100mg/L inositol, 0.5mg/L nicotinic acid, 0.5mg/L pyridoxine hydrochloride, 0.1mg/L thiamine hydrochloride, 2mg/L glycine, 0.83mg/L KI, 6.2mg/L H3BO3，22.3mg/L MnSO4·4H2O，8.6mg/L ZnSO4·7H2O，0.25mg/L Na2MoO4·2H2O，0.025mg/L CuSO4·5H2O，0.025mg/L CoCl2·6H2O, 30g/L of sucrose, 3g/L of gel and the balance of water, and the pH value of the MS solid culture medium is 5.8.
3) The pre-culture medium is MS solid medium containing 171.15g/L sucrose and 1.5mg/L picloram.
4) Loading liquid: comprises 145.83mL/L of glycerol, 136.92g/L of sucrose and 1g/L of KNO3The MS culture solution of (1).
5) Cryoprotectant: 1/2MS culture solution containing 136.92g/L of sucrose, 300g/L of glycerol, 150g/L of ethylene glycol, 136.4mL/L of dimethyl sulfoxide and agapanthus cystatin.
6) Washing liquid: contains 410.76g/L sucrose and 1g/L KNO3The MS culture solution of (1).
7) Recovering the culture medium: MS solid medium containing 30g/L sucrose.
The agapanthus cystatin adopted in the embodiment is obtained by performing prokaryotic expression by using escherichia coli Transetta competence (Beijing holotype gold biotechnology, Inc., catalog number CD801-01), wherein the prokaryotic expression conditions are that the temperature is 16-25 ℃, the IPTG concentration is 0.1-1 mM, and the protein expression induction time is 18-24 h; and (3) carrying out GST-tag purification after protein induction expression.
The agapanthus cystatin is mixed with a phosphate buffer solution and then added into each solution.
The specific implementation method comprises the following steps:
1) preculturing the agapanthus embryonic callus subcultured for 20 days on a preculture medium at 4 ℃ for 2 days;
2) transferring the mixture into a loading solution and soaking the mixture for 60 minutes at room temperature;
3) transferring into cryoprotectant, and dehydrating at 0 deg.C for 40 min;
4) directly placing in liquid nitrogen for ultralow temperature preservation.
And 3) directly placing the agapanthus embryonic callus soaked in the vitrification solution in liquid nitrogen for ultralow temperature preservation without removing the vitrification solution after the step 3).
The agapanthus embryonic callus was divided into experimental group and control group according to the above procedure.
In the experimental group 1, 0.4mg/L, 0.8mg/L, 1.6mg/L and 3.2mg/L of agapanthus cystatin is respectively added into the loading liquid, in the experimental group 2, 0.4mg/L, 0.8mg/L, 1.6mg/L and 3.2mg/L of agapanthus cystatin are respectively added into the freezing protective agent, in the experimental group 3, 0.4mg/L, 0.8mg/L, 1.6mg/L and 3.2mg/L of agapanthus cystatin are respectively added into the washing liquid, in the experimental group 4, 0.4mg/L of agapanthus cystatin is simultaneously added into the loading liquid, 1.6mg/L of agapanthus cystatin is added into the freezing protective agent, and 1.6mg/L of agapanthus cystatin is added into the washing liquid. All solutions in the control group do not contain agapanthus cystatin, and other conditions are the same as those in the experimental group.
Preserving in liquid nitrogen for 24 hr, taking out, quickly placing into 40 deg.C water bath, thawing for 90s, and shaking gently; removing the cryoprotectant by suction, adding a washing solution, treating at room temperature for 30min, and replacing the washing solution every 10 min; after the washed agapanthus embryonic callus is transferred to a recovery medium for 1 day, the relative survival rate of the agapanthus embryonic callus in the experimental group and the control group is calculated and compared.
The relative survival rates of the agapanthus embryogenic callus in the experimental group and the control group are shown in Table 1.
TABLE 1
The experimental results are as follows:
as can be seen from Table 1, the survival rate of the agapanthus embryonic callus is improved from 33% to 55% by adding the agapanthus cystatin into the loading liquid alone, wherein the optimal concentration is 0.4 mg/L. The single addition of the freeze protectant can improve the survival rate of the agapanthus embryonic callus to 75% with the optimal concentration of 1.6 mg/L. The single addition of the extract in the washing solution with the optimal concentration of 1.6mg/L can improve the survival rate of the agapanthus embryonic callus to 70%. The three solutions with the optimal concentration are simultaneously added for carrying out the ultralow-temperature preservation treatment on the agapanthus embryonic callus, and the survival rate can be improved to 80%. This shows that the effect of agapanthus cystatin on improving the cryopreservation rate of agapanthus embryonic callus is very obvious.
Example 2
The formula of the experimental reagent in this example is as follows:
1) the MS culture solution is as follows: MS culture solution containing 1900mg/L KNO3，1650mg/L NH4NO3，170mg/L KH2PO4，370mg/L MgSO4·7H2O，440mg/L CaCl2·2H2O，37.3mg/L Na2-EDTA，27.8mg/L FeSO4·7H2O, 100mg/L inositol, 0.5mg/L nicotinic acid, 0.5mg/L pyridoxine hydrochloride, 0.1mg/L thiamine hydrochloride, 2mg/L glycine, 0.83mg/L KI, 6.2mg/L H3BO3，22.3mg/L MnSO4·4H2O，8.6mg/L ZnSO4·7H2O，0.25mg/LNa2MoO4·2H2O，0.025mg/L CuSO4·5H2O，0.025mg/L CoCl2·6H2O and the balance of water. The pH of the MS culture solution is 5.8.
2) The MS solid culture medium is: MS solid culture medium containing 1900mg/L KNO3，1650mg/L NH4NO3，170mg/LKH2PO4，370mg/L MgSO4·7H2O，440mg/L CaCl2·2H2O，37.3mg/L Na2-EDTA，27.8mg/LFeSO4·7H2O, 100mg/L inositol, 0.5mg/L nicotinic acid, 0.5mg/L pyridoxine hydrochloride, 0.1Thiamine hydrochloride in mg/L, glycine in 2mg/L, KI in 0.83mg/L and KI in 6.2mg/L H3BO3，22.3mg/L MnSO4·4H2O，8.6mg/L ZnSO4·7H2O，0.25mg/L Na2MoO4·2H2O，0.025mg/L CuSO4·5H2O，0.025mg/L CoCl2·6H2O, 30g/L of sucrose, 10g/L of agar powder and the balance of water, wherein the pH value of the MS solid culture medium is 5.8.
3) Loading liquid: MS culture medium containing 145.83mL/L of glycerol and 136.92g/L of sucrose.
4) Cryoprotectant: 1/2MS culture solution containing 300g/L of glycerol, 150g/L of ethylene glycol, 136.4mL/L of dimethyl sulfoxide, 136.92g/L of cane sugar and agapanthus cystatin.
5) Washing liquid: MS culture medium containing 410.76g/L sucrose.
6) Recovering the culture medium: MS solid medium containing 30g/L sucrose.
The agapanthus cystatin used in this example was the same as in example 1 and was added in the same manner as in example 1.
The specific implementation method comprises the following steps:
1) sowing Arabidopsis seeds on an MS solid culture medium, placing the MS solid culture medium in a refrigerator at 4 ℃ for vernalization for 2 days, then placing the MS solid culture medium in a lighting incubator at 25 ℃, and continuously lighting and culturing for 60 hours;
2) transferring the mixture into a loading solution and soaking the mixture for 30 minutes at room temperature;
3) transferring into cryoprotectant, and dehydrating at 4 deg.C for 50 min;
4) and finally placing the mixture in liquid nitrogen for ultralow temperature preservation.
And 3) directly placing the arabidopsis seedlings soaked in the vitrification solution in liquid nitrogen for ultralow temperature preservation without removing the vitrification solution after the step 3).
Arabidopsis seedlings were divided into experimental and control groups according to the above procedure.
In the experimental group 1, 0.4mg/L, 0.8mg/L, 1.6mg/L and 3.2mg/L of agapanthus cystatin is respectively added into the loading liquid, in the experimental group 2, 0.4mg/L, 0.8mg/L, 1.6mg/L and 3.2mg/L of agapanthus cystatin are respectively added into the freezing protective agent, in the experimental group 3, 0.4mg/L, 0.8mg/L, 1.6mg/L and 3.2mg/L of agapanthus cystatin are respectively added into the washing liquid, in the experimental group 4, 0.4mg/L of agapanthus cystatin is simultaneously added into the loading liquid, 1.6mg/L of agapanthus cystatin is added into the freezing protective agent, and 1.6mg/L of agapanthus cystatin is added into the washing liquid. All solutions in the control group do not contain agapanthus cystatin, and other conditions are the same as those in the experimental group.
Preserving in liquid nitrogen for 24 hr, taking out, quickly placing into 40 deg.C water bath, thawing for 90s, and shaking gently; removing the vitrification solution by suction, adding a washing solution, treating at room temperature for 40min, and replacing the washing solution every 10 min; after the washed Arabidopsis seedlings were transferred to a recovery medium and cultured for 14 days, the survival rates of Arabidopsis seedlings in the experimental group and the control group were calculated and compared.
The survival rates of Arabidopsis seedlings in the experimental group and the control group are shown in Table 2.
TABLE 2
The experimental results are as follows:
as can be seen from Table 2, the survival rate of 60h seedlings germinated from Arabidopsis is improved from 30% to 40% by adding the agapanthus cystatin into the loading liquid alone for survival, wherein the optimal concentration is 0.4 mg/L. The freeze protectant is added separately, the optimal concentration is 1.6mg/L, and the survival rate of seedlings which germinate 60 hours in arabidopsis thaliana can be improved to 37%. The growth promoter is added into a washing solution independently, the optimal concentration is 1.6mg/L, and the survival rate of seedlings which germinate 60 hours from arabidopsis thaliana can be improved to 45%. Three solutions with optimal concentrations are simultaneously added for carrying out ultralow-temperature preservation treatment on seedlings of arabidopsis thaliana germinated for 60 hours, and the survival rate can be improved to 50%. This shows that the effect of the agapanthus cystatin on improving the ultralow temperature preservation rate of 60h seedlings germinated from arabidopsis thaliana is very obvious.
The foregoing description of specific embodiments of the present invention has been presented. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes or modifications may be made by one skilled in the art within the scope of the appended claims without departing from the spirit of the invention. The embodiments and features of the embodiments of the present application may be combined with each other arbitrarily without conflict.
Claims (10)
1. Application of agapanthus cystatin in improving survival rate of plant material after ultralow temperature preservation is provided.
2. The use according to claim 1, wherein the agapanthus cystatin is added as an additive to at least one of a loading liquid, a cryoprotectant, a washing liquid.
3. The use according to claim 2, wherein the concentration of baizi cystatin in the loading solution, cryoprotectant or washing solution is 0.4-1.6 mg/L.
4. The use according to claim 2 or 3, wherein the agapanthus cystatin is added to each solution after mixing with phosphate buffer.
5. The use of claim 2 or 3, wherein the agapanthus cystatin is obtained by prokaryotic expression using escherichia coli Transetta competence (Beijing hologold biotechnology, Inc., catalog number CD801-01), and the prokaryotic expression conditions are 16-25 ℃, IPTG concentration is 0.1-1 mM, and protein expression induction time is 18-24 h; performing His-tag purification after protein induction expression.
6. A method for improving the survival rate of plant materials after ultralow temperature preservation is characterized by comprising the following steps:
A. pretreating plant materials, and soaking in a loading solution;
B. transferring the soaked plant material into a cryoprotectant, dehydrating, and then placing in liquid nitrogen for ultralow temperature preservation;
C. taking out the plant material from liquid nitrogen, thawing in water bath, removing cryoprotectant, and treating with washing solution;
D. removing the washing liquid, and transferring to a recovery culture medium for recovery culture;
at least one solution of the loading liquid, the freezing protective agent and the washing liquid contains agapanthus cystatin; the concentration of the agapanthus cystatin in the loading liquid, the cryoprotectant or the washing liquid is 0.4-1.6mg/L respectively.
7. The method of increasing survival rate of plant material after ultra-low temperature storage according to claim 6, wherein the loading liquid comprises the following components: 4.3g/L MS powder, 12mol/L glycerol, 0.4mol/L sucrose and 10mmol/L KNO3(ii) a The pH value of the loading liquid is 5.6-6.2.
8. The method of increasing survival rate of plant material after cryopreservation as claimed in claim 6 wherein the cryoprotectant comprises the following components: 2.15g/L MS powder, 136.92g/L sucrose, 300g/L glycerol, 150g/L ethylene glycol and 136.4mL/L dimethyl sulfoxide; the pH value of the cryoprotectant is 5.6-6.2.
9. The method of increasing survival rate of plant material after ultra-low temperature storage according to claim 6, wherein the washing solution comprises the following components: 4.3g/L MS powder, 1.2mol/L sucrose and 10mmol/L KNO3(ii) a The pH value of the washing liquid is 5.6-6.2.
10. The method for improving survival rate of plant materials after ultralow temperature preservation according to claim 6, wherein the recovery medium comprises 4.3g/L MS powder, 30g/L sucrose and 10g/L agar, and has a pH value of 5.6-6.2.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911108062.3A CN110959330B (en) | 2019-11-13 | 2019-11-13 | Application of agapanthus cystatin in improving survival rate of plant cells after ultralow-temperature preservation |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911108062.3A CN110959330B (en) | 2019-11-13 | 2019-11-13 | Application of agapanthus cystatin in improving survival rate of plant cells after ultralow-temperature preservation |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110959330A true CN110959330A (en) | 2020-04-07 |
| CN110959330B CN110959330B (en) | 2021-07-13 |
Family
ID=70030475
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911108062.3A Active CN110959330B (en) | 2019-11-13 | 2019-11-13 | Application of agapanthus cystatin in improving survival rate of plant cells after ultralow-temperature preservation |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110959330B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104170818A (en) * | 2014-09-15 | 2014-12-03 | 上海交通大学 | Method for optimizing vitrified cryopreservation effect of agapanthus embryonic calluses |
| CN104255706A (en) * | 2014-09-15 | 2015-01-07 | 上海交通大学 | Method for optimizing vitrification ultra-low temperature preservation effect of arabidopsis seedlings |
-
2019
- 2019-11-13 CN CN201911108062.3A patent/CN110959330B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104170818A (en) * | 2014-09-15 | 2014-12-03 | 上海交通大学 | Method for optimizing vitrified cryopreservation effect of agapanthus embryonic calluses |
| CN104255706A (en) * | 2014-09-15 | 2015-01-07 | 上海交通大学 | Method for optimizing vitrification ultra-low temperature preservation effect of arabidopsis seedlings |
Non-Patent Citations (2)
| Title |
|---|
| GUAN-QUN CHEN 等: "Cloning and characterization of ApCystatin, a plant cystatin gene from Agapanthus praecox ssp. orientalis responds to abiotic stress", 《PROTEIN EXPRESSION AND PURIFICATION》 * |
| LI REN: "Transcriptomic profiling revealed the regulatory mechanism of Arabidopsis seedlings response to oxidative stress from cryopreservation", 《PLANT CELL REP》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110959330B (en) | 2021-07-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Sakai | Plant cryopreservation | |
| JP5679219B2 (en) | Flowering inducer | |
| CN109553671B (en) | Poncirus trifoliata cold-resistant gene PtrTZF1 and application thereof in plant cold-resistant genetic improvement | |
| GEZELIUS et al. | Seasonal variation in ribulose bisphosphate carboxylase activity in Pinus silvestris | |
| Huang et al. | Plant regeneration from rice (Oryza sativa L.) embryogenic suspension cells cryopreserved by vitrification | |
| CN111493066A (en) | Peony cut flower vase preservative and use method thereof | |
| CN113773137B (en) | Universal low-temperature-resistant liquid fertilizer and preparation method thereof | |
| CN110959330B (en) | Application of agapanthus cystatin in improving survival rate of plant cells after ultralow-temperature preservation | |
| CN111972402A (en) | Vitrification cryopreservation and thawing method for agate red cherry embryogenic callus | |
| CN109182359B (en) | Pear cold-resistant gene PbrBAM3, expression vector and application thereof, encoded protein and application thereof | |
| CN110066810B (en) | Tamarix chinensis plasma membrane Na+/H+Antiporter gene and application thereof | |
| CN108094020A (en) | A kind of jujube tree engrafting method | |
| CN110627568B (en) | Seed soaking composition and method for improving germination rate and seedling rate of cherry seeds | |
| Weinberger et al. | Glutamic carboxylase of the mature wheat leaf | |
| Heszky et al. | Effect of cooling rate, cryoprotectant and holding time at different transfer temperatures on the survival of cryopreserved cell suspension culture (Puccinellia distans (L.) Parl.) | |
| CN107488669B (en) | CauliflowerBoTLP1Coding sequence of gene and application thereof in cultivating salt-tolerant drought-resistant transgenic plant | |
| CN111493064A (en) | Bovine cloned embryo refrigerating fluid, thawing fluid, kit and bovine cloned embryo freezing and thawing method | |
| Jogaiah et al. | Rootstock induced changes in enzymes activity and biochemical constituents during budbreak in ‘Thompson Seedless’ grapevine | |
| CN117378599B (en) | Ultralow temperature cryopreservation and activation method for tetraploid sperm of Pacific oyster | |
| CN109619094B (en) | Baizilian SK3Application of dehydrin protein in reducing cell stress injury and improving ultralow temperature preservation effect | |
| RU2248121C1 (en) | Method for cryogenic in vitro keeping of meristems isolated from red raspberry plants | |
| CN101637124A (en) | Pre-culture medium for storing Chinese yam idioplasm by embedding vitrifaction at ultralow temperature and cultivation method thereof | |
| FI95042C (en) | Storage procedure for plant sprouts | |
| Chang¹ et al. | Cold acclimation improves the cryopreservation of in vitro-grown Pyrus and Rubus meristems | |
| AU2020103457A4 (en) | Composition for improving cold resistance of peppers at a seedling stage, and preparation method and use thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |