CN108077077B - Hair removing method and in-vitro culture method for top-growing cluster hair seeds - Google Patents
Hair removing method and in-vitro culture method for top-growing cluster hair seeds Download PDFInfo
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- CN108077077B CN108077077B CN201711429587.8A CN201711429587A CN108077077B CN 108077077 B CN108077077 B CN 108077077B CN 201711429587 A CN201711429587 A CN 201711429587A CN 108077077 B CN108077077 B CN 108077077B
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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- 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
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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Abstract
the invention provides a hair removing method for a clumpy hair-like seed growing at the top end, which comprises the following steps: 1) cutting off the cluster hair of the cluster hair seed growing at the top end to the base part to obtain the seed with the cut cluster hair; 2) immersing the seeds cut off the cluster hair obtained in the step 1) in a sodium chloride solution to obtain clean and plump seeds with hair removed; the mass concentration of the sodium chloride solution is 10-16 g/L. The invention removes the cluster hair of the cluster hair seed growing on the top, so that the seed is more thoroughly disinfected in the in vitro culture, and the pollution rate of the seed in the in vitro culture is greatly reduced.
Description
Technical Field
The invention relates to the technical field of tissue culture, in particular to a hair removing method and an in vitro culture method for a top-growing tufted hair seed.
Background
Desert plants have irreplaceable effects in ecological restoration in arid desert regions. Like other dry-grown and sandy plants, the sandy tamarisk forms typical sandy dry-grown characteristics when adapting to external environmental conditions such as drought, sand burying, strong light and the like for a long time, so that the method has strong adaptability, and the main habitat types of the sandy tamarisk are flowing and semi-flowing sand, and are dominant species and main population building species of a sandy plant community. However, the amount of the psammosilene tamarisk bearing is very small, the seeds are ovoid and thin, the length of the seeds is about 3-4 mm, the seeds are small and light, the top end of the seeds are tufted and have the hair length of about 5mm, the seeds are easy to be blown off and hung on other plants or float to the ground along with wind after being mature, the seeds are difficult to germinate, the psammosilene tamarisk is difficult to reproduce and update under the natural condition of the field by the self-force of the seeds, and seedlings are less and less. In addition, with the global warming in recent years, the rainfall area of the desert area is greatly changed, and the wild sand-grown tamarix chinensis resources are greatly damaged due to the serious desertification and the excessive grazing of the land. Therefore, it is urgently needed to accelerate the protection of germplasm resources by means of an artificial propagation method. The in vitro rapid propagation of the plants utilizes the asexual propagation method to solve the problem that the plants are difficult to propagate under the natural condition on the basis of keeping the excellent characteristics of the plants.
the seed is an explant material widely adopted in the process of aseptic seedling culture in the early stage of plant in vitro rapid propagation. The sterilization and disinfection of seeds is the first step of the in vitro rapid propagation of plants and is also the key for determining the success of the asexual propagation of the plants. The sand tamarix chinensis seeds are small and light and have hair, so that the seeds are easy to float in the air or adhere to an operation table top, a vessel and clothes of operators due to large air flow after picking, and the environmental pollution of an operation room and the great waste of the seeds are caused.
at present, no patent or research report on a hair removing method and an in vitro culture method of the Saxago chinensis or other apical tufted hair seeds is available in China.
Disclosure of Invention
The invention aims to provide a method for removing hairs of a top-growing cluster hair seed, which can reduce the pollution rate in the in vitro culture process.
In order to achieve the above object, the present invention provides the following technical solutions:
The invention provides a hair removing method for a clumpy hair-like seed growing at the top end, which comprises the following steps:
1) Cutting off the cluster hair of the cluster hair seed growing at the top end to the base part to obtain the seed with the cut cluster hair;
2) Immersing the seeds cut off the cluster hair obtained in the step 1) in a sodium chloride solution to obtain clean and plump seeds with hair removed; the mass concentration of the sodium chloride solution is 10-16 g/L.
preferably, the top-growing bushy seeds are soaked before cutting off the bushy, the soaking comprising: mixing the seed raw material with the clumpy hair growing on the top end with water, and sequentially carrying out first soaking and second soaking; the first soaking temperature is 35-45 ℃, and the second soaking temperature is 1-8 ℃.
Preferably, the first soaking time is 3-5 hours, and the second soaking time is 1-3 hours.
The invention also provides an isolated culture method of the top-growing tufted hair seed, which comprises the following steps:
a. Disinfecting the seed without hair obtained by the method for removing hair in the technical scheme to obtain disinfected seed;
b. And c, placing the sterilized seeds obtained in the step a on an in vitro culture medium for culture.
Preferably, the sterilization process comprises the steps of:
I. Sequentially treating the seed without the seed hair with an ethanol solution and washing with sterile water to obtain the seed treated with the ethanol solution;
And II, treating the seeds treated by the obtained ethanol solution by using a mercuric chloride solution and washing the seeds by using sterile water in sequence to obtain the sterilized seeds.
Preferably, the volume percentage content of the ethanol solution in the step I is 70-80%, and the treatment time of the ethanol solution is 25-35 s.
Preferably, the mass percentage of the mercuric chloride solution in the step II is 0.05-0.15%, and the treatment time of the mercuric chloride solution is 4-8 min.
Preferably, the in vitro culture medium takes an MS culture medium as a basic culture medium and comprises the following components in concentration: 0.1-0.5 mg/L gibberellin, 20-40 g/L sucrose and 4-6 g/L agar.
Preferably, the culture conditions include: the cultivation temperature is 23-27 ℃, the cultivation illumination intensity is 1400-1800 lx, and the cultivation illumination time is 10-14 h/d.
The invention provides a hair removing method for a clumpy hair-like seed growing at the top end, which comprises the following steps: 1) cutting off the cluster hair of the cluster hair seed growing at the top end to the base part to obtain the seed with the cut cluster hair; 2) immersing the seeds cut off the cluster hair obtained in the step 1) in a sodium chloride solution to obtain clean and plump seeds with hair removed; the mass concentration of the sodium chloride solution is 10-16 g/L.
the invention removes the hair of the cluster hair seeds growing on the top, so that the seeds are thoroughly disinfected in the in vitro culture, and the pollution rate of the seeds in the in vitro culture is greatly reduced.
The results of the embodiments of the present invention show that: according to the method for removing the hair of the bush-like hair seeds growing on the top end, the hair removal rate of the Sasa chinensis seeds is 85-92%, mechanical damage to the Sasa chinensis seeds cannot be caused in the operation process, the pollution rate of a culture medium in-vitro culture of the seeds is 5-15%, and compared with the unhaired seeds, the pollution rate of the culture medium is reduced by about 30-40%.
Detailed Description
The invention provides a hair removing method for a clumpy hair-like seed growing at the top end, which comprises the following steps:
1) Cutting off the cluster hair of the cluster hair seed growing at the top end to the base part to obtain the seed with the cut cluster hair;
2) immersing the seeds cut off the cluster hair obtained in the step 1) in a sodium chloride solution to obtain clean and plump seeds with hair removed; the mass concentration of the sodium chloride solution is 10-16 g/L.
In the present invention, the top-grown tufted tamarisk or goose-down vine is preferably selected as the seed.
The invention cuts off the cluster hair of the cluster hair seed growing at the top end to the base part to obtain the seed with the cut cluster hair to the base part. The present invention preferably uses a scalpel blade or razor blade to cut clumpy hair to the base.
In the invention, the top end growing bush-shaped hair seeds are soaked before cutting off the bush-shaped hair, and the soaking comprises the following steps: mixing the top-end-growing cluster hair seed raw material with water, and sequentially carrying out first soaking and second soaking; the first soaking temperature is 35-45 ℃, and the second soaking temperature is 1-8 ℃.
In the present invention, the mode of mixing the top-end-growing clumpy hair seed material with water is preferably to soak the seed material in water.
In the present invention, the process of the first soaking and the second soaking is preferably repeated, and the number of repetitions is preferably 1.
In the invention, the first soaking temperature is preferably 35-45 ℃, more preferably 38-42 ℃, and most preferably 40 ℃; the first soaking time is preferably 3-5 h, more preferably 3.5-4.5 h, and most preferably 4 h. In the invention, the second soaking temperature is preferably 1-8 ℃, more preferably 2-6 ℃, and most preferably 4 ℃; the second soaking time is preferably 1-3 h, more preferably 1.5-2.5 h, and most preferably 2 h. In the invention, the first soaking and the second soaking can ensure that the tufted seed with the hair growing at the top end fully absorbs water to expand, increases the flexibility, reduces the mechanical damage during cutting the hair, and simultaneously, the temperature-variable treatment of the first soaking and the second soaking can break the dormancy of the seed, so that the seed without the hair can germinate more quickly during in vitro culture.
The obtained seeds with the cut cluster-shaped hair are immersed in a sodium chloride solution to obtain clean and plump seeds with the removed hair; the mass concentration of the sodium chloride solution is 10-16 g/L.
In the invention, the mass concentration of the sodium chloride solution is preferably 10-16 g/L, more preferably 12-14 g/L, and most preferably 13 g/L. In the invention, the principle that the buoyancy of the sodium chloride solution is larger than the buoyancy of water is utilized in the soaking process of the sodium chloride solution, so that residual seeds, unsaturated seeds, impurities and the like float on the water surface, and are discarded, and the seeds which sink to the water bottom are clean, full and thoroughly unhatched seeds. The seeds submerged in the water are preferably filtered through a filter funnel, and then the water is sucked dry by using filter paper.
The invention also provides an isolated culture method of the top-growing tufted hair seed, which comprises the following steps:
a. disinfecting the seed without hair obtained by the method for removing hair in the technical scheme to obtain disinfected seed;
b. And c, placing the sterilized seeds obtained in the step a on an in vitro culture medium for culture.
in the present invention, the sterilization treatment preferably includes the steps of:
I. sequentially treating the seed without the seed hair with an ethanol solution and washing with sterile water to obtain the seed treated with the ethanol solution;
And II, treating the seeds treated by the obtained ethanol solution by using a mercuric chloride solution and washing the seeds by using sterile water in sequence to obtain the sterilized seeds.
in the invention, the time for washing the steps I and II with sterile water is preferably 50-70 s, more preferably 55-65 s, and most preferably 60 s.
In the invention, the time for treating the ethanol solution is preferably 25-35 s, more preferably 28-32 s, and most preferably 30 s; the volume percentage content of the ethanol solution is preferably 70-80%, more preferably 72-78%, and most preferably 75%. According to the invention, after the ethanol solution treatment, the hair removed seeds are preferably washed by sterile water. In the invention, the process of the step I is preferably repeated, and the number of times of repeatedly washing by the sterile water is preferably 5-7 times.
In the invention, the time for treating the mercuric chloride solution is preferably 4-8 min, more preferably 5-7 min, and most preferably 6 min; the mass percentage of the mercuric chloride solution is preferably 0.05-0.15%, more preferably 0.08-0.12%, and most preferably 0.1%. According to the invention, the seeds treated by the ethanol solution are preferably washed by sterile water after being treated by the mercuric chloride solution. In the invention, the process of the step II is preferably repeated, and the number of times of repeatedly washing by the sterile water is preferably 5-7 times.
The invention puts the disinfected seeds on an isolated culture medium for culture.
In the present invention, the in vitro culture medium preferably uses an MS culture medium as a basic culture medium, and comprises the following components in concentration: 0.1-0.5 mg/L gibberellin, 20-40 g/L sucrose and 4-6 g/L agar; more preferably, it comprises: 0.2-0.4 mg/L gibberellin, 25-35 g/L sucrose and 4.5-5.5 g/L agar; most preferably comprising: 0.3mg/L gibberellin, 30g/L sucrose and 5g/L agar. In the invention, the pH value of the isolated culture medium is preferably 5.8-6.0. The method of adjusting the pH is not particularly limited in the present invention, and the pH can be adjusted by using a pH adjusting agent well known to those skilled in the art, for example, a sodium hydroxide solution with a mass concentration of 4% or a hydrochloric acid solution with a mass concentration of 8.3%.
In the present invention, the conditions of the culture include: the culture temperature is preferably 23-27 ℃, more preferably 24-26 ℃, and most preferably 25 ℃; the illumination intensity of the culture is preferably 1400-1800 lx, more preferably 1500-1700 lx and most preferably 1600 lx; the illumination time of the culture is preferably 10-14 h/d, more preferably 11-13 h/d, and most preferably 12 h/d.
The following examples are provided to describe in detail the method for removing and in vitro culturing the top-grown tufted hair seed according to the present invention, but they should not be construed as limiting the scope of the present invention.
example 1
Shasheng tamarix chinensis hair removal treatment
1. Seed harvesting
Collecting mature and plump seeds of the psammosilene tamarisk in 10 months of the year, placing the seeds in an indoor pure cotton cloth bag for drying in the shade, and then filling the seeds in a self-sealing bag for sealing so as to avoid pollution and waste caused by flying the seeds into the air due to the fact that the seeds are too light and grow into clumpy-shaped hairs.
2. Soaking and unhairing
(1) Soaking: clamping a plurality of the sand tamarix chinensis seeds by using forceps, putting the sand tamarix chinensis seeds into a beaker, adding warm water of 40 ℃ into the beaker, putting the beaker and the seeds into a constant-temperature incubator, setting the temperature to be 40 ℃, soaking for 4 hours, then transferring into a refrigerator, standing for 2 hours at the temperature of 4 ℃, and then repeating the constant-temperature incubator and the refrigerator for 1 time. The method comprises the following steps of soaking seeds, stirring by using a glass rod, so as to avoid mechanical damage to the seeds, pouring the seeds floating on the surface of a container and not full after the treatment is finished, keeping the seeds full at the bottom of the container, pouring the full seeds and residual moisture into a filter paper funnel, filtering most of the moisture, absorbing the residual moisture on the surface of the seeds by using dry filter paper, and obtaining the full Tamarix psammophila seeds with cluster-shaped hair.
(2) Unhairing: gently clamping the soaked plump seeds with the cluster hair on clean filter paper, gently clamping and neatly placing the seeds with the tweezers, then cutting off the cluster hair to the base by using a surgical blade to obtain the bush-removed seeds of the Tamarix psammophila, then placing the seeds in a 100ml small beaker, and pouring 2/3 distilled water.
3. Seed cleaning
And (3) soaking the tamarix chinensis seeds with the clustered hairy sand removed in a sodium chloride solution, wherein the mass concentration of the sodium chloride solution is 12 g/L. After being soaked in the sodium chloride solution, the seeds of the Tamarix psammophila which are not full and have extremely high purity can be further screened out by floating residual hairs, impurities and the like on the upper part of the sodium chloride solution and discarding the hairs and the impurities which are not full and are remained in the hair cutting process.
4. disinfection
The whole disinfection operation is carried out on a superclean workbench, the seeds of the Tamarix psammophila obtained in the step 3 are placed into a 10ml plastic test tube with a cover, 2/375% of ethanol is poured into the test tube, the cover is screwed down and then gently shaken for 30s, then a pipettor is used for quickly sucking off the ethanol in the test tube so as to prevent the seeds from being dead due to inactivation caused by overlong ethanol sterilization time, then 2/3 sterile water is poured, the cover is screwed down and then gently shaken for 20s, and the operations are repeated for 5 times; then 2/30.1% mercuric chloride is poured into the test tube, the cap is screwed down and then gently shaken for 5min, then the mercuric chloride in the test tube is sucked off by a pipette, then 2/3 sterile water is poured in, the cap is screwed down and then gently shaken for 20s, and the operation is repeated for 5 times. After the operations are finished, pouring a little sterile water into the test tube, pouring the sterile water and the seeds into a sterilized filter paper funnel, and then washing the test tube with the sterile water for multiple times until the seeds adhered to the wall of the test tube are washed clean; and finally, making a plurality of filter paper funnels with the same size, superposing until the water on the surfaces of the seeds is sucked dry to obtain the disinfected seeds, and spreading the filter paper and the seeds in a culture dish for later use.
5. In vitro culture
Clamping disinfected Chinese tamarisk seeds by using sterilized tweezers, placing the Chinese tamarisk seeds on an in-vitro culture medium, wherein the formula of the culture medium is MS +0.3mg/L GA3+ 30g/L sucrose + 5g/L agar powder, screwing a bottle cap, and placing the Chinese tamarisk seeds in a tissue culture room for culturing aseptic seedlings. The culture conditions are 12h/d of illumination, 25 ℃ of temperature and 1500lx of illumination intensity.
The results were: by adopting the unhairing method disclosed by the invention, after aseptic culture is carried out for 15 days, the unhairing rate of the tamarix arenarium seeds is 89.16%, and the pollution rate of a culture medium is 10.26%.
Example 2
Comparison example of hairless treatment of Shasheng tamarix seeds
1. seed harvesting
Collecting mature and plump seeds of the Sasa chinensis in 10 months of the year, placing the seeds in an indoor pure cotton bag, drying in the shade, and then packaging in a self-sealing bag and sealing for later use.
2. Soaking
Clamping a plurality of the sand tamarix chinensis seeds by using forceps, putting the sand tamarix chinensis seeds into a beaker, adding warm water of 40 ℃ into the beaker, putting the beaker and the seeds into a constant-temperature incubator, setting the temperature to be 40 ℃, soaking for 4 hours, then transferring into a refrigerator, standing for 3 hours at the temperature of 4 ℃, and then repeating the constant-temperature incubator and the refrigerator once. The method comprises the following steps of soaking seeds, stirring by using a glass rod, so as to avoid mechanical damage to the seeds, pouring the seeds floating on the surface of a container and not full after the seeds are treated, keeping the seeds full at the bottom of the container, pouring the full seeds and residual moisture into a filter paper funnel, filtering most of the moisture, absorbing the residual moisture on the surface of the seeds by using dry filter paper, and obtaining clean Tamarix psammophila seeds with cluster-shaped hairs at one end.
3. disinfection
The whole disinfection operation is carried out on a superclean workbench, the soaked Tamarix psammophila seeds are placed into a 10ml plastic test tube with a cover, ethanol accounting for 2/375% of the volume of the test tube is poured in, the cover is screwed down and then gently shaken for 30s, then the ethanol in the test tube is quickly sucked off by a pipette so as to prevent the seeds from being dead due to inactivation caused by overlong ethanol sterilization time, then 2/3 sterile water is poured in, the cover is screwed down and gently shaken for 20s, and the sterile water washing operation is repeated for 5 times; then 2/30.1% mercuric chloride is poured into the test tube, the cap is screwed down and then gently shaken for 6min, then the mercuric chloride in the test tube is sucked off by a pipette, 2/3 sterile water is poured in, the cap is screwed down and then gently shaken for 20s, and the above sterile water washing operation is repeated for 5 times. Pouring a little sterile water into the test tube after washing, pouring the sterile water and the seeds into a sterilized filter paper funnel, and then washing the test tube with the sterile water for multiple times until the seeds are washed clean; and finally, making a plurality of filter paper funnels with the same size, superposing until the water on the surfaces of the seeds is sucked dry to obtain disinfected hairy seeds, and spreading the filter paper and the seeds in a culture dish for later use.
4. in vitro culture
Clamping disinfected sand-bearing tamarisk seeds by using sterilized tweezers, placing the seeds on an in-vitro culture medium, wherein the formula of the culture medium is MS +0.25mg/L GA3+ 28g/L sucrose + 4.6g/L agar powder, screwing a bottle cap, and placing the bottle cap in a tissue culture room for culturing aseptic seedlings. The culture conditions are 14h/d of illumination, 26 ℃ of temperature and 1700lx of illumination intensity.
The results were: the method directly inoculates the sand tamarix chinensis seeds which are not subjected to the hair treatment on the culture medium, and after the sand tamarix chinensis seeds are subjected to aseptic culture for 15 days, the pollution rate of the culture medium is 41.26%.
Example 3
Comparative example of goose down vine seed hair-removing treatment
1. Seed harvesting
Mature and full goose down rattan seeds are collected in 9 months of the year, placed in an indoor pure cotton bag to be dried in the shade, and then packaged in a self-sealing bag to be sealed, so that pollution and waste caused by flying into the air due to the fact that the seeds are too light and have cluster-shaped hairs are avoided.
2. Soaking and unhairing
(1) Soaking: clamping a plurality of goose down vine seeds by using tweezers, putting the goose down vine seeds into a beaker, adding warm water of 38 ℃ into the beaker, putting the beaker and the seeds into a constant-temperature incubator, setting the temperature to be 38 ℃, soaking for 4 hours, putting the beaker into a refrigerator, standing for 2.5 hours at the temperature of 4 ℃, and repeating the operations in the constant-temperature incubator and the refrigerator once. The soaking process cannot be used for stirring by using a glass rod so as to avoid mechanical damage to seeds, the seeds floating on the surface of the container and not full are poured after the treatment is completed, the full seeds at the bottom of the container are kept, the full seeds are poured into a filter paper funnel together with residual moisture, after most of the moisture is filtered, the residual moisture on the surface of the seeds is absorbed by dry filter paper, and then the clean and full goose down vine seeds can be obtained.
(2) Unhairing: gently clamping clean and full seeds after soaking on filter paper, gently clamping and neatly placing the seeds with forceps, then cutting off the tufted hairs with an operation blade until the bases of the tufted hairs are cut off to obtain the gooseberry seeds, then placing the seeds in a 100ml small beaker, and pouring 2/3 distilled water.
3. Seed cleaning
And soaking the goose down vine seeds without the clumpy hair in a sodium chloride solution, wherein the mass concentration of the sodium chloride solution is 10 g/L. After being soaked in the sodium chloride solution, the goose down vines which are not full and have extremely high purity can be further screened out by floating residual hairs, impurities and the like on the upper part of the sodium chloride solution and discarding the hairs and the impurities which are not full and are left in the hair cutting process.
4. Disinfection
Putting the goose down vine seeds obtained in the step 3 into a 10ml plastic test tube with a cover, pouring 2/370% ethanol into the test tube, screwing the cover, slightly shaking for 33s, quickly sucking off the ethanol in the test tube by using a pipette so as to prevent the seeds from being dead due to inactivation caused by overlong ethanol sterilization time, then pouring 2/3 sterile water, slightly shaking for 20s after screwing the cover, and repeating the operation for 5 times; then 2/30.12% mercuric chloride is poured into the test tube, the cap is screwed down and then gently shaken for 5min, then the mercuric chloride in the test tube is sucked off by a pipette, then 2/3 sterile water is poured in, the cap is screwed down and then gently shaken for 20s, and the operation is repeated for 5 times. After the operations are finished, pouring a little sterile water into the test tube, pouring the sterile water and the seeds into a sterilized filter paper funnel, and then washing the test tube with the sterile water for multiple times until the seeds adhered to the wall of the test tube are washed clean; and finally, making a plurality of filter paper funnels with the same size, superposing until the water on the surfaces of the seeds is sucked dry to obtain the disinfected seeds, and spreading the filter paper and the seeds in a culture dish for later use.
5. In vitro culture
Clamping the sterilized goose down vine seeds by using sterilized tweezers, placing the goose down vine seeds on an in-vitro culture medium, wherein the formula of the culture medium is MS +0.4mg/L GA3+ 33g/L sucrose + 4.5g/L agar powder, screwing a bottle cap, and placing the goose down vine seeds in a tissue culture room for culturing aseptic seedlings. The culture conditions are that the illumination is 13h/d, the temperature is 26 ℃, and the illumination intensity is 1600 lx.
the results were: by adopting the dehairing method, the dehairing rate of the goose down vine seeds is 90.68 percent, and the pollution rate of the dehaired goose down vine seeds in a culture medium for 15 days is less than 10 percent after the unhaired goose down vine seeds are subjected to aseptic culture.
Example 4
Comparison example of goose down vine seed without unhairing treatment
1. Seed harvesting
Collecting mature and full goose down rattan seeds in 9 months of the year, placing the goose down rattan seeds in an indoor pure cotton bag for drying in the shade, then placing the goose down rattan seeds in a self-sealing bag, and screwing a bottle cap so as to avoid pollution and waste caused by flying into the air due to the fact that the seeds are too light and have cluster-shaped hairs.
2. soaking
Clamping a plurality of goose down vine seeds by using tweezers, putting the goose down vine seeds into a beaker, adding warm water of 36 ℃, putting the beaker and the seeds into a constant-temperature incubator, setting the temperature to be 40 ℃, soaking for 4 hours, then transferring into a refrigerator, standing for 1 hour at the temperature of 6 ℃, and then repeating the operations in the constant-temperature incubator and the refrigerator once. The soaking process cannot be used for stirring by using a glass rod so as to avoid mechanical damage to seeds, the seeds floating on the surface of the container and not full are poured after the treatment is completed, the full seeds at the bottom of the container are kept, the full seeds are poured into a filter paper funnel together with residual moisture, after most of the moisture is filtered, the residual moisture on the surface of the seeds is absorbed by dry filter paper, and then the clean and full goose down vine seeds can be obtained.
3. disinfection
The whole disinfection operation is carried out on a superclean bench, the soaked goose down vine seeds are placed into a 10ml plastic test tube with a cover, 2/375% ethanol is poured into the test tube, the cover is screwed down and then gently shaken for 35s, then a pipettor is used for quickly sucking off the ethanol in the test tube so as to prevent the seeds from being dead due to the inactivation of the seeds caused by overlong ethanol sterilization time, then 2/3 sterile water is poured, the cover is screwed down and then gently shaken for 20s, and the operations are repeated for 5 times; then 2/30.1% mercuric chloride is poured into the test tube, the cap is screwed down and then gently shaken for 6min, then the mercuric chloride in the test tube is sucked off by a pipette, then 2/3 sterile water is poured into the test tube, the cap is screwed down and then gently shaken for 20s, and the operation is repeated for 5 times. After the operations are finished, pouring a little sterile water into the test tube, pouring the sterile water and the seeds into a sterilized filter paper funnel, and then washing the test tube with the sterile water for multiple times until the seeds remained on the wall of the test tube are washed clean; and finally, making a plurality of filter paper funnels with the same size, superposing until the water on the surfaces of the seeds is sucked dry to obtain the disinfected seeds, and spreading the filter paper and the seeds in a culture dish for later use.
4. In vitro culture
Clamping the sterilized goose down vine seeds by using sterilized tweezers, placing the goose down vine seeds on an in-vitro culture medium, wherein the formula of the culture medium is MS +0.2.5mg/L GA3+ sucrose 25g/L + agar powder 5.5g/L, screwing a bottle cap, and placing the bottle cap in a tissue culture room for culturing aseptic seedlings. The culture conditions are 14h/d of illumination, 25 ℃ of temperature and 1600lx of illumination intensity.
The invention directly inoculates the goose down vine seeds without the hair treatment on the culture medium, and after the goose down vine seeds are aseptically cultured for 15 days, the pollution rate of the culture medium is 38.25 percent.
according to the embodiments, the method for removing the hairline of the top-end-growth tufted tamarisk seeds provided by the invention has the advantages that the hairline removal rate of the seeds of the Saxago tamarisk is 85-92%, the seeds of the Saxago tamarisk cannot be mechanically damaged in the operation process, the pollution rate of a culture medium in the in-vitro culture of the seeds is about 10%, and compared with the unhairing-free culture medium, the pollution rate of the culture medium is reduced by about 30-40%. The unhairing rate of the goose down vine seeds is 90.68 percent, the mechanical damage to the goose down vine seeds can not be caused in the operation process, and the pollution rate of a culture medium is less than 10 percent in the in vitro culture process of the seeds.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (3)
1. An isolated culture method of a top-growing tufted hair seed comprises the following steps:
1) Cutting off the cluster hair of the cluster hair seed growing at the top end to the base part to obtain the seed with the cut cluster hair;
2) Immersing the seeds cut off the cluster hair obtained in the step 1) in a sodium chloride solution to obtain clean and plump seeds with hair removed; the mass concentration of the sodium chloride solution is 10-16 g/L; the top-end grown clumpy hair seeds are soaked before cutting clumpy hair, the soaking comprises: mixing the top-end-growing cluster hair seed raw material with water, and sequentially carrying out first soaking and second soaking; the first soaking temperature is 35-45 ℃, and the second soaking temperature is 1-8 ℃; the first soaking time is 3-5 hours, and the second soaking time is 1-3 hours;
3) Sterilizing the haired seed to obtain sterilized seed;
The sterilization process comprises the steps of:
I. Sequentially treating the seed without the seed hair with an ethanol solution and washing with sterile water to obtain the seed treated with the ethanol solution; the volume percentage content of the ethanol solution in the step I is 70-80%, and the treatment time of the ethanol solution is 25-35 s;
II, treating the seeds treated by the obtained ethanol solution by a mercuric chloride solution and washing the seeds by sterile water in sequence to obtain sterilized seeds; the mass percentage of the mercuric chloride solution in the step II is 0.05-0.15%, and the treatment time of the mercuric chloride solution is 4-8 min;
4) And placing the sterilized seeds on an isolated culture medium for culture.
2. The ex vivo culture method according to claim 1, wherein the ex vivo culture medium is a basal medium based on MS culture medium, comprising the following components in the concentrations: 0.1-0.5 mg/L gibberellin, 20-40 g/L sucrose and 4-6 g/L agar.
3. The ex vivo culture method according to claim 1 or 2, wherein the culture conditions comprise: the cultivation temperature is 23-27 ℃, the cultivation illumination intensity is 1400-1800 lx, and the cultivation illumination time is 10-14 h/d.
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