CN109729977B - Method for inhibiting browning of explant pollution in process of cultivating juglans mandshurica stem segments - Google Patents
Method for inhibiting browning of explant pollution in process of cultivating juglans mandshurica stem segments Download PDFInfo
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- 235000014075 Juglans mandschurica Nutrition 0.000 title claims abstract description 35
- 241000305529 Juglans mandshurica Species 0.000 title claims abstract description 33
- 230000002401 inhibitory effect Effects 0.000 title claims abstract description 24
- 239000001963 growth medium Substances 0.000 claims abstract description 32
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 12
- SQGYOTSLMSWVJD-UHFFFAOYSA-N silver(1+) nitrate Chemical compound [Ag+].[O-]N(=O)=O SQGYOTSLMSWVJD-UHFFFAOYSA-N 0.000 claims abstract description 12
- 229930024421 Adenine Natural products 0.000 claims abstract description 8
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 claims abstract description 8
- 229960000643 adenine Drugs 0.000 claims abstract description 8
- 229910001961 silver nitrate Inorganic materials 0.000 claims abstract description 6
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- 235000019345 sodium thiosulphate Nutrition 0.000 claims abstract description 6
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- 238000005406 washing Methods 0.000 claims description 20
- 238000012258 culturing Methods 0.000 claims description 18
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 12
- 230000001954 sterilising effect Effects 0.000 claims description 12
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- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 6
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- 238000005286 illumination Methods 0.000 claims description 6
- 238000005057 refrigeration Methods 0.000 claims description 6
- 238000002791 soaking Methods 0.000 claims description 6
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- 239000005720 sucrose Substances 0.000 claims description 6
- RCTYPNKXASFOBE-UHFFFAOYSA-M chloromercury Chemical compound [Hg]Cl RCTYPNKXASFOBE-UHFFFAOYSA-M 0.000 claims description 5
- 238000004140 cleaning Methods 0.000 claims description 5
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- 239000000022 bacteriostatic agent Substances 0.000 claims description 3
- 229940086763 ascorbic acid 100 mg Drugs 0.000 claims description 2
- 229910052799 carbon Inorganic materials 0.000 claims description 2
- 238000011109 contamination Methods 0.000 claims 1
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 abstract description 6
- 235000010323 ascorbic acid Nutrition 0.000 abstract description 3
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- 210000001099 axilla Anatomy 0.000 description 1
- JNPZQRQPIHJYNM-UHFFFAOYSA-N carbendazim Chemical compound C1=C[CH]C2=NC(NC(=O)OC)=NC2=C1 JNPZQRQPIHJYNM-UHFFFAOYSA-N 0.000 description 1
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- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 1
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
- Y02A40/22—Improving land use; Improving water use or availability; Controlling erosion
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/40—Afforestation or reforestation
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Abstract
A method for inhibiting browning of explant pollution in a juglans mandshurica stem section culture process is characterized in that an explant is disinfected by using Eikek, then the disinfected explant is inoculated on a DKW culture medium added with 10mg/L adenine AD for low-temperature dark culture, and then conventional culture is carried out. The method for inhibiting the browning of the explant in the process of cultivating the juglans mandshurica stem section has the following advantages: the DKW culture medium is used as a basic culture medium, activated carbon, sodium thiosulfate, silver nitrate, 2% PVP and ascorbic acid with different concentrations are added into the culture medium, and explants are transplanted every 1 day in the culture process, so that browning of the juglans mandshurica explants can be effectively inhibited; compared with the existing substances for reducing browning, the invention does not need to add special equipment, has low cost and simple and quick operation, and can realize the browning resistance rate of more than 92 percent which is far higher than the prior art.
Description
Technical Field
The invention belongs to the technical field of forest seedling propagation, and particularly relates to a method for inhibiting browning of explants in a juglans mandshurica stem section culture process.
Background
Juglans mandshurica Maxim belongs to Juglandaceae Juglans, is one of three hard broad trees in northeast forest region, is mainly distributed in northeast mountain region of China in a centralized way, has sporadic distribution in northeast China region, and extends to great Xingan mountain region and Russian far east region from northeast south to north of Korea and Japan. But due to long-term excessive felling, the natural forest resources are in imminent danger; the material is hard and compact, the texture is straight and beautiful, and the product has the advantages of toughness, non-cracking, corrosion resistance and the like, and simultaneously, the immature epicarp, the root branch bark, the shell and the leaves can be used as medicines, so the product has wide application and high economic value.
At present, the research on the juglans mandshurica is mainly focused on the aspects of distribution, site afforestation, fostering renewal, material properties and the like, and relatively few in the aspect of reproduction. The conventional breeding mode of the juglans mandshurica mainly comprises two ways of seed breeding and sprout breeding, the transport and gnawing of rodents cause the loss of a juglans mandshurica seed bank, and the natural germination rate is low; the sprouting shoot propagation often causes the dry type and the unsatisfactory material quality of the juglans mandshurica maxim.
The juglans mandshurica belongs to the gradual-growing variety, the juglans mandshurica seeds are usually propagated by adopting seeds in production, but the juglans mandshurica seeds belong to dormant seeds, the seeds need to be subjected to germination accelerating treatment, the period is long, the emergence rate is low, the quality of seedlings is uneven, the seedling propagation speed is slow in production practice and market requirements and hardly reaches the required quantity, and the development of forestry production is seriously hindered. Therefore, tissue culture of juglans mandshurica becomes particularly important. Although the domestic report about the tissue culture of the juglans mandshurica maxim exists, the juglans mandshurica maxim plants are rich in polysaccharide polyphenol substances, so that a brown necrotic layer is easily generated at the cut of the explant in the tissue culture process, the germination of the juglans mandshurica maxim explant is difficult, and the browning and the degeneration of an isolated culture are serious, so that the tissue culture of the juglans mandshurica maxim is a main factor for inhibiting the tissue culture of the juglans mandshurica maxim. Therefore, the research of inhibiting the browning of the explant in the tissue culture process of the juglans mandshurica has important guiding significance for the tissue culture and the asexual propagation of the juglans mandshurica.
Disclosure of Invention
The invention aims to provide a method for inhibiting browning of an explant in a juglans mandshurica stem section culture process, which is simple and convenient to operate and has a good browning inhibiting effect.
A method for inhibiting browning of explants in a juglans mandshurica stem section culture process comprises the steps of disinfecting the explants by using Ailock, then inoculating the disinfected explants on a DKW culture medium added with 10mg/L adenine AD to perform low-temperature dark culture, and then performing conventional culture.
A method for inhibiting browning of explants polluted in the process of cultivating the stem segments of juglans mandshurica maxim comprises the following steps:
(1) respectively placing bacteriostatic agents Aiike A and Aiike B into 1000mL of water to prepare Aiike disinfection solution with the concentration of 250 mg/L;
(2) selecting a stem section which is robust and has no disease or insect pest and is provided with axillary buds or terminal buds and grows for 1-2 years as an explant, slightly brushing off bacteria at axillary positions by using a soft brush, then soaking the explant in the Ailock disinfection solution prepared in the step (1) for 30min, continuously stirring, and then placing the sterilized explant under running water for washing for 2 h;
(3) transferring to a clean bench, sterilizing with ultraviolet rays for 20min, cleaning the explant treated in the step (2) with 70% ethanol for 2 times, 30s each time, and then washing with sterile water for 1-2 times, wherein the time is 1 min;
(4) with 0.1% HgCl 2 Sterilizing the explant treated in the step (3) for 10min, and then washing with sterile water for 5-7 times, wherein the time for each time is 1 min;
(5) placing the explant sterilized in the step (4) on high-temperature sterilized filter paper to absorb water, and then cutting off two ends of the explant by using a sterile scalpel to enable the size of the explant to be about 2.0 cm;
(6) inoculating the explant treated in the step (5) into a culture bottle, wherein a culture medium in the culture bottle is a DKW culture medium added with 10mg/L adenine AD, and IBA0.1mg/L, BA1.0mg/L, sucrose 30g/L and agar 6g/L are added into the DKW culture medium, and the pH value is 5.8-6.0;
(7) placing the inoculated culture bottle in a refrigerator at 0-4 ℃ for refrigeration, and culturing for 5d under the dark condition;
(8) transferring the culture bottle treated in the step (7) into a culture room for conventional culture; the conventional culture conditions are as follows: continuously culturing for 12h at the temperature of 23-25 ℃ and the humidity of 70-80% under the illumination intensity of 1500Lux every day, and then continuously culturing for 12h under the dark condition;
(9) and (4) performing subculture on the explants cultured in the culture bottle for 2d in the step (8), and replacing the positions in the culture bottle for 1 time every 1 day to play a role in inhibiting browning.
Preferably, the DKW culture medium is added with 1.0mg/L of sodium thiosulfate, 1.5mg/L of silver nitrate, 1.0mg/L of 2% PVP, 0.5g/L of active carbon and 100mg/L of ascorbic acid.
More preferably, the browning rate of the method for inhibiting the browning of the explant in the process of cultivating the juglans mandshurica stem sections is more than 92%.
The method for inhibiting browning of explant pollution in the juglans mandshurica stem culture process has the following advantages:
firstly, on one hand, the explant is transplanted every 1 day in the culture process by taking a DKW culture medium as a basic culture medium, so that the browning of the explant can be effectively relieved;
secondly, a DKW culture medium is used as a basic culture medium, and activated carbon, sodium thiosulfate, silver nitrate, 2% PVP and ascorbic acid with different concentrations can be added into the culture medium, so that browning of the juglans mandshurica maxim explant is effectively inhibited;
compared with the existing substances for reducing browning, the method of the invention does not need to add special equipment, has low cost and simple, convenient and quick operation;
the method can realize the anti-browning rate of more than 92 percent, which is far higher than the prior art.
Detailed Description
To make the objects, technical solutions and advantages of the embodiments of the present invention clearer and more complete description of the technical solutions in the embodiments of the present invention, it is obvious that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
A method for inhibiting browning of explants polluted in the process of cultivating the stem segments of juglans mandshurica maxim comprises the following steps:
(1) respectively placing bacteriostatic agents Aiike A and Aiike B into 1000mL of water to prepare Aiike disinfection solution with the concentration of 250 mg/L;
(2) selecting a stem section which grows vigorously and is free of diseases and insect pests and has axillary buds or terminal buds and is 1-2 years old as an explant, slightly brushing bacteria at axillary positions with a pappus brush, then soaking the explant in the Ailike disinfection solution prepared in the step (1) for 30min, continuously stirring, and then placing the sterilized explant under running water for washing for 2 h;
(3) transferring to a clean bench, sterilizing with ultraviolet rays for 20min, washing the explant treated in step (2) with 70% ethanol for 2 times, each time for 30s, and then washing with sterile water for 2 times, each time for 1 min;
(4) with 0.1% HgCl 2 Sterilizing the explants processed in the step (3) for 10min, and then washing the explants with sterile water for 5 times, wherein the time of each time is 1 min;
(5) placing the explant sterilized in the step (4) on high-temperature sterilized filter paper to absorb water, and then cutting off two ends of the explant by using a sterile scalpel to enable the size of the explant to be about 2.0 cm;
(6) inoculating the explant treated in the step (5) into a culture bottle, wherein a culture medium in the culture bottle is a DKW culture medium added with 10mg/L adenine AD, IBA0.1mg/L, BA 1.0.0 mg/L, sucrose 30g/L and agar 6g/L are further added into the DKW culture medium, the pH value is 5.8-6.0, and the DKW culture medium is added with sodium thiosulfate 1.0mg/L, silver nitrate 1.5mg/L, 2% PVP 1.0mg/L, activated carbon 0.5g/L and ascorbic acid 100 mg/L;
(7) placing the inoculated culture bottle in a refrigerator at 0-4 ℃ for refrigeration, and culturing for 5d under the dark condition;
(8) transferring the culture bottle treated in the step (7) into a culture chamber for conventional culture; the conventional culture conditions are as follows: continuously culturing for 12h at the temperature of 23-25 ℃ and the humidity of 70-80% under the illumination intensity of 1500Lux every day, and then continuously culturing for 12h under the dark condition;
(9) and (4) subculturing the explants cultured in the culture bottle for 2d in the step (8), and replacing the positions in the culture bottle for 1 time every 1 day to play a role in inhibiting browning.
The detection result shows that the anti-browning rate of the sample can reach more than 92%.
Comparative example 1
A method for inhibiting browning of explant pollution in the process of cultivating juglans mandshurica stem segments comprises the following steps:
(1) selecting a stem section which is strong and has no disease or insect pest and grows 1-2 years of juglans mandshurica and has axillary buds or terminal buds as an explant, slightly brushing off bacteria at axillary parts of leaves by using a pappus brush, and then placing the explant under running water for washing for 2 hours;
(2) soaking the explants processed in the step (1) in a solution containing 0.1% carbendazim inhibitor for 15min, continuously stirring, and then placing the disinfected explants under running water for washing for 2 h;
(3) transferring to a clean bench, ultraviolet sterilizing for 10min, cleaning the explant treated in step (2) with 70% ethanol for 2 times and 30 s/time, washing with sterile water for 1 time, and standing for 1 min;
(4) with 0.1% HgCl 2 Sterilizing the explant treated in the step (3) for 10min, and then washing with sterile waterStanding for 1min for 5 times;
(5) placing the explant sterilized in the step (4) on high-temperature sterilized filter paper to absorb water, and then cutting off two ends of the explant by using a sterile scalpel to enable the size of the explant to be about 2.0 cm;
(6) inoculating the explant treated in the step (5) into a culture bottle, wherein a culture medium in the culture bottle is an MS culture medium; IBA0.1mg/L, BA 1.0.0 mg/L, sucrose 30g/L and agar 6g/L are also added into the MS culture medium, and the pH value is 5.8-6.0;
(7) placing the inoculated culture bottle in a refrigerator at 0-4 ℃ for refrigeration, and culturing for 5d under the dark condition;
(8) transferring the culture bottle treated in the step (7) into a culture room for conventional culture; the conventional culture conditions are as follows: continuously culturing for 12h at the temperature of 23-25 ℃ and the humidity of 70-80% under the illumination intensity of 2000Lux every day, and then continuously culturing for 12h under the dark condition;
(9) and (4) subculturing the explants cultured in the culture bottle for 2d in the step (8), and replacing the positions in the culture bottle for 1 time every 1 day to play a role in inhibiting browning.
The comparative example was tested for a brown change resistance of 84.6%.
Comparative example 2
A method for inhibiting browning of explants polluted in the process of cultivating the stem segments of juglans mandshurica maxim comprises the following steps:
(1) selecting a stem section which is vigorous and has no disease or insect pest and is provided with axillary buds or terminal buds and is grown for 1-2 years as an explant, and slightly brushing off bacteria at the axillary part of a leaf by using a soft brush;
(2) soaking the explant treated in the step (1) in 84 disinfectant containing 0.2% for 10-15 min, continuously stirring, and then placing the disinfected explant under running water for washing for 2 h;
(3) transferring to a clean bench, sterilizing with ultraviolet for 20min, cleaning the explant treated in step (2) with 70% ethanol for 2 times, 30s each time, washing with sterile water for 3 times, standing for 1min each time, and irradiating with ultraviolet for 10 min;
(4) With 0.1% HgCl 2 Sterilizing the explant treated in the step (3) for 5min, and then washing with sterile water for 5 times, wherein the time for each time is 1 min;
(5) placing the explant sterilized in the step (4) on high-temperature sterilized filter paper to absorb water, and then cutting off two ends of the explant by using an aseptic scalpel to enable the size of the explant to be about 2.0 cm;
(6) inoculating the explant treated in the step (5) into a culture bottle, wherein a culture medium in the culture bottle is a DKW culture medium added with 10mg/L adenine AD, IBA0.1mg/L, BA 1.0.0 mg/L, sucrose 30g/L and agar 6g/L are additionally added into the DKW culture medium, the pH value is 5.8-6.0, and silver nitrate 1.0mg/L and activated carbon 2.0g/L are added into the DKW culture medium;
(7) placing the inoculated culture bottle in a refrigerator at 0-4 ℃ for refrigeration, and culturing for 5d under a dark condition;
(8) transferring the culture bottle treated in the step (7) into a culture room for conventional culture; the conventional culture conditions are as follows: continuously culturing for 12 hours at the temperature of 23-25 ℃ and the humidity of 70-80% under the illumination intensity of 2000Lux every day, and then continuously culturing for 12 hours under the dark condition;
(9) and (4) subculturing the explants cultured in the culture bottle for 2d in the step (8), and replacing the positions in the culture bottle for 1 time every 3 days to play a role in inhibiting browning.
The comparative example was tested for a brown change resistance of 86.7%.
Comparative example 3
A method for inhibiting browning of explant pollution in the process of cultivating juglans mandshurica stem segments comprises the following steps:
(1) selecting a stem section which is vigorous and free from diseases and insect pests and is provided with axillary buds or terminal buds and grows for 1-2 years as an explant, and refrigerating the explant at the low temperature of 1-2 ℃ for 24 h;
(2) then soaking the explant in a 20% sodium thiosulfate solution for 30min, then slightly brushing off bacteria at the axilla of the leaf by using a soft brush, and then putting the explant under running water for washing for 2 h;
(3) transferring to a clean bench, ultraviolet sterilizing for 20min, cleaning the explant treated in step (2) with 70% ethanol for 2 times (30 s each time), washing with sterile water for 2 times (1 min each time), and ultraviolet irradiating for 10 min;
(4) sterilizing the explant treated in the step (3) by using 10% sodium hypochlorite for 10min, and then washing the explant by using sterile water for 5 times, wherein the explant stays for 1min each time;
(5) placing the explant sterilized in the step (4) on high-temperature sterilized filter paper to absorb water, and then cutting off two ends of the explant by using an aseptic scalpel to enable the size of the explant to be about 1.5 cm;
(6) inoculating the explant treated in the step (5) into a culture bottle, wherein a culture medium in the culture bottle is a DKW culture medium added with 10mg/L adenine AD, IBA0.1mg/L, BA 1.0.0 mg/L, sucrose 30g/L and agar 6g/L are additionally added into the DKW culture medium, the pH value is 5.8-6.0, and 2% PVP 0.1mg/L, activated carbon 0.5g/L and ascorbic acid 50mg/L are added into the DKW culture medium;
(7) placing the inoculated culture bottle in a refrigerator at 0-4 ℃ for refrigeration, and culturing for 5d under a dark condition;
(8) transferring the culture bottle treated in the step (7) into a culture room for conventional culture; the conventional culture conditions are as follows: continuously culturing for 12h at the temperature of 23-25 ℃ and the humidity of 70-80% under the illumination intensity of 2000Lux every day, and then continuously culturing for 12h under the dark condition;
(9) and (4) subculturing the explants in the culture bottle cultured in the step (8) for 2d, and replacing the positions in the culture bottle for 1 time every 1 week to play a role in inhibiting browning.
The comparative example was tested to have a brown change resistance of 80.7%.
It should be understood that the above-described specific embodiments are merely illustrative of the present invention and are not intended to limit the present invention. Obvious variations or modifications which are within the spirit of the invention are possible within the scope of the invention.
Claims (2)
1. A method for inhibiting browning of explants in the process of cultivating the stem segments of juglans mandshurica maxim is characterized in that the explants are disinfected by using Aiike, then the disinfected explants are inoculated on a DKW culture medium added with 10mg/L adenine AD for low-temperature dark cultivation, and then conventional cultivation is carried out;
the method comprises the following steps:
(1) respectively adding bacteriostatic agents Aiike A and Aiike B into 1000mL of water to prepare Aiike disinfection solution with the concentration of 250 mg/L;
(2) selecting a stem section which is robust and has no disease or insect pest and is provided with axillary buds or terminal buds and grows for 1-2 years as an explant, slightly brushing off bacteria at axillary positions by using a soft brush, then soaking the explant in the Ailock disinfection solution prepared in the step (1) for 30min, continuously stirring, and then placing the sterilized explant under running water for washing for 2 h;
(3) transferring the explant on a super-clean workbench, sterilizing the explant for 20min by ultraviolet rays, cleaning the explant treated in the step (2) by using 70% ethanol for 2 times, 30s each time, and then washing the explant by using sterile water for 1-2 times, wherein the explant stays for 1min each time;
(4) with 0.1% HgCl 2 Sterilizing the explant treated in the step (3) for 10min, and then washing with sterile water for 5-7 times, wherein the time for each time is 1 min;
(5) placing the explant sterilized in the step (4) on high-temperature sterilized filter paper to absorb water, and then cutting off two ends of the explant by using a sterile scalpel to enable the size of the explant to be about 2.0 cm;
(6) inoculating the explant treated in the step (5) into a culture bottle, wherein the culture medium in the culture bottle is a DKW culture medium, adenine AD 10mg/L, IBA0.1mg/L, BA1.0mg/L, sucrose 30g/L, agar 6g/L, sodium thiosulfate 1.0mg/L, silver nitrate 1.5mg/L, 2% PVP 1.0mg/L, active carbon 0.5g/L and ascorbic acid 100mg/L, and the pH is 5.8-6.0;
(7) placing the inoculated culture bottle in a refrigerator at 0-4 ℃ for refrigeration, and culturing for 5d under a dark condition;
(8) transferring the culture bottle treated in the step (7) into a culture chamber for conventional culture; the conventional culture conditions are as follows: continuously culturing for 12h at the temperature of 23-25 ℃ and the humidity of 70-80% under the illumination intensity of 1500Lux every day, and then continuously culturing for 12h under the dark condition;
(9) and (4) subculturing the explants cultured in the culture bottle for 2d in the step (8), and replacing the positions in the culture bottle for 1 time every 1 day to play a role in inhibiting browning.
2. The method of claim 1, wherein the browning of the explant contamination during the stem section culture of juglans mandshurica has a browning resistance of 92%.
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