CN112544446A - Production method of dendrobium officinale Kimura et Migo mother bottle seedlings - Google Patents
Production method of dendrobium officinale Kimura et Migo mother bottle seedlings Download PDFInfo
- Publication number
- CN112544446A CN112544446A CN202011430986.8A CN202011430986A CN112544446A CN 112544446 A CN112544446 A CN 112544446A CN 202011430986 A CN202011430986 A CN 202011430986A CN 112544446 A CN112544446 A CN 112544446A
- Authority
- CN
- China
- Prior art keywords
- culture medium
- mother liquor
- dendrobium officinale
- bottle
- culture
- 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.)
- Pending
Links
Images
Classifications
-
- 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
-
- 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
Abstract
The invention discloses a production method of dendrobium officinale Kimura et Migo bottle seedlings, which comprises the following steps: step 1): sterilizing and drying Dendrobium officinale capsules; step 2): inoculating seeds in a dendrobium officinale capsule into a 1-generation semi-solid culture medium for culture; wherein the 1-generation semisolid culture medium is prepared from the following raw materials: using 1/2MS culture medium as basic culture medium, 30g/L sucrose, 0.1g/L inositol and 1-2 g/L agar, pH 5.8-6.0; step 3): transferring the dendrobium officinale seed suspension in the step 2) and uniformly spreading the dendrobium officinale seed suspension in a 2-generation mother bottle seedling culture medium for culture; the 2-generation mother bottle seedling culture medium is prepared from the following raw materials: 1/2MS culture medium is used as a basic culture medium, 0.2mg/L NAA, 30g/L sucrose, 0.1g/L inositol, 5g/L agar and 8-10% banana puree, and the pH value is 6.0-6.2. By adopting the production method provided by the invention, the production efficiency of the dendrobium officinale mother bottle can be improved, and the production cost is greatly reduced.
Description
Technical Field
The invention relates to the technical field of plant culture, and particularly relates to a production method of dendrobium officinale mother bottle seedlings.
Background
Dendrobium officinale (Dendrobium officinale Kimura et Migo) belonging to the genus Dendrobium of the family Orchidaceae, also known as Equisetum nigrum. It is recorded in Shen nong Ben Cao Jing (Shen nong's herbal) at the head of Jiu Da Xian Cao. It has sweet taste and slightly cold nature, and has effects of promoting fluid production, invigorating stomach, nourishing yin, clearing heat, moistening lung, invigorating kidney, improving eyesight, and strengthening waist. Modern pharmacological studies show that dendrobium officinale has the effects of promoting salivary secretion, resisting gastric ulcer, enhancing immunity, resisting oxidation, resisting aging, resisting fatigue, resisting tumors, reducing blood sugar, blood pressure and blood fat, protecting liver and the like, and is paid more and more attention. Dendrobium officinale is called as panda in the pharmaceutical kingdom by the international medicinal plant kingdom. In a natural state, due to immature development and specific requirements on the environment, the propagation rate of seeds is always low in the natural state, the seeds grow slowly, and wild dendrobium officinale is much less and less due to artificial over-mining. At present, dendrobium officinale seedlings for artificial cultivation are mainly obtained by cultivating through a plant tissue culture technology, so that the optimization of the dendrobium officinale tissue culture technology is the key of large-scale production.
The seeds of the orchids are tiny like dust, often one capsule contains tens of thousands of tiny seeds, and most of the seeds of the orchids can carry out symbiotic germination under the action of bacteria. The dendrobium officinale seeds are used as explants for tissue culture, so that the propagation coefficient can be greatly improved. At present, dendrobium officinale seeds are taken as explants for tissue culture, and the main steps are seed germination, protocorm transfer, mother flask seedling culture and mother flask seedling transfer culture. Inoculating dendrobium officinale seeds to a solid culture medium for culture, transferring protocorms generated by seed germination to a mother bottle seedling culture medium for dendrobium officinale mother bottle seedling culture after the protocorms are cultured for a period of time, and transferring the seedlings to a strong seedling rooting culture medium for culture after the seedlings in the mother bottle culture medium grow to a certain stage. In the stage of transferring protocorms generated by seed germination, the transfer efficiency is low and the labor cost is high.
Disclosure of Invention
To the technical problem that exists among the prior art, this application provides a dendrobium officinale mother bottle seedling production method.
A production method of dendrobium officinale Kimura et Migo mother bottle seedlings comprises the following steps:
step 1): sterilizing and drying Dendrobium officinale capsules;
step 2): inoculating seeds in a dendrobium officinale capsule into a 1-generation semi-solid culture medium for culture; wherein the 1-generation semisolid culture medium is prepared from the following raw materials: using 1/2MS culture medium as basic culture medium, 30g/L sucrose, 0.1g/L inositol and 1-2 g/L agar, pH 5.8-6.0;
step 3): transferring the dendrobium officinale seed suspension in the step 2) and uniformly spreading the dendrobium officinale seed suspension in a 2-generation mother bottle seedling culture medium for culture; the 2-generation mother bottle seedling culture medium is prepared from the following raw materials: 1/2MS culture medium is used as a basic culture medium, 0.2mg/L NAA, 30g/L sucrose, 0.1g/L inositol, 5g/L agar and 8-10% banana puree, and the pH value is 6.0-6.2. By adopting the production method provided by the invention, the production efficiency of the dendrobium officinale mother bottle can be improved, and the production cost is greatly reduced.
Preferably, the dendrobium officinale capsule disinfection and drying method in the step 1) comprises the following specific steps: soaking capsule of Tinospora glabra in washing powder for 10-15min, washing with tap water for 5min, sterilizing with 75% ethanol for 3min, washing with sterile water for 3 times, sterilizing with 0.1% mercuric chloride solution for 25min, washing with sterile water to remove residual mercuric chloride, sterilizing, air drying on a clean bench, and packaging in sterile paper bags; drying the sterile paper bag in an oven at 30 ℃ until the weight is unchanged; and (4) placing the seed bag in a cool, dry and tidy environment for storage for later use.
Preferably, when preparing the 1-generation semisolid culture medium in the step 2), mother liquor preparation needs to be performed in advance according to an MS culture medium formula, and the preparation comprises the steps of preparing MS macroelement mother liquor, MS calcium salt mother liquor, MS vitamin mother liquor, MS microelement mother liquor and MS iron salt mother liquor; then accurately measuring 25mLMS macroelement mother liquor, 25mLMS calcium salt mother liquor, 10mLMS vitamin mother liquor, 10mLMS microelement mother liquor and 5mLMS iron salt mother liquor, adding 30g of cane sugar and 0.1g of inositol for dissolving, weighing 1-2 g of agar powder, adding a proper amount of water for boiling and dissolving, then adding and mixing, and fixing the volume to 1L; then regulating the pH value to 5.8-6.0 by titrating 1mol/L NaoH or 1mol/L HCl; subpackaging the culture medium with the adjusted pH into wide-mouth bottles, subpackaging each bottle with 75mL, placing into a high-pressure steam sterilization pot for sterilization under 117 deg.C for 30 min; and placing the sterilized culture medium in an inoculation buffer room for cooling for later use.
Preferably, the specific operation steps of inoculating and culturing the dendrobium officinale seeds in the step 2) are as follows: inoculating Dendrobium officinale seeds on a sterile super clean bench, wherein the super clean bench is opened for ultraviolet irradiation for more than 0.5h before use, and the ultraviolet lamp is turned off and the common illuminating lamp is turned on when the super clean bench is used; before inoculation of the dendrobium officinale seeds, firstly, igniting an alcohol lamp in an ultra-clean bench, and then wiping and disinfecting hands and arms which extend into the ultra-clean bench and the tabletop of the operation by using alcohol cotton; on the basis, tools such as tweezers, scissors, a spoon and the like required by inoculation are placed on an alcohol lamp for sterilization, and the sterilization time is 15-30 s; placing the sterilized and dried inoculation disc on a super clean bench, placing a spoon in the super clean bench, taking out the sterilized and dried dendrobium officinale capsules with tweezers, placing the disinfected and dried dendrobium officinale capsules on the spoon, then clamping the stems with the tweezers with the left hand, shearing the capsules with scissors with the right hand, shaking the seeds off the spoon, and inoculating the dendrobium officinale seeds in a 1-generation semisolid culture medium for culture; after inoculation, slowly shaking the wide-mouth bottle by hand to enable the seeds to be fully and uniformly mixed with the culture medium, and finally placing the wide-mouth bottle in a culture chamber for culture.
Preferably, when the 2 generation mother bottle seedling culture medium is prepared, firstly, mother liquor of each component in an MS culture medium is prepared in advance, wherein the mother liquor comprises MS macroelement mother liquor, MS calcium salt mother liquor, MS vitamin mother liquor, MS microelement mother liquor, MS ferric salt mother liquor, NAA mother liquor and slurry banana; then accurately measuring 25mLMS major element mother liquor, 25mLMS calcium salt mother liquor, 10mLMS vitamin mother liquor, 10mLMS trace element mother liquor, 5mLMS ferric salt mother liquor, banana puree made of 80-100g bananas, 2mL NAA mother liquor, 30g cane sugar and 0.1g inositol, adding and dissolving, accurately weighing 5g agar powder, adding a proper amount of water, boiling and dissolving, adding and mixing, and fixing the volume to 1L; adjusting the pH value to 6.0-6.2 by titrating 1mol/L NaoH or 1mol/L HCl; subpackaging the culture medium with the adjusted pH into orchid bottles with each bottle containing 150-200mL of the culture medium, and then placing the bottles in a high-pressure steam sterilization pot for sterilization under the sterilization conditions of 117 ℃ for 30 min; placing the sterilized culture medium in an inoculation buffer room for cooling for later use; the preparation method of the NAA mother liquor comprises the following steps: weighing 100.0mg of NAA, dissolving with a small amount of 95% ethanol, and then diluting with deionized water to 1000ml to prepare NAA mother liquor with the concentration of 100 mg/L; slurry banana: accurately weighing 80-100g of peeled banana, placing in a beater, adding appropriate amount of water, and beating into slurry.
Preferably, the suspension transfer of the dendrobium officinale seed in the step 3) is carried out on a sterile super clean bench, wherein the super clean bench is opened for ultraviolet irradiation for more than 0.5h before use, and the ultraviolet lamp is turned off and a common light illuminator is turned on when the super clean bench is used; wherein the liquid-transfering gun is always placed on a super clean bench for ultraviolet disinfection and sterilization; before transfer, wiping the mouth of the inoculated culture bottle with alcohol cotton, opening the wiped wide-mouth bottle, and then placing the mouth of the wide-mouth bottle outside an alcohol lamp for surrounding sterilization for about 15 s; then a liquid transfer gun is used for sucking 2.5ml of dendrobium officinale seed suspension to be inoculated into a 2-generation mother bottle seedling culture medium, the orchid bottle is gently shaken after the transfer is finished, so that the seed suspension is uniformly paved on the surface of the culture medium, and finally the orchid bottle is placed in a culture room for culture; culturing for about 120 days in a culture room to obtain mother bottle seedlings for transfer culture.
The invention is further illustrated below:
the method skillfully utilizes the distribution characteristics of the dendrobium officinale seeds in the semi-solid culture medium, greatly improves the production efficiency of the dendrobium officinale mother bottle, greatly reduces the labor cost, and has advantages in scale and industrial development of the dendrobium officinale. (the preparation method can improve the production efficiency of the dendrobium officinale mother bottle, and the embodiment can be seen in detail)
At present, no research report for fast production of mother bottle seedlings by using a semi-solid culture medium as a carrier of dendrobium officinale seeds or dendrobium officinale protocorms exists at home and abroad. In the early research of the research and development team of the company, the distribution characteristics of the dendrobium officinale seeds in the semisolid culture medium are successfully and skillfully utilized, a large amount of dendrobium officinale mother bottle seedlings for transfer are produced in a very short time, and a solid foundation is laid for the further development of the dendrobium officinale industry.
The invention aims to provide a method for producing a mother bottle by taking dendrobium officinale seeds as explants. The invention adopts the following specific technical scheme:
(1) and (3) sterilizing and drying the dendrobium officinale capsules: soaking capsule of herba Dendrobii in washing powder for 10-15min, and washing with tap water for 5 min. Then sterilizing with 75% ethanol for 3min, washing with sterile water for 3 times, sterilizing with 0.1% mercuric chloride solution for 25min, washing residual mercuric chloride with sterile water, air drying on a superclean bench, and packaging into sterile paper bags. The sterile paper bags were placed in an oven at 30 ℃ and dried until no weight change occurred. And (4) placing the seed bag in a cool, dry and tidy environment for storage for later use.
(2) 1-generation semisolid culture medium preparation: the formula of the culture medium is 1/2MS +30g/L sucrose +0.1g/L inositol + 1-2 g/L agar. The specific configuration process is as follows: preparing mother liquor according to MS culture medium formula, including macroelements, calcium salt, vitamins, microelements and iron salt; preparing 1L of culture medium requires accurately measuring 25mL of macroelements, 25mL of calcium salt, 10mL of vitamins, 10mL of trace elements and 5mL of ferric salt, accurately weighing 30g of sucrose (generally white sugar is used for replacing) and 0.1g of inositol, adding a proper amount of water, boiling and dissolving, accurately weighing 1-2 g of agar powder, and mixing all the materials to a constant volume of 1L. Adjusting the pH value to 5.8-6.0 by titrating 1mol/L NaoH or 1mol/L HCl; subpackaging the culture medium with the adjusted pH into wide-mouth bottles, subpackaging each bottle with 75mL, and placing into a high-pressure steam sterilization pot for sterilization under 117 deg.C for 30 min; and placing the sterilized culture medium in an inoculation buffer room for cooling for later use.
(3) Inoculating and culturing the dendrobium officinale seeds: the inoculation of Dendrobium officinale seeds is carried out on a sterile super clean bench, wherein the super clean bench is opened for ultraviolet irradiation for more than 0.5h before use, and the ultraviolet lamp and the common illuminating lamp are turned off and turned on when in use. Before inoculation of the dendrobium officinale seeds, firstly, igniting an alcohol lamp in an ultra-clean bench, and then wiping and disinfecting hands and arms which extend into the ultra-clean bench and the tabletop of the operation by using alcohol cotton; on the basis, tools such as tweezers, scissors, spoons and the like (soaking in 75% alcohol before sterilization) required for inoculation are placed on the outer flame of an alcohol lamp for sterilization for 15-30 s; placing the sterilized and dried inoculation disc on a clean bench, placing a spoon in the sterilization and dried inoculation disc, taking out the sterilized and dried Dendrobium officinale capsules with tweezers, placing the disinfected and dried Dendrobium officinale capsules on the spoon, then clamping the stems with the tweezers with the left hand, shearing the capsules with scissors with the right hand, shaking the seeds on the spoon, and finally inoculating the seeds in the 1-generation culture medium; after inoculation, slowly shaking the wide-mouth bottle by hand to enable the seeds to be fully and uniformly mixed with the culture medium, and finally placing the wide-mouth bottle in a culture chamber for culture.
(4) Preparing a culture medium for the 2 generation mother bottle seedlings: the formula of the culture medium is 1/2MS +0.2mg/L NAA +30g/L sucrose +0.1g/L inositol +5g/L agar + 8% -10% banana puree. The specific configuration process is as follows: firstly, preparing mother liquor of each component of the MS formula; then preparing mother liquid of hormone plant growth regulator (hormone). Specifically, NAA: 100.0mg NAA is accurately weighed, dissolved by a small amount of 95% ethanol, and then the volume of the solution is determined to 1000ml by deionized water, so as to prepare a mother solution with the concentration of 100 mg/L. Pulping the bananas into paste: accurately weighing 80-100g of peeled banana, placing in a beater, adding appropriate amount of water, and beating into slurry. Preparing 1L of culture medium requires accurately measuring 25mL of macroelements, 25mL of calcium salt, 10mL of vitamins, 10mL of trace elements and 5mL of iron salt, slurry formed by beating bananas with 80-100g of bananas and 2mL of NAA, accurately weighing 30g of cane sugar (generally substituted by white sugar) and 0.1g of inositol, adding the cane sugar and the inositol for dissolving, accurately weighing 5g of agar powder, adding an appropriate amount of water, boiling and dissolving, and mixing all the components to a constant volume of 1L. Adjusting the pH value to 6.0-6.2 by titrating 1mol/L NaoH or 1mol/L HCl; subpackaging the culture medium with the adjusted pH into orchid bottles, wherein each bottle is preferably subpackaged with 150-200mL, and then placing the bottles in a high-pressure steam sterilization pot for sterilization under the sterilization condition of 117 ℃ for 30 min; and placing the sterilized culture medium in an inoculation buffer room for cooling for later use.
(5) Transfer culture by a pipette: transferring the dendrobium officinale seed suspension mixed with the culture medium to a 2-generation mother bottle seedling culture medium by using a liquid transfer gun on a super clean bench, slightly shaking the orchid bottle after the transfer is finished, uniformly paving the seed suspension on the surface of the culture medium, and finally placing the orchid bottle in a culture room for culture. The specific operation process is as follows: sterile pipette tips were prepared prior to transfer. The dendrobium officinale seed suspension transfer is carried out on a sterile super clean bench, wherein the super clean bench is opened for over 0.5h of ultraviolet irradiation before use, and the ultraviolet lamp is turned off and the common illuminating lamp is turned on when the dendrobium officinale seed suspension transfer is used. Wherein the liquid-transfering gun is always placed on a super clean bench for ultraviolet disinfection and sterilization. Other details are as described above, ready for the relevant operation. Before transfer, the mouth of the seed culture flask (i.e., jar) was wiped with alcohol cotton (without opening the cap). Opening the wiped wide-mouth bottle, placing the bottle mouth of the wide-mouth bottle in an alcohol burner to perform surrounding sterilization for about 15s, then using a liquid transfer gun to absorb 2.5ml of dendrobium officinale seed suspension to inoculate the suspension in a 2-generation mother bottle seedling culture medium, slightly shaking the orchid bottle after transfer is completed to enable the seed suspension to be uniformly spread on the surface of the culture medium, finally placing the orchid bottle in a culture room for culture, and culturing in the culture room for about 120d to obtain the orchid bottle seedling serving as a mother bottle seedling for transfer culture.
Drawings
FIG. 1 is a flow chart of a conventional cultivation method;
FIG. 2 is a flow chart of the culturing method of the present invention.
Detailed Description
The invention provides a production method of dendrobium officinale Kimura et Migo bottle seedlings, which comprises the following steps:
step 1): sterilizing and drying Dendrobium officinale capsules; the method comprises the following specific steps: soaking capsule of Tinospora glabra in washing powder for 10-15min, washing with tap water for 5min, sterilizing with 75% ethanol for 3min, washing with sterile water for 3 times, sterilizing with 0.1% mercuric chloride solution for 25min, washing with sterile water to remove residual mercuric chloride, sterilizing, air drying on a clean bench, and packaging in sterile paper bags; drying the sterile paper bag in an oven at 30 ℃ until the weight is unchanged; and (4) placing the seed bag in a cool, dry and tidy environment for storage for later use.
Step 2): inoculating seeds in a dendrobium officinale capsule into a 1-generation semi-solid culture medium for culture; wherein the 1-generation semisolid culture medium is prepared from the following raw materials: using 1/2MS culture medium as basic culture medium, 30g/L sucrose, 0.1g/L inositol and 1-2 g/L agar, pH 5.8-6.0;
preparing mother liquor in advance according to an MS culture medium formula when preparing the 1-generation semisolid culture medium in the step 2), wherein the preparation comprises preparing MS macroelement mother liquor, MS calcium salt mother liquor, MS vitamin mother liquor, MS microelement mother liquor and MS iron salt mother liquor; then accurately measuring 25mLMS macroelement mother liquor, 25mLMS calcium salt mother liquor, 10mLMS vitamin mother liquor, 10mLMS microelement mother liquor and 5mLMS iron salt mother liquor, adding 30g of cane sugar and 0.1g of inositol for dissolving, additionally weighing 1-2 g of agar powder, adding a proper amount of water for boiling, dissolving, adding and mixing, and fixing the volume to 1L; then regulating the pH value to 5.8-6.0 by titrating 1mol/L NaoH or 1mol/L HCl; subpackaging the culture medium with the adjusted pH into wide-mouth bottles, subpackaging each bottle with 75mL, placing into a high-pressure steam sterilization pot for sterilization under 117 deg.C for 30 min; and placing the sterilized culture medium in an inoculation buffer room for cooling for later use.
The specific operation steps of inoculating and culturing the dendrobium officinale seeds in the step 2) are as follows: the inoculation of Dendrobium officinale seeds is carried out on a sterile super clean bench, wherein the super clean bench is opened for ultraviolet irradiation for more than 0.5h before use, and the ultraviolet lamp and the common illuminating lamp are turned off and turned on when in use. Before inoculation of the dendrobium officinale seeds, firstly, igniting an alcohol lamp in an ultra-clean bench, and then wiping and disinfecting hands and arms which extend into the ultra-clean bench and the tabletop of the operation by using alcohol cotton; on the basis, tools such as tweezers, scissors, spoons and the like (soaking in 75% alcohol before sterilization) required for inoculation are placed on the outer flame of an alcohol lamp for sterilization for 15-30 s; placing the sterilized and dried inoculation disc on a super clean bench, placing a spoon in the super clean bench, taking out the sterilized and dried dendrobium officinale capsules with tweezers, placing the disinfected and dried dendrobium officinale capsules on the spoon, then clamping the stems with the tweezers with the left hand, shearing the capsules with scissors with the right hand, shaking the seeds off the spoon, and inoculating the dendrobium officinale seeds in a 1-generation semisolid culture medium for culture; after inoculation, slowly shaking the wide-mouth bottle by hand to enable the seeds to be fully and uniformly mixed with the culture medium, and finally placing the wide-mouth bottle in a culture chamber for culture.
Step 3): transferring the dendrobium officinale seed suspension in the step 2) and uniformly spreading the dendrobium officinale seed suspension in a 2-generation mother bottle seedling culture medium for culture; the 2-generation mother bottle seedling culture medium is prepared from the following raw materials: 1/2MS culture medium is used as a basic culture medium, 0.2mg/L NAA, 30g/L sucrose, 0.1g/L inositol, 5g/L agar and 8-10% banana puree are used, and the pH value is 6.0-6.2;
when the 2 generation mother bottle seedling culture medium is prepared, firstly, mother liquor of each component in an MS culture medium is prepared in advance, wherein the mother liquor comprises MS macroelement mother liquor, MS calcium salt mother liquor, MS vitamin mother liquor, MS microelement mother liquor, MS ferric salt mother liquor, NAA mother liquor and slurry banana; then accurately measuring 25mLMS major element mother liquor, 25mLMS calcium salt mother liquor, 10mLMS vitamin mother liquor, 10mLMS trace element mother liquor, 5mLMS ferric salt mother liquor, banana puree made of 80-100g bananas, 2mL NAA mother liquor, 30g cane sugar and 0.1g inositol, adding and dissolving, accurately weighing 5g agar powder, adding a proper amount of water, boiling and dissolving, adding and mixing, and fixing the volume to 1L; adjusting the pH value to 6.0-6.2 by titrating 1mol/L NaoH or 1mol/L HCl; subpackaging the culture medium with the adjusted pH into orchid bottles, wherein each bottle is preferably 150-200mL, and then placing the bottles in a high-pressure steam sterilization pot for sterilization under the sterilization condition of 117 ℃ for 30 min; placing the sterilized culture medium in an inoculation buffer room for cooling for later use; the preparation method of the NAA mother liquor comprises the following steps: weighing 100.0mg of NAA, dissolving with a small amount of 95% ethanol, and then diluting with deionized water to 1000ml to prepare NAA mother liquor with the concentration of 100 mg/L; pulping the bananas into paste: accurately weighing 80-100g of peeled banana, placing in a beater, adding appropriate amount of water, and beating into slurry.
The suspension transfer of the dendrobium officinale seed in the step 3) is carried out on a sterile super clean bench, wherein the super clean bench is opened for ultraviolet irradiation for more than 0.5h before use, and the ultraviolet lamp is turned off and a common light illuminator is turned on when the super clean bench is used; wherein the liquid-transfering gun is always placed on a super clean bench for ultraviolet disinfection and sterilization; before transfer, wiping the mouth of a seed culture bottle (without opening a bottle cap) with alcohol cotton, opening the wiped wide-mouth bottle, and then placing the mouth of the wide-mouth bottle outside an alcohol lamp for surrounding sterilization for about 15 s; then a liquid transfer gun is used for sucking 2.5ml of dendrobium officinale seed suspension to be inoculated into a 2-generation mother bottle seedling culture medium, the orchid bottle is gently shaken after the transfer is finished, so that the seed suspension is uniformly paved on the surface of the culture medium, and finally the orchid bottle is placed in a culture room for culture; culturing for about 120 days in a culture room to obtain mother bottle seedlings for transfer culture.
Compared with the traditional culture mode of the dendrobium officinale mother bottle seedlings, the traditional culture mode of the dendrobium officinale mother bottle seedlings has a great difference; comprises the difference of 1 generation of culture medium and the difference of transferring mode of transferring 1 generation of germinated dendrobium officinale to 2 generation of culture medium. The difference between the two types of seedlings enables the cost of the dendrobium officinale mother bottle seedling culture method to be saved by about 1.0 yuan per bottle compared with the traditional method.
A flow chart of a conventional culture method, as shown in FIG. 1;
the flow chart of the culture method of the present invention, as shown in FIG. 2,
TABLE 1 comparison of the cost of producing Dendrobium officinale Kimura et Migo mother bottle seedlings by different culture methods
Note: the total cost does not contain the cost of dendrobium officinale fruit pods.
Claims (6)
1. A production method of dendrobium officinale Kimura et Migo mother bottle seedlings comprises the following steps:
step 1): sterilizing and drying Dendrobium officinale capsules;
step 2): inoculating seeds in a dendrobium officinale capsule into a 1-generation semi-solid culture medium for culture; wherein the 1-generation semisolid culture medium is prepared from the following raw materials: using 1/2MS culture medium as basic culture medium, 30g/L sucrose, 0.1g/L inositol and 1-2 g/L agar, pH 5.8-6.0;
step 3): transferring the dendrobium officinale seed suspension in the step 2) and uniformly spreading the dendrobium officinale seed suspension in a 2-generation mother bottle seedling culture medium for culture; the 2-generation mother bottle seedling culture medium is prepared from the following raw materials: 1/2MS culture medium is used as a basic culture medium, 0.2mg/L NAA, 30g/L sucrose, 0.1g/L inositol, 5g/L agar and 8-10% banana puree, and the pH value is 6.0-6.2.
2. The method of claim 1, wherein: the method comprises the following specific steps of sterilizing and drying the dendrobium officinale capsules in the step 1): soaking capsule of Tinospora glabra in washing powder for 10-15min, washing with tap water for 5min, sterilizing with 75% ethanol for 3min, washing with sterile water for 3 times, sterilizing with 0.1% mercuric chloride solution for 25min, washing with sterile water to remove residual mercuric chloride, sterilizing, air drying on a clean bench, and packaging in sterile paper bags; drying the sterile paper bag in an oven at 30 ℃ until the weight is unchanged; and (4) placing the seed bag in a cool, dry and tidy environment for storage for later use.
3. The method of claim 1, wherein: when preparing the 1-generation semisolid culture medium in the step 2), mother liquor preparation is required to be carried out in advance according to an MS culture medium formula, and the preparation comprises the steps of preparing MS macroelement mother liquor, MS calcium salt mother liquor, MS vitamin mother liquor, MS microelement mother liquor and MS iron salt mother liquor; then accurately measuring 25mLMS macroelement mother liquor, 25mLMS calcium salt mother liquor, 10mLMS vitamin mother liquor, 10mLMS microelement mother liquor and 5mLMS iron salt mother liquor, adding 30g of cane sugar and 0.1g of inositol for dissolving, weighing 1-2 g of agar powder, adding a proper amount of water for boiling and dissolving, then adding and mixing, and fixing the volume to 1L; then regulating the pH value to 5.8-6.0 by titrating 1mol/L NaoH or 1mol/L HCl; subpackaging the culture medium with the adjusted pH into wide-mouth bottles, subpackaging each bottle with 75mL, placing into a high-pressure steam sterilization pot for sterilization under 117 deg.C for 30 min; and placing the sterilized culture medium in an inoculation buffer room for cooling for later use.
4. The method of claim 1, wherein: the specific operation steps of inoculating and culturing the dendrobium officinale seeds in the step 2) are as follows: inoculating Dendrobium officinale seeds on a sterile super clean bench, wherein the super clean bench is opened for ultraviolet irradiation for more than 0.5h before use, and the ultraviolet lamp is turned off and the common illuminating lamp is turned on when the super clean bench is used; before inoculation of the dendrobium officinale seeds, firstly, igniting an alcohol lamp in an ultra-clean bench, and then wiping and disinfecting hands and arms which extend into the ultra-clean bench and the tabletop of the operation by using alcohol cotton; on the basis, tools such as tweezers, scissors, a spoon and the like required by inoculation are placed on an alcohol lamp for sterilization, and the sterilization time is 15-30 s; placing the sterilized and dried inoculation disc on a super clean bench, placing a spoon in the super clean bench, taking out the sterilized and dried dendrobium officinale capsules with tweezers, placing the disinfected and dried dendrobium officinale capsules on the spoon, then clamping the stems with the tweezers with the left hand, shearing the capsules with scissors with the right hand, shaking the seeds off the spoon, and inoculating the dendrobium officinale seeds in a 1-generation semisolid culture medium for culture; after inoculation, slowly shaking the wide-mouth bottle by hand to enable the seeds to be fully and uniformly mixed with the culture medium, and finally placing the wide-mouth bottle in a culture chamber for culture.
5. The method of claim 1, wherein: when the 2 generation mother bottle seedling culture medium is prepared, firstly, mother liquor of each component in an MS culture medium is prepared in advance, wherein the mother liquor comprises MS macroelement mother liquor, MS calcium salt mother liquor, MS vitamin mother liquor, MS microelement mother liquor, MS ferric salt mother liquor, NAA mother liquor and slurry banana; then accurately measuring 25mLMS major element mother liquor, 25mLMS calcium salt mother liquor, 10mLMS vitamin mother liquor, 10mLMS trace element mother liquor, 5mLMS ferric salt mother liquor, banana puree made of 80-100g bananas, 2mL NAA mother liquor, 30g cane sugar and 0.1g inositol, adding and dissolving, accurately weighing 5g agar powder, adding a proper amount of water, boiling and dissolving, adding and mixing, and fixing the volume to 1L; adjusting the pH value to 6.0-6.2 by titrating 1mol/L NaoH or 1mol/L HCl; subpackaging the culture medium with the adjusted pH into orchid bottles with each bottle containing 150-200mL of the culture medium, and then placing the bottles in a high-pressure steam sterilization pot for sterilization under the sterilization conditions of 117 ℃ for 30 min; placing the sterilized culture medium in an inoculation buffer room for cooling for later use; the preparation method of the NAA mother liquor comprises the following steps: weighing 100.0mg of NAA, dissolving with a small amount of 95% ethanol, and then diluting with deionized water to 1000ml to prepare NAA mother liquor with the concentration of 100 mg/L; slurry banana: accurately weighing 80-100g of peeled banana, placing in a beater, adding appropriate amount of water, and beating into slurry.
6. The method of claim 1, wherein: the suspension transfer of the dendrobium officinale seed in the step 3) is carried out on a sterile super clean bench, wherein the super clean bench is opened for ultraviolet irradiation for more than 0.5h before use, and the ultraviolet lamp is turned off and a common light illuminator is turned on when the super clean bench is used; wherein the liquid-transfering gun is always placed on a super clean bench for ultraviolet disinfection and sterilization; before transfer, wiping the mouth of the inoculated culture bottle with alcohol cotton, opening the wiped wide-mouth bottle, and then placing the mouth of the wide-mouth bottle outside an alcohol lamp for surrounding sterilization for about 15 s; then a liquid transfer gun is used for sucking 2.5ml of dendrobium officinale seed suspension to be inoculated into a 2-generation mother bottle seedling culture medium, the orchid bottle is gently shaken after the transfer is finished, so that the seed suspension is uniformly paved on the surface of the culture medium, and finally the orchid bottle is placed in a culture room for culture; culturing for about 120 days in a culture room to obtain mother bottle seedlings for transfer culture.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011430986.8A CN112544446A (en) | 2020-12-09 | 2020-12-09 | Production method of dendrobium officinale Kimura et Migo mother bottle seedlings |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011430986.8A CN112544446A (en) | 2020-12-09 | 2020-12-09 | Production method of dendrobium officinale Kimura et Migo mother bottle seedlings |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112544446A true CN112544446A (en) | 2021-03-26 |
Family
ID=75059950
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011430986.8A Pending CN112544446A (en) | 2020-12-09 | 2020-12-09 | Production method of dendrobium officinale Kimura et Migo mother bottle seedlings |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112544446A (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102301953A (en) * | 2011-07-20 | 2012-01-04 | 江苏农林职业技术学院 | Method for efficiently and rapidly propagating Dendrobium candidum test-tube seedlings in large scale |
| WO2015003408A1 (en) * | 2013-07-08 | 2015-01-15 | 中国科学院华南植物园 | Dendrobium in vitro crossbreeding method |
| CN104705188A (en) * | 2015-04-03 | 2015-06-17 | 南京工业大学 | Method for culturing dendrobium candidum protocorms |
| CN111990254A (en) * | 2020-08-31 | 2020-11-27 | 南京农业大学 | Dendrobium nobile culture medium and application thereof |
-
2020
- 2020-12-09 CN CN202011430986.8A patent/CN112544446A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102301953A (en) * | 2011-07-20 | 2012-01-04 | 江苏农林职业技术学院 | Method for efficiently and rapidly propagating Dendrobium candidum test-tube seedlings in large scale |
| WO2015003408A1 (en) * | 2013-07-08 | 2015-01-15 | 中国科学院华南植物园 | Dendrobium in vitro crossbreeding method |
| CN104705188A (en) * | 2015-04-03 | 2015-06-17 | 南京工业大学 | Method for culturing dendrobium candidum protocorms |
| CN111990254A (en) * | 2020-08-31 | 2020-11-27 | 南京农业大学 | Dendrobium nobile culture medium and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 宋顺等: ""铁皮石斛兰种子萌发及其原球茎的快繁培养条件研究"", 《安徽农业科学》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101455180B (en) | Open type plant tissue culture seedlings-raising method | |
| CN105145352B (en) | A kind of bletilla seed height of seedling imitates group culturation rapid propagating technology | |
| CN104145816B (en) | Bletilla striata tissue culture method | |
| CN102835318B (en) | Technology for rapid sugar-free micropropagation of dendrobium officinale kimura et migo under non-aseptic condition | |
| CN107047320B (en) | A kind of bigflower centranthera root method for tissue culture | |
| CN102246695A (en) | Method for preparing common bletilla pseudobulb in culture vessel and special culture media thereof | |
| CN106561450B (en) | It is a kind of using Lycoris radiata rachis as the method for explant evoking adventive bud | |
| CN104920065A (en) | Preparation method for mother strain of wild dictyophora rubrovolvatain of Mount Fanjing | |
| CN107047310A (en) | A kind of cultural method of bletilla striata seeds culture seedling | |
| CN103210842A (en) | Dendrobium candidum test-tube flowering and seed setting method | |
| CN102668990B (en) | Plant tissue culturing method and device | |
| CN111990254A (en) | Dendrobium nobile culture medium and application thereof | |
| CN103688860B (en) | Culture medium for rapid propagation and seedling of dendrobium officinale protocorm like-bodies and tissue culture method | |
| CN105191803B (en) | A kind of candidum tissue culturing bag seedling production method | |
| CN105349438A (en) | Functional red yeast rice strain and production process thereof | |
| CN110663555B (en) | Rapid propagation method of odorous jasminum grandiflorum | |
| CN110663549B (en) | Sterile sowing and seedling raising method for dendrobium hybrid seeds | |
| CN112544446A (en) | Production method of dendrobium officinale Kimura et Migo mother bottle seedlings | |
| CN108243959A (en) | It is a kind of using yellow fine strain of millet wood stem section as the highly efficient regeneration method of explant | |
| CN110839530A (en) | Method for inducing flowering of Chinese rose in test tube | |
| CN104737916B (en) | Suspension culture method for producing quiquefolium saponins | |
| CN107182782A (en) | A kind of method of acceleration sunflower generation process | |
| CN111374055A (en) | Method for preparing artificial seeds of elaeagnus mollis | |
| CN111528098A (en) | Angelica sinensis tissue culture seedling rooting culture method | |
| CN112167064A (en) | Tissue culture and rapid propagation method for salvia miltiorrhiza |
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 | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20210326 |
|
| RJ01 | Rejection of invention patent application after publication |