CN116941528B - Method for in-vitro preservation of uncaria - Google Patents
Method for in-vitro preservation of uncaria Download PDFInfo
- Publication number
- CN116941528B CN116941528B CN202310852641.9A CN202310852641A CN116941528B CN 116941528 B CN116941528 B CN 116941528B CN 202310852641 A CN202310852641 A CN 202310852641A CN 116941528 B CN116941528 B CN 116941528B
- Authority
- CN
- China
- Prior art keywords
- culture
- temperature
- seeds
- illumination
- day
- 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.)
- Active
Links
- 238000000338 in vitro Methods 0.000 title claims abstract description 94
- 238000004321 preservation Methods 0.000 title claims abstract description 77
- 241000157352 Uncaria Species 0.000 title claims abstract description 57
- 238000000034 method Methods 0.000 title claims abstract description 34
- 238000005286 illumination Methods 0.000 claims abstract description 181
- 239000001963 growth medium Substances 0.000 claims abstract description 84
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 claims abstract description 67
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 59
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 21
- 230000035784 germination Effects 0.000 claims abstract description 17
- 239000008223 sterile water Substances 0.000 claims description 45
- 238000005406 washing Methods 0.000 claims description 45
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 45
- 230000001954 sterilising effect Effects 0.000 claims description 30
- 238000004659 sterilization and disinfection Methods 0.000 claims description 30
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 15
- 235000013601 eggs Nutrition 0.000 claims description 15
- 239000005556 hormone Substances 0.000 claims description 15
- 229940088597 hormone Drugs 0.000 claims description 15
- 229960002523 mercuric chloride Drugs 0.000 claims description 15
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 claims description 15
- 238000005728 strengthening Methods 0.000 claims description 6
- 241000238631 Hexapoda Species 0.000 claims description 2
- 238000012258 culturing Methods 0.000 claims 1
- 230000004083 survival effect Effects 0.000 abstract description 25
- 230000001902 propagating effect Effects 0.000 abstract description 2
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 59
- 230000000638 stimulation Effects 0.000 description 18
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 13
- 229930006000 Sucrose Natural products 0.000 description 13
- 239000002609 medium Substances 0.000 description 13
- 239000005720 sucrose Substances 0.000 description 13
- 230000008261 resistance mechanism Effects 0.000 description 11
- 230000007704 transition Effects 0.000 description 11
- 230000000052 comparative effect Effects 0.000 description 8
- 206010010904 Convulsion Diseases 0.000 description 3
- 230000036461 convulsion Effects 0.000 description 3
- RMOGWMIKYWRTKW-UONOGXRCSA-N (S,S)-paclobutrazol Chemical compound C([C@@H]([C@@H](O)C(C)(C)C)N1N=CN=C1)C1=CC=C(Cl)C=C1 RMOGWMIKYWRTKW-UONOGXRCSA-N 0.000 description 2
- 239000005985 Paclobutrazol Substances 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000002035 prolonged effect Effects 0.000 description 2
- 230000035899 viability Effects 0.000 description 2
- 241000345998 Calamus manan Species 0.000 description 1
- 206010019233 Headaches Diseases 0.000 description 1
- 241001107098 Rubiaceae Species 0.000 description 1
- 229930187904 Uncarine Natural products 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 230000036772 blood pressure Effects 0.000 description 1
- 230000001914 calming effect Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 231100000869 headache Toxicity 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000004161 plant tissue culture Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 235000012950 rattan cane Nutrition 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
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
- A01H4/008—Methods for regeneration to complete plants
-
- 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
- A01H4/002—Culture media for tissue culture
Abstract
The invention discloses an in-vitro preservation method of uncaria, which belongs to the technical field of uncaria tissue culture and comprises the following steps: hookSterile germination culture is carried out on vine seeds to obtain seed seedlings; the seed seedlings are subjected to propagation culture to obtain propagation seedlings; the propagating seedlings are placed in a strong seedling culture medium containing 0.01-0.1 mg/L proline for strong seedling culture, and a low-temperature culture period of 15-21 ℃ is set at the later stage of strong seedling culture; placing the tissue culture seedling subjected to the strong seedling culture in an in vitro culture medium for in vitro culture, wherein the in vitro culture medium contains 1/2MS+6-BA0.02mg/L+PPP 333 1.0-2.0 mg/L of active carbon, 0.5g/L of active carbon and 0.01-0.1 mg/L of proline, the culture temperature is 15-21 ℃, the illumination intensity is 1200Lx, and the illumination duration is 10 hours/day. The invention prolongs the in-vitro preservation period of the uncaria and obviously improves the in-vitro preservation survival rate of more than 200 days.
Description
Technical Field
The invention relates to the technical field of plant tissue culture, in particular to the technical field of uncaria tissue culture, and more particularly relates to an in-vitro uncaria preservation method.
Background
Ramulus Uncariae cum Uncis [ Uncaria rhynchopylla (Miq.) Jacks ] is ramulus Uncariae cum Uncis belonging to Rubiaceae, belonging to woody vine, namely Geng Magou rattan, double ramulus Uncariae cum Uncis, and ramulus Uncariae cum Uncis, produced in Guangdong, guangxi, yunnan, guizhou, fujian, hunan, etc., and commonly used in the forest or brush of mountain valley stream, and is a well-known Chinese medicine in China. The uncaria has the functions of clearing blood and calming liver, stopping wind and arresting convulsion and the like, can be used for treating wind-heat headache, cold and clamping convulsion, convulsion and the like, and the uncarine contained in the uncaria has the function of reducing blood pressure, is a better medicinal material, and can also be used as a medicine. In the production process of uncaria seedlings, the most rapid and effective propagation path is tissue culture. A large number of seedlings can be obtained through a tissue culture technology, and meanwhile, good germplasm can be preserved for a long time, in the long-term preservation process of the uncaria tissue culture seedlings, the isolated preservation time of uncaria is short by adopting a conventional MS culture medium in the prior art, and the uncaria tissue culture seedlings are often required to be transferred, and the continuous preservation can be realized by preparing the culture medium for subculture in a short time, so that the long-term isolated preservation of uncaria germplasm resources is not facilitated.
Disclosure of Invention
The invention aims to provide an in-vitro preservation method of uncaria, which comprises the steps of adding proline into a culture medium in a strong seedling culture stage, introducing low-temperature stimulation, and then selecting normal-growth buds to be placed in a low-temperature and low-light environment and a culture medium containing paclobutrazol and proline for in-vitro preservation, so that the in-vitro preservation period is prolonged, and the survival rate is improved.
To achieve these objects and other advantages and in accordance with the purpose of the invention, a method for preserving uncaria in vitro is provided, comprising: the uncaria seeds are germinated and cultured to obtain sterile seed seedlings; the seed seedlings are subjected to propagation culture to obtain propagation seedlings; the propagating seedlings are placed in a strong seedling culture medium containing 0.01-0.1 mg/L proline for strong seedling culture, and a low-temperature culture period of 15-21 ℃ is set at the later stage of strong seedling culture;
placing the tissue culture seedling subjected to the strong seedling culture in an in vitro culture medium for in vitro culture, wherein the in vitro culture medium contains 1/2MS+6-BA0.02mg/L+PPP 333 1.0-2.0 mg/L of active carbon, 0.5g/L of active carbon and 0.01-0.1 mg/L of proline, the culture temperature is 15-21 ℃, the illumination intensity is 1200Lx, and the illumination duration is 10 hours/day.
Preferably, in the method for preserving uncaria in vitro, the culture temperature in the low-temperature culture period is 17 ℃, the illumination intensity is 1200-1800 Lx, the illumination duration is 10-12 hours/day, and the duration is 6-8 days.
Preferably, in the method for in vitro preservation of uncaria, the early stage of strong seedling culture is normal culture, the culture temperature of normal culture is 24-26 ℃, the illumination intensity is 1800Lx, and the illumination time is 10-12 hours/day; the low-temperature culture period is 7 days after normal culture, the culture temperature in the low-temperature culture period is 17 ℃, the illumination intensity is 1200-1800 Lx, and the illumination duration is 10-12 hours/day.
Preferably, in the method for in vitro preservation of uncaria, the culture temperature in the low-temperature culture period is 17 ℃, the illumination intensity is 1800Lx, and the illumination duration is 12 hours/day.
Preferably, in the method for in vitro preservation of uncaria, the seedling-strengthening culture medium contains MS+IBA0.5mg/L+NAA0.1mg/L+activated carbon 1 g/L+proline 0.05mg/L.
Preferably, in the method for preserving uncaria in vitro, 1/2MS+6-BA of the isolated culture medium is 0.02mg/L+PPP 333 1.5 mg/L+0.5 g/L of activated carbon+0.05 mg/L of proline, the culture temperature is 17 ℃, the illumination intensity is 1200Lx, and the illumination duration is 10 hours/day.
Preferably, the method for preserving uncaria in vitro comprises the following steps:
s1, taking uncinate ramulus Uncariae cum Uncis seeds which are full, uniform in texture and do not carry eggs of insects;
s2: taking seeds of S1, washing the seeds for 3-5 times by using sterile water, then placing the seeds into 75% alcohol for sterilization for 30 seconds, washing the seeds for 3-5 times by using sterile water, then placing the seeds into 0.1% mercuric chloride solution for sterilization for 7 minutes, washing the seeds for 3-5 times by using sterile water, finally inoculating the seeds into an MS culture medium without any hormone, and carrying out germination culture to obtain sterile seedlings, wherein the culture time is about 14 days, the culture temperature is 24 ℃, the illumination intensity is 1800Lx, and the illumination duration is 10 hours/day;
s3: inoculating the seed seedling in S2 into a culture medium containing MS+6-BA0.5mg/L+NAA0.20mg/L for propagation culture, wherein the culture temperature is 24 ℃, the illumination intensity is 1800Lx, and the illumination duration is 12 hours/day;
s4: inoculating the bud in S3 into a seedling strengthening culture medium of MS+IBA0.5mg/L+NAA0.1mg/L+active carbon 1 g/L+proline 0.01-0.1 mg/L for seedling strengthening culture; wherein the culture temperature in the early stage of the strong seedling culture is 24-26 ℃, the illumination intensity is 1800Lx, and the illumination time is 12 hours/day; in the last 6-8 days of strong seedling cultivation, the cultivation temperature is 15-21 ℃, the illumination intensity is 1200-1800 Lx, and the illumination time is 12 hours/day;
s5: taking normal tissue culture seedling of S4, inoculating to 1/2MS+6-BA0.02mg/L+PPP 333 In-vitro preservation culture is carried out in an in-vitro preservation culture medium of 1.0-2.0 mg/L+0.5 g/L of activated carbon+0.01-0.1 mg/L of proline, the culture temperature is 15-21 ℃, the illumination intensity is 1200Lx, and the illumination duration is 10 hours/day.
The invention at least comprises the following beneficial effects:
1. according to the invention, proline is added into the strong seedling culture medium to carry out strong seedling culture on the propagation buds, so that proline is accumulated in the propagation buds, when low-temperature stimulation is introduced into the latter half of the strong seedling culture period, the accumulated proline improves the resistance of the propagation seedlings to smoothly transition, and the tissue culture seedling resistance mechanism is activated to generate stronger stress resistance due to the low-temperature stimulation, so that the storage life and the survival rate of in-vitro preservation culture under the subsequent low-temperature weak light condition are improved.
2. The low-temperature weak light culture condition in the in-vitro culture can slow down the growth speed of the tissue culture seedling, and simultaneously, paclobutrazol and proline are added to improve the resistance of the tissue culture seedling to the low-temperature weak light, so that the survival rate of the in-vitro culture seedling is improved while the preservation time of the tissue culture seedling is prolonged.
3. In the embodiment of the method, 0.05mg/L of proline is added into the strong seedling culture medium, and the low-temperature stimulation is carried out at the temperature of 17 ℃ after 7 days of strong seedling culture, so that the survival rate of the strong seedling culture reaches 100 percent, and compared with the survival rate of the strong seedling culture under normal conditions, the survival rate of the strong seedling culture can be effectively improved in comparison with the low-temperature stimulation without adding proline. In one embodiment, the material is subjected to cryogenic stimulation and then placed in PPP 333 The survival rate of 1.5 mg/L+proline is 0.05 mg/L+the in vitro preservation culture medium with the culture temperature of 17 ℃ is up to 95.0% after the in vitro preservation culture medium is finally preserved for 200 days, and the survival rate of 47.5% after the in vitro preservation culture medium is preserved for 400 days.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.
Drawings
FIG. 1 is a graph showing the growth condition of the tissue culture seedling of example 2 of the present invention at 200 days;
FIG. 2 is a graph showing the growth of the tissue culture seedling of example 2 of the present invention at 400 days.
Detailed Description
The present invention is described in further detail below with reference to examples to enable those skilled in the art to practice the same by referring to the description.
Example 1
The invention discloses an in-vitro preservation method of uncaria, which comprises the following steps:
(1) And (3) selecting seeds with excellent uncaria germplasm resources, and picking out the seeds which are not damaged, full in seeds, uniform in texture and free of carrying eggs for later use.
(2) Taking seeds of the step (1), washing the seeds with sterile water for 3-5 times, then placing the seeds into 75% alcohol for sterilization for 30 seconds, washing the seeds with sterile water for 3-5 times, placing the seeds into 0.1% mercuric chloride solution for sterilization for 7 minutes, washing the seeds with sterile water for 3-5 times, finally inoculating the seeds into an MS culture medium without any hormone for germination culture, wherein the culture time is about 14 days, the culture temperature is 24 ℃, the illumination intensity is 1800Lx, and the illumination duration is 10 hours/day.
(3) Inoculating the aseptic seed seedlings germinated in the step (2) into a culture medium of MS+6-BA0.5mg/L+NAA0.20mg/L for propagation culture, wherein the culture temperature is 24 ℃, the culture time is about 14 days, the illumination intensity is 1800Lx, and the illumination duration is 12 hours/day; the temperature is proper, the illumination is sufficient, so that the seedlings can grow normally.
(4) Inoculating the bud obtained in the step (3) into a culture medium of MS+IBA0.5mg/L+NAA0.1mg/L+active carbon 1 g/L+proline 0.01mg/L for strong seedling culture for about 14 days; wherein the culture temperature is 24 ℃ in the first 7 days, the illumination intensity is 1800Lx, and the illumination duration is 12 hours/day; the culture temperature is 17 ℃ in the latter 7 days, the illumination intensity is 1800Lx, and the illumination duration is 12 hours/day; the propagation seedlings are firstly cultivated by proline, the proline is accumulated in the propagation seedlings in advance, the resistance is improved to cope with the smooth transition of low-temperature stimulation cultivation, and after the propagation seedlings are cultivated at low temperature and under sufficient illumination, the propagation seedlings generate a stronger resistance mechanism, so that the survival rate of in-vitro preservation under the subsequent low-temperature weak light condition is improved.
(5) Immediately taking the normal tissue culture seedling which is subjected to the strong seedling culture in the step (4), and inoculating the normal tissue culture seedling to 1/2MS+6-BA0.02mg/L+PPP 333 In an in vitro preservation culture medium of 1.5 mg/L+0.5 g/L of activated carbon+0.05 mg/L of proline, carrying out in vitro preservation culture at 17 ℃ under the illumination intensity of 1200Lx for 10 hours/day. The time interval from the completion of the seedling strengthening culture to the inoculation to the in-vitro preservation culture medium is as short as possible, the time interval is not more than 1 day, and the same applies.
The medium in each step contained 25g/L sucrose, ph=5.8.
Example 2
The invention discloses an in-vitro preservation method of uncaria, which comprises the following steps:
(1) Selecting seeds with good hooked uncaria germplasm resources, which are not damaged, full in seeds, uniform in texture and free of carrying eggs for later use;
(2) Taking seeds of the step (1), washing the seeds with sterile water for 3-5 times, then placing the seeds into 75% alcohol for sterilization for 30 seconds, washing the seeds with sterile water for 3-5 times, placing the seeds into 0.1% mercuric chloride solution for sterilization for 7 minutes, washing the seeds with sterile water for 3-5 times, finally inoculating the seeds into an MS culture medium without any hormone for germination culture, wherein the culture time is about 14 days, the culture temperature is 24 ℃, the illumination intensity is 1800Lx, and the illumination duration is 10 hours/day;
(3) Inoculating the aseptic seed seedlings germinated in the step (2) into a culture medium of MS+6-BA0.5mg/L+NAA0.20mg/L for propagation culture, wherein the culture temperature is 24 ℃, the culture time is about 14 days, the illumination intensity is 1800Lx, and the illumination duration is 12 hours/day; the temperature is proper, the illumination is sufficient, so that the seedlings can grow normally.
(4) Inoculating the bud obtained in the step (3) into a culture medium of MS+IBA0.5mg/L+NAA0.1mg/L+active carbon 1 g/L+proline 0.05mg/L for strong seedling culture for about 14 days; wherein the culture temperature is 24 ℃ in the first 7 days, the illumination intensity is 1800Lx, and the illumination duration is 12 hours/day; the culture temperature is 17 ℃ in the latter 7 days, the illumination intensity is 1800Lx, and the illumination duration is 12 hours/day; the propagation seedlings are firstly cultivated by proline, the proline is accumulated in the propagation seedlings in advance, the resistance is improved to cope with the smooth transition of low-temperature stimulation cultivation, and after the propagation seedlings are cultivated at low temperature and under sufficient illumination, the propagation seedlings generate a stronger resistance mechanism, so that the survival rate of in-vitro preservation under the subsequent low-temperature weak light condition is improved.
(5) Taking the normal tissue culture seedling which is subjected to the strong seedling culture in the step (4), and inoculating to 1/2MS+6-BA0.02mg/L+PPP 333 In an in vitro preservation culture medium of 1.5 mg/L+0.5 g/L of activated carbon+0.05 mg/L of proline, carrying out in vitro preservation culture at 17 ℃ under the illumination intensity of 1200Lx for 10 hours/day.
The medium in each step contained 25g/L sucrose, ph=5.8.
Example 3
The invention discloses an in-vitro preservation method of uncaria, which comprises the following steps:
(1) Selecting seeds with good hooked uncaria germplasm resources, which are not damaged, full in seeds, uniform in texture and free of carrying eggs for later use;
(2) Taking seeds of the step (1), washing the seeds with sterile water for 3-5 times, then placing the seeds into 75% alcohol for sterilization for 30 seconds, washing the seeds with sterile water for 3-5 times, placing the seeds into 0.1% mercuric chloride solution for sterilization for 7 minutes, washing the seeds with sterile water for 3-5 times, finally inoculating the seeds into an MS culture medium without any hormone for germination culture, wherein the culture time is about 14 days, the culture temperature is 24 ℃, the illumination intensity is 1800Lx, and the illumination duration is 10 hours/day;
(3) Inoculating the aseptic seed seedlings germinated in the step (2) into a culture medium of MS+6-BA0.5mg/L+NAA0.20mg/L for propagation culture, wherein the culture temperature is 24 ℃, the culture time is about 14 days, the illumination intensity is 1800Lx, and the illumination duration is 12 hours/day; the temperature is proper, the illumination is sufficient, so that the seedlings can grow normally.
(4) Inoculating the bud obtained in the step (3) into a culture medium of MS+IBA0.5mg/L+NAA0.1mg/L+active carbon 1 g/L+proline 0.1mg/L for strong seedling culture for about 14 days; wherein the culture temperature in the first 7 days is 24 ℃, the illumination intensity is 1800Lx, and the illumination duration is 12 hours/day; the culture temperature is 17 ℃ in the latter 7 days, the illumination intensity is 1800Lx, and the illumination duration is 12 hours/day; the propagation seedlings are firstly cultivated by proline, the proline is accumulated in the propagation seedlings in advance, the resistance is improved to cope with the smooth transition of low-temperature stimulation cultivation, and after the propagation seedlings are cultivated at low temperature and under sufficient illumination, the propagation seedlings generate a stronger resistance mechanism, so that the survival rate of in-vitro preservation under the subsequent low-temperature weak light condition is improved.
(5) Immediately taking the normal tissue culture seedling which is subjected to the strong seedling culture in the step (4), and inoculating the normal tissue culture seedling to 1/2MS+6-BA0.02mg/L+PPP 333 In an in vitro preservation culture medium of 1.5 mg/L+0.5 g/L of activated carbon+0.05 mg/L of proline, carrying out in vitro preservation culture at 17 ℃ under the illumination intensity of 1200Lx for 10 hours/day.
The medium in each step contained 25g/L sucrose, ph=5.8.
Example 4
The invention discloses an in-vitro preservation method of uncaria, which comprises the following steps:
(1) Selecting seeds with good hooked uncaria germplasm resources, which are not damaged, full in seeds, uniform in texture and free of carrying eggs for later use;
(2) Taking the seeds of the step (1), washing the seeds with sterile water for 3-5 times, then placing the seeds into 75% alcohol for sterilization for 30 seconds, then washing the seeds with sterile water for 3-5 times, then placing the seeds into 0.1% mercuric chloride solution for sterilization for 7 minutes, then washing the seeds with sterile water for 3-5 times, finally inoculating the seeds into an MS culture medium without any hormone for germination culture, wherein the culture time is about 14 days, the culture temperature is 24 ℃, and the illumination intensity is 1800L X The illumination time is 10 hours/day;
(3) Inoculating the aseptic seed seedling germinated in step (2) into a culture medium of MS+6-BA0.5mg/L+NAA0.20mg/L for propagation culture at 24deg.C for about 14 days with illumination intensity of 1800L X The illumination time is 12 hours/day; the temperature is proper, the illumination is sufficient, so that the seedlings can grow normally.
(4) Inoculating the bud obtained in the step (3) into a culture medium of MS+IBA0.5mg/L+NAA0.1mg/L+active carbon 1 g/L+proline 0.05mg/L for strong seedling culture for about 14 days; wherein the culture temperature in the first 7 days is 24 ℃, the illumination intensity is 1800Lx, and the illumination duration is 12 hours/day; the culture temperature is 17 ℃ in the latter 7 days, the illumination intensity is 1800Lx, and the illumination duration is 12 hours/day; the propagation seedlings are firstly cultivated by proline, the proline is accumulated in the propagation seedlings in advance, the resistance is improved to cope with the smooth transition of low-temperature stimulation cultivation, and after the propagation seedlings are cultivated at low temperature and under sufficient illumination, the propagation seedlings generate a stronger resistance mechanism, so that the survival rate of in-vitro preservation under the subsequent low-temperature weak light condition is improved.
(5) Immediately taking the normal tissue culture seedling which is subjected to the strong seedling culture in the step (4), and inoculating the normal tissue culture seedling to 1/2MS+6-BA0.02mg/L+PPP 333 In an in vitro preservation culture medium of 1.5 mg/L+0.5 g/L of activated carbon+0.01 mg/L of proline, carrying out in vitro preservation culture at 17 ℃ under the illumination intensity of 1200Lx for 10 hours/day.
The medium in each step contained 25g/L sucrose, ph=5.8.
Example 5
The invention discloses an in-vitro preservation method of uncaria, which comprises the following steps:
(1) Selecting seeds with good hooked uncaria germplasm resources, which are not damaged, full in seeds, uniform in texture and free of carrying eggs for later use;
(2) Taking the seeds of the step (1), washing the seeds with sterile water for 3-5 times, then placing the seeds into 75% alcohol for sterilization for 30 seconds, then washing the seeds with sterile water for 3-5 times, then placing the seeds into 0.1% mercuric chloride solution for sterilization for 7 minutes, then washing the seeds with sterile water for 3-5 times, finally inoculating the seeds into an MS culture medium without any hormone for germination culture, wherein the culture time is about 14 days, the culture temperature is 24 ℃, and the illumination intensity is 1800L X The illumination time is 10 hours/day;
(3) Inoculating the aseptic seed seedlings germinated in the step (2) into a culture medium of MS+6-BA0.5mg/L+NAA0.20mg/L for propagation culture, wherein the culture temperature is 24 ℃, the culture time is about 14 days, the illumination intensity is 1800Lx, and the illumination duration is 12 hours/day; the temperature is proper, the illumination is sufficient, so that the seedlings can grow normally.
(4) Inoculating the bud obtained in the step (3) into a culture medium of MS+IBA0.5mg/L+NAA0.1mg/L+active carbon 1 g/L+proline 0.05mg/L for strong seedling culture for about 14 days; wherein the culture temperature in the first 7 days is 24 ℃, the illumination intensity is 1800Lx, and the illumination duration is 12 hours/day; the culture temperature is 17 ℃ in the latter 7 days, the illumination intensity is 1800Lx, and the illumination duration is 12 hours/day; the propagation seedlings are firstly cultivated by proline, the proline is accumulated in the propagation seedlings in advance, the resistance is improved to cope with the smooth transition of low-temperature stimulation cultivation, and after the propagation seedlings are cultivated at low temperature and under sufficient illumination, the propagation seedlings generate a stronger resistance mechanism, so that the survival rate of in-vitro preservation under the subsequent low-temperature weak light condition is improved.
(5) Immediately taking the normal tissue culture seedling which is subjected to the strong seedling culture in the step (4), and inoculating the normal tissue culture seedling to 1/2MS+6-BA0.02mg/L+PPP 333 In an in vitro preservation culture medium of 1.5 mg/L+0.5 g/L of activated carbon+0.1 mg/L of proline, carrying out in vitro preservation culture at 17 ℃ under the illumination intensity of 1200Lx for 10 hours/day.
The medium in each step contained 25g/L sucrose, ph=5.8.
Example 6
The invention discloses an in-vitro preservation method of uncaria, which comprises the following steps:
(1) Selecting seeds with good hooked uncaria germplasm resources, which are not damaged, full in seeds, uniform in texture and free of carrying eggs for later use;
(2) Taking the seeds of the step (1), washing the seeds with sterile water for 3-5 times, then placing the seeds into 75% alcohol for sterilization for 30 seconds, then washing the seeds with sterile water for 3-5 times, then placing the seeds into 0.1% mercuric chloride solution for sterilization for 7 minutes, then washing the seeds with sterile water for 3-5 times, finally inoculating the seeds into an MS culture medium without any hormone for germination culture, wherein the culture time is about 14 days, the culture temperature is 24 ℃, and the illumination intensity is 1800L X The illumination time is 10 hours/day;
(3) Inoculating the aseptic seed seedling germinated in step (2) into a culture medium of MS+6-BA0.5mg/L+NAA0.20mg/L for propagation culture at 24deg.C for about 14 days with illumination intensity of 1800L X The illumination time is 12 hours/day; the temperature is proper, the illumination is sufficient, so that the seedlings can grow normally.
(4) Inoculating the bud obtained in the step (3) into a culture medium of MS+IBA0.5mg/L+NAA0.1mg/L+active carbon 1 g/L+proline 0.05mg/L for strong seedling culture for about 14 days; wherein the culture temperature in the first 7 days is 24 ℃, the illumination intensity is 1800Lx, and the illumination duration is 12 hours/day; the culture temperature is 17 ℃ in the latter 7 days, the illumination intensity is 1800Lx, and the illumination duration is 12 hours/day; the propagation seedlings are firstly cultivated by proline, the proline is accumulated in the propagation seedlings in advance, the resistance is improved to cope with the smooth transition of low-temperature stimulation cultivation, and after the propagation seedlings are cultivated at low temperature and under sufficient illumination, the propagation seedlings generate a stronger resistance mechanism, so that the survival rate of in-vitro preservation under the subsequent low-temperature weak light condition is improved.
(5) Immediately taking the normal tissue culture seedling which is subjected to the strong seedling culture in the step (4), and inoculating the normal tissue culture seedling to 1/2MS+6-BA0.02mg/L+PPP 333 In an in vitro preservation culture medium of 1.0 mg/L+0.5 g/L of activated carbon+0.05 mg/L of proline, carrying out in vitro preservation culture at 17 ℃ under the illumination intensity of 1200Lx for 10 hours/day.
The medium in each step contained 25g/L sucrose, ph=5.8.
Example 7
The invention discloses an in-vitro preservation method of uncaria, which comprises the following steps:
(1) Selecting seeds with good hooked uncaria germplasm resources, which are not damaged, full in seeds, uniform in texture and free of carrying eggs for later use;
(2) Taking seeds of the step (1), washing the seeds with sterile water for 3-5 times, then placing the seeds into 75% alcohol for sterilization for 30 seconds, washing the seeds with sterile water for 3-5 times, placing the seeds into 0.1% mercuric chloride solution for sterilization for 7 minutes, washing the seeds with sterile water for 3-5 times, finally inoculating the seeds into an MS culture medium without any hormone for germination culture, wherein the culture time is about 14 days, the culture temperature is 24 ℃, the illumination intensity is 1800Lx, and the illumination duration is 10 hours/day;
(3) Inoculating the aseptic seed seedlings germinated in the step (2) into a culture medium of MS+6-BA0.5mg/L+NAA0.20mg/L for propagation culture, wherein the culture temperature is 24 ℃, the culture time is about 14 days, the illumination intensity is 1800Lx, and the illumination duration is 12 hours/day; the temperature is proper, the illumination is sufficient, so that the seedlings can grow normally.
(4) Inoculating the bud obtained in the step (3) into a culture medium of MS+IBA0.5mg/L+NAA0.1mg/L+active carbon 1 g/L+proline 0.05mg/L for strong seedling culture for about 14 days; wherein the culture temperature in the first 7 days is 24 ℃, the illumination intensity is 1800Lx, and the illumination duration is 12 hours/day; the culture temperature is 17 ℃ in the latter 7 days, the illumination intensity is 1800Lx, and the illumination duration is 12 hours/day; the propagation seedlings are firstly cultivated by proline, the proline is accumulated in the propagation seedlings in advance, the resistance is improved to cope with the smooth transition of low-temperature stimulation cultivation, and after the propagation seedlings are cultivated at low temperature and under sufficient illumination, the propagation seedlings generate a stronger resistance mechanism, so that the survival rate of in-vitro preservation under the subsequent low-temperature weak light condition is improved.
(5) Immediately taking the normal tissue culture seedling which is subjected to the strong seedling culture in the step (4), and inoculating the normal tissue culture seedling to 1/2MS+6-BA0.02mg/L+PPP 333 In an in vitro preservation culture medium of 2.0 mg/L+0.5 g/L of activated carbon+0.05 mg/L of proline, carrying out in vitro preservation culture at 17 ℃ under the illumination intensity of 1200Lx for 10 hours/day.
The medium in each step contained 25g/L sucrose, ph=5.8.
Example 8
The invention discloses an in-vitro preservation method of uncaria, which comprises the following steps:
(1) Selecting seeds with good hooked uncaria germplasm resources, which are not damaged, full in seeds, uniform in texture and free of carrying eggs for later use;
(2) Taking seeds of the step (1), washing the seeds with sterile water for 3-5 times, then placing the seeds into 75% alcohol for sterilization for 30 seconds, washing the seeds with sterile water for 3-5 times, placing the seeds into 0.1% mercuric chloride solution for sterilization for 7 minutes, washing the seeds with sterile water for 3-5 times, finally inoculating the seeds into an MS culture medium without any hormone for germination culture, wherein the culture time is about 14 days, the culture temperature is 24 ℃, the illumination intensity is 1800Lx, and the illumination duration is 10 hours/day;
(3) Inoculating the aseptic seed seedlings germinated in the step (2) into a culture medium of MS+6-BA0.5mg/L+NAA0.20mg/L for propagation culture, wherein the culture temperature is 24 ℃, the culture time is about 14 days, the illumination intensity is 1800Lx, and the illumination duration is 12 hours/day; the temperature is proper, the illumination is sufficient, so that the seedlings can grow normally.
(4) Inoculating the bud obtained in the step (3) into a culture medium of MS+IBA0.5mg/L+NAA0.1mg/L+active carbon 1 g/L+proline 0.05mg/L for strong seedling culture for about 14 days; wherein the culture temperature in the first 7 days is 24 ℃, the illumination intensity is 1800Lx, and the illumination duration is 12 hours/day; the culture temperature is 17 ℃ in the latter 7 days, the illumination intensity is 1800Lx, and the illumination duration is 12 hours/day; the propagation seedlings are firstly cultivated by proline, the proline is accumulated in the propagation seedlings in advance, the resistance is improved to cope with the smooth transition of low-temperature stimulation cultivation, and after the propagation seedlings are cultivated at low temperature and under sufficient illumination, the propagation seedlings generate a stronger resistance mechanism, so that the survival rate of in-vitro preservation under the subsequent low-temperature weak light condition is improved.
(5) Immediately taking the normal tissue culture seedling which is subjected to the strong seedling culture in the step (4), and inoculating the normal tissue culture seedling to 1/2MS+6-BA0.02mg/L+PPP 333 In an in vitro preservation culture medium of 1.5 mg/L+0.5 g/L of activated carbon+0.05 mg/L of proline, carrying out in vitro preservation culture at 15 ℃ under 1200Lx of illumination intensity for 10 hours/day.
The medium in each step contained 25g/L sucrose, ph=5.8.
Example 9
The invention discloses an in-vitro preservation method of uncaria, which comprises the following steps:
(1) Selecting seeds with good hooked uncaria germplasm resources, which are not damaged, full in seeds, uniform in texture and free of carrying eggs for later use;
(2) Taking seeds of the step (1), washing the seeds with sterile water for 3-5 times, then placing the seeds into 75% alcohol for sterilization for 30 seconds, washing the seeds with sterile water for 3-5 times, placing the seeds into 0.1% mercuric chloride solution for sterilization for 7 minutes, washing the seeds with sterile water for 3-5 times, finally inoculating the seeds into an MS culture medium without any hormone for germination culture, wherein the culture time is about 14 days, the culture temperature is 24 ℃, the illumination intensity is 1800Lx, and the illumination duration is 10 hours/day;
(3) Inoculating the aseptic seed seedlings germinated in the step (2) into a culture medium of MS+6-BA0.5mg/L+NAA0.20mg/L for propagation culture, wherein the culture temperature is 24 ℃, the culture time is about 14 days, the illumination intensity is 1800Lx, and the illumination duration is 12 hours/day; the temperature is proper, the illumination is sufficient, so that the seedlings can grow normally.
(4) Inoculating the bud obtained in the step (3) into a culture medium of MS+IBA0.5mg/L+NAA0.1mg/L+active carbon 1 g/L+proline 0.05mg/L for strong seedling culture for about 14 days; wherein the culture temperature in the first 7 days is 24 ℃, the illumination intensity is 1800Lx, and the illumination duration is 12 hours/day; the culture temperature is 17 ℃ in the latter 7 days, the illumination intensity is 1800Lx, and the illumination duration is 12 hours/day; the propagation seedlings are firstly cultivated by proline, the proline is accumulated in the propagation seedlings in advance, the resistance is improved to cope with the smooth transition of low-temperature stimulation cultivation, and after the propagation seedlings are cultivated at low temperature and under sufficient illumination, the propagation seedlings generate a stronger resistance mechanism, so that the survival rate of in-vitro preservation under the subsequent low-temperature weak light condition is improved.
(5) Immediately taking the normal tissue culture seedling which is subjected to the strong seedling culture in the step (4), and inoculating the normal tissue culture seedling to 1/2MS+6-BA0.02mg/L+PPP 333 In an in vitro preservation culture medium of 1.5 mg/L+0.5 g/L of activated carbon+0.05 mg/L of proline, carrying out in vitro preservation culture at a culture temperature of 21 ℃ and an illumination intensity of 1200Lx for 10 hours/day.
The medium in each step contained 25g/L sucrose, ph=5.8.
Example 10
The invention discloses an in-vitro preservation method of uncaria, which comprises the following steps:
(1) Selecting seeds with good hooked uncaria germplasm resources, which are not damaged, full in seeds, uniform in texture and free of carrying eggs for later use;
(2) Taking the seeds of the step (1), washing the seeds with sterile water for 3-5 times, then placing the seeds into 75% alcohol for sterilization for 30 seconds, then washing the seeds with sterile water for 3-5 times, then placing the seeds into 0.1% mercuric chloride solution for sterilization for 7 minutes, then washing the seeds with sterile water for 3-5 times, finally inoculating the seeds into an MS culture medium without any hormone for germination culture, wherein the culture time is about 14 days, the culture temperature is 24 ℃, and the illumination intensity is 24 DEG C1800L X The illumination time is 10 hours/day;
(3) Inoculating the aseptic seed seedlings germinated in the step (2) into a culture medium of MS+6-BA0.5mg/L+NAA0.20mg/L for propagation culture, wherein the culture temperature is 24 ℃, the culture time is about 14 days, the illumination intensity is 1800Lx, and the illumination duration is 12 hours/day; the temperature is proper, the illumination is sufficient, so that the seedlings can grow normally.
(4) Inoculating the bud obtained in the step (3) into a culture medium of MS+IBA0.5mg/L+NAA0.1mg/L+active carbon 1 g/L+proline 0.05mg/L for strong seedling culture for about 14 days; wherein the culture temperature in the first 7 days is 24 ℃, the illumination intensity is 1800Lx, and the illumination duration is 12 hours/day; the subsequent 7 days, the culture temperature is 17 ℃, the illumination intensity is 1200Lx, and the illumination duration is 10 hours/day; the propagation seedlings are firstly cultivated by proline, the proline is accumulated in the propagation seedlings in advance, the resistance is improved to cope with the smooth transition of low-temperature stimulation cultivation, and after the propagation seedlings are cultivated at low temperature and under sufficient illumination, the propagation seedlings generate a stronger resistance mechanism, so that the survival rate of in-vitro preservation under the subsequent low-temperature weak light condition is improved.
(5) Immediately taking the normal tissue culture seedling which is subjected to the strong seedling culture in the step (4), and inoculating the normal tissue culture seedling to 1/2MS+6-BA0.02mg/L+PPP 333 In an in vitro preservation culture medium of 1.5 mg/L+0.5 g/L of activated carbon+0.05 mg/L of proline, carrying out in vitro preservation culture at 17 ℃ under the illumination intensity of 1200Lx for 10 hours/day.
The medium in each step contained 25g/L sucrose, ph=5.8.
Comparative example 1
An in-vitro preservation method of uncaria comprises the following steps:
(1) And (3) taking excellent uncaria germplasm resources, and picking out seeds which are not damaged, full in seeds, uniform in texture and free of carrying eggs for later use.
(2) Taking seeds of the step (1), washing the seeds with sterile water for 3-5 times, then placing the seeds into 75% alcohol for sterilization for 30 seconds, washing the seeds with sterile water for 3-5 times, placing the seeds into 0.1% mercuric chloride solution for sterilization for 7 minutes, washing the seeds with sterile water for 3-5 times, finally inoculating the seeds into an MS culture medium without any hormone for germination culture, wherein the culture time is about 14 days, the culture temperature is 24 ℃, the illumination intensity is 1800Lx, and the illumination duration is 10 hours/day.
(3) Inoculating the aseptic seed seedlings germinated in the step (2) into a culture medium of MS+6-BA0.5mg/L+NAA0.20mg/L for propagation culture, wherein the culture temperature is 24 ℃, the culture time is about 14 days, the illumination intensity is 1800Lx, and the illumination duration is 12 hours/day; the temperature is proper, the illumination is sufficient, so that the seedlings can grow normally.
(4) Inoculating the bud obtained in the step (3) into a culture medium of MS+IBA0.5mg/L+NAA0.1mg/L+active carbon 1g/L for strong seedling culture, wherein the culture time is about 14 days; the culture temperature is 24 ℃, the illumination intensity is 1800Lx, and the illumination duration is 12 hours/day.
(5) Immediately taking the tissue culture seedling subjected to the strong seedling culture in the step (4), and inoculating the tissue culture seedling to 1/2MS+6-BA0.02mg/L+PPP 333 In an in vitro preservation culture medium of 1.5 mg/L+0.5 g/L of activated carbon+0.05 mg/L of proline, carrying out in vitro preservation culture at 17 ℃ under the illumination intensity of 1200Lx for 10 hours/day. The medium in each step contained 25g/L sucrose, ph=5.8.
Comparative example 2
An in-vitro preservation method of uncaria comprises the following steps:
(1) And (3) taking excellent uncaria germplasm resources, and picking out seeds which are not damaged, full in seeds, uniform in texture and free of carrying eggs for later use.
(2) Taking seeds of the step (1), washing the seeds with sterile water for 3-5 times, then placing the seeds into 75% alcohol for sterilization for 30 seconds, washing the seeds with sterile water for 3-5 times, placing the seeds into 0.1% mercuric chloride solution for sterilization for 7 minutes, washing the seeds with sterile water for 3-5 times, finally inoculating the seeds into an MS culture medium without any hormone for germination culture, wherein the culture time is about 14 days, the culture temperature is 24 ℃, the illumination intensity is 1800Lx, and the illumination duration is 10 hours/day.
(3) Inoculating the aseptic seed seedlings germinated in the step (2) into a culture medium of MS+6-BA0.5mg/L+NAA0.20mg/L for propagation culture, wherein the culture temperature is 24 ℃, the culture time is about 14 days, the illumination intensity is 1800Lx, and the illumination duration is 12 hours/day; the temperature is proper, the illumination is sufficient, so that the seedlings can grow normally.
(4) Inoculating the bud obtained in the step (3) into a culture medium of MS+IBA0.5mg/L+NAA0.1mg/L+active carbon 1g/L for strong seedling culture, wherein the culture time is about 14 days; the culture temperature is 17 ℃, the illumination intensity is 1800Lx, and the illumination duration is 12 hours/day.
(5) Immediately taking the tissue culture seedling subjected to the strong seedling culture in the step (4), and inoculating the tissue culture seedling to 1/2MS+6-BA0.02mg/L+PPP 333 In an in vitro preservation culture medium of 1.5 mg/L+0.5 g/L of activated carbon+0.05 mg/L of proline, carrying out in vitro preservation culture at 17 ℃ under the illumination intensity of 1200Lx for 10 hours/day. The medium in each step contained 25g/L sucrose, ph=5.8.
Comparative example 3
An in-vitro preservation method of uncaria comprises the following steps:
(1) And (3) taking excellent uncaria germplasm resources, and picking out seeds which are not damaged, full in seeds, uniform in texture and free of carrying eggs for later use.
(2) Taking seeds of the step (1), washing the seeds with sterile water for 3-5 times, then placing the seeds into 75% alcohol for sterilization for 30 seconds, washing the seeds with sterile water for 3-5 times, placing the seeds into 0.1% mercuric chloride solution for sterilization for 7 minutes, washing the seeds with sterile water for 3-5 times, finally inoculating the seeds into an MS culture medium without any hormone for germination culture, wherein the culture time is about 14 days, the culture temperature is 24 ℃, the illumination intensity is 1800Lx, and the illumination duration is 10 hours/day.
(3) Inoculating the aseptic seed seedlings germinated in the step (2) into a culture medium of MS+6-BA0.5mg/L+NAA0.20mg/L for propagation culture, wherein the culture temperature is 24 ℃, the culture time is about 14 days, the illumination intensity is 1800Lx, and the illumination duration is 12 hours/day; the temperature is proper, the illumination is sufficient, so that the seedlings can grow normally.
(4) Inoculating the bud obtained in the step (3) into a culture medium of MS+IBA0.5mg/L+NAA0.1mg/L+active carbon 1g/L for strong seedling culture, wherein the culture time is about 14 days; the culture temperature is 24 ℃, the illumination intensity is 1800Lx, and the illumination duration is 12 hours/day.
(5) Immediately taking the tissue culture seedling subjected to the strong seedling culture in the step (4), and inoculating the tissue culture seedling to 1/2MS+6-BA0.02mg/L+PPP 333 In-vitro preservation culture is carried out in an in-vitro preservation culture medium with the concentration of 1.5mg/L and 0.5g/L of activated carbon, wherein the culture temperature is 17 ℃, the illumination intensity is 1200Lx, and the illumination duration is 10 hours/day. The medium in each step contained 25g/L sucrose, ph=5.8.
Examples 1 to 10 and comparative examples 1 to 3 each statistically observed survival rate of 40 seedlings from propagation and strong seedling culture, and each statistically observed survival rate of 40 seedlings from tissue culture in-vitro preservation culture for 200 days and 400 days.
The conditions and viability of the in vitro conservation culture of examples 1 to 10 and comparative examples 1 to 3 are shown in Table 1 below:
table 1 culture conditions and viability of examples and comparative examples
/>
As can be seen from the above Table 1, the 200-day survival rate in examples 1 to 10 can reach 95% at the highest, and 77.5% at the lowest, and can meet the seedling and seed protection requirements below 200 days. The 400-day survival rates of examples 1 to 3 and examples 5 to 10 are 30% or more, and the requirements of seedling and seed conservation for up to 400 days can be met.
Examples 2 to 10 add 0.05mg/L or 0.1mg/L of proline to the strong seedling culture medium and set the culture temperature at 17℃for low-temperature stimulation 7 days after the strong seedling culture, and as a result, the survival rate of the strong seedling culture was found to be 100% which was the same as that of the strong seedling culture under the normal conditions of comparative example 1. Compared with the low-temperature stimulation comparative example 2 without adding proline, the survival rate of the strong seedling culture can be effectively improved.
Although embodiments of the invention have been disclosed above, they are not limited to the use listed in the specification and embodiments. It can be applied to various fields suitable for the present invention. Additional modifications will readily occur to those skilled in the art.
Claims (7)
1. The method for in-vitro preservation of uncaria is characterized by comprising the following steps: culturing ramulus Uncariae cum Uncis seed by aseptic germination to obtain seed seedling; the seed seedlings are subjected to propagation culture to obtain propagation seedlings; placing the propagation seedlings in a strong seedling culture medium containing 0.01-0.1 mg/L proline for strong seedling culture, wherein the strong seedling culture time is 14 days, and setting a low-temperature culture period of 15-21 ℃ 7 days after the strong seedling culture;
placing the tissue culture seedling subjected to the strong seedling culture in an in vitro culture medium for in vitro culture, wherein the in vitro culture medium contains 1/2MS+6-BA0.02mg/L+PPP 333 1.0-2.0 mg/L of active carbon, 0.5g/L of active carbon and 0.01-0.1 mg/L of proline, the culture temperature is 15-21 ℃, the illumination intensity is 1200Lx, and the illumination duration is 10 hours/day.
2. The method for preserving uncaria in vitro according to claim 1, wherein the culture temperature in the low-temperature culture period is 17 ℃, the illumination intensity is 1200-1800 lx, the illumination duration is 10-12 hours/day, and the duration is 6-8 days.
3. The method for in vitro conservation of uncaria as claimed in claim 1, wherein the first 7 days of strong seedling cultivation are normal cultivation, the cultivation temperature of normal cultivation is 24-26 ℃, the illumination intensity is 1800Lx, and the illumination time is 10-12 hours/day; the culture period is a low-temperature culture period of 7 days after normal culture, the culture temperature in the low-temperature culture period is 17 ℃, the illumination intensity is 1200-1800 Lx, and the illumination duration is 10-12 hours/day.
4. The method for preserving uncaria in vitro according to claim 2 or 3, wherein the culture temperature in the low-temperature culture period is 17 ℃, the illumination intensity is 1800Lx, and the illumination duration is 12 hours/day.
5. The method for preserving uncaria according to claim 1, wherein the strong culture medium contains ms+iba0.5mg/l+naa0.1 mg/l+activated carbon 1 g/l+proline 0.05mg/L.
6. The method for preserving uncaria in vitro according to claim 1, 3 or 5, wherein the culture medium in vitro is 1/2MS+6-BA0.02mg/L+PPP 333 1.5 mg/L+0.5 g/L of activated carbon+0.05/mg/L of proline, the culture temperature is 17 ℃, the illumination intensity is 1200Lx, and the illumination duration is 10 hours/day.
7. The method for preserving uncaria in vitro according to claim 1, comprising the steps of:
s1: taking uncinate ramulus Uncariae cum Uncis seeds with no damage, plump seeds, uniform texture and no carrier of insect eggs;
s2: taking seeds of S1, washing the seeds for 3-5 times by using sterile water, then placing the seeds into 75% alcohol for sterilization for 30 seconds, washing the seeds for 3-5 times by using sterile water, then placing the seeds into 0.1% mercuric chloride solution for sterilization for 7 minutes, washing the seeds for 3-5 times by using sterile water, finally inoculating the seeds into an MS culture medium without any hormone, and carrying out germination culture to obtain sterile seedlings, wherein the culture time is 14 days, the culture temperature is 24 ℃, the illumination intensity is 1800Lx, and the illumination duration is 10 hours/day;
s3: inoculating the seed seedling in S2 into a culture medium containing MS+6-BA0.5mg/L+NAA0.2mg/L for propagation culture, wherein the culture temperature is 24 ℃, the illumination intensity is 1800Lx, and the illumination duration is 12 hours/day;
s4: inoculating the bud in S3 into a seedling strengthening culture medium of MS+IBA0.5mg/L+NAA0.1mg/L+active carbon 1 g/L+proline 0.01-0.1 mg/L for seedling strengthening culture; the culture temperature in the early stage of strong seedling culture is 24-26 ℃, the illumination intensity is 1800Lx, and the illumination time is 12 hours/day; in the last 6-8 days of strong seedling cultivation, the cultivation temperature is 15-21 ℃, the illumination intensity is 1200-1800 Lx, and the illumination time is 12 hours/day;
s5: taking normal tissue culture seedling of S4, inoculating to 1/2MS+6-BA0.02mg/L+PPP 333 In-vitro preservation culture is carried out in an in-vitro preservation culture medium with the concentration of 1.0-2.0 mg/L, 0.5g/L of activated carbon and 0.01-0.1 mg/L of proline, the culture temperature is 15-21 ℃, the illumination intensity is 1200Lx, and the illumination duration is 10 hours/day.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310852641.9A CN116941528B (en) | 2023-07-12 | 2023-07-12 | Method for in-vitro preservation of uncaria |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310852641.9A CN116941528B (en) | 2023-07-12 | 2023-07-12 | Method for in-vitro preservation of uncaria |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116941528A CN116941528A (en) | 2023-10-27 |
| CN116941528B true CN116941528B (en) | 2024-04-09 |
Family
ID=88459680
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310852641.9A Active CN116941528B (en) | 2023-07-12 | 2023-07-12 | Method for in-vitro preservation of uncaria |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116941528B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116941530A (en) * | 2023-07-28 | 2023-10-27 | 广西壮族自治区药用植物园 | In-vitro preservation method of artemisia annua |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107873516A (en) * | 2017-11-14 | 2018-04-06 | 广西壮族自治区药用植物园 | The breeding method of yncaria stem with hooks |
| CN108834894A (en) * | 2018-07-10 | 2018-11-20 | 云南省农业科学院药用植物研究所 | A kind of method for tissue culture of uncaria |
-
2023
- 2023-07-12 CN CN202310852641.9A patent/CN116941528B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107873516A (en) * | 2017-11-14 | 2018-04-06 | 广西壮族自治区药用植物园 | The breeding method of yncaria stem with hooks |
| CN108834894A (en) * | 2018-07-10 | 2018-11-20 | 云南省农业科学院药用植物研究所 | A kind of method for tissue culture of uncaria |
Non-Patent Citations (1)
| Title |
|---|
| 钩藤生根壮苗培养基优化及移栽基质的筛选;潘丽梅等;《江苏农业科学》;20141125;第42卷(第11期);第83-85页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116941528A (en) | 2023-10-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN116941528B (en) | Method for in-vitro preservation of uncaria | |
| CN111886961B (en) | Application of gamma-aminobutyric acid as dormancy breaking promoter in breaking seed dormancy | |
| CN113331055A (en) | Cutting method of stingless pepper tissue culture seedlings | |
| CN104871679A (en) | Balance dormancy breaking and seedling cultivation method of cerasus campanulata seeds | |
| CN110150147B (en) | Tissue culture and rapid propagation method for populus euphratica | |
| CN113875749B (en) | Ultralow temperature preservation method for tamarix seeds | |
| CN105475129A (en) | Tissue-culture rapid propagation method for arundina graminifolia | |
| CN111226796B (en) | Sugar-free tissue culture method for dendrobium officinale | |
| CN107278414B (en) | Method for promoting germination of rare endangered plant water chestnut seeds | |
| CN102919122A (en) | High-efficiency method for inducing pinellia in vitro bulb | |
| CN102210229B (en) | Non-test tube rapid propagation method of Chinese cymbidium | |
| CN103155866B (en) | Malus zumi tissue culture rapid propagation seedling raising method | |
| CN111202002B (en) | Tissue culture and rapid propagation method of clerodendrum japonicum | |
| CN111919751B (en) | Tissue culture method for murraya paniculata seeds | |
| CN108521929B (en) | Application of ascorbic acid as dormancy breaking promoter in breaking seed dormancy | |
| CN111771450A (en) | Low-temperature preservation process for taxus mairei seeds | |
| CN105993871A (en) | Rapid seedling-raising method capable of promoting high germination rate of pseudo-ginseng by utilizing acremonium strictum | |
| CN111802380A (en) | Ultralow-temperature in-vitro preservation method for stem tips of dormant buds of peaches | |
| CN110999914A (en) | Chinese medicinal material seed pretreatment agent and application thereof | |
| CN104170738A (en) | Rapid propagation method of acer ginnala maxim. | |
| QingYuan et al. | Germination of Azolla filiculoides Lam. sporocarps and factors affecting their growth | |
| CN110432153B (en) | Summer-autumn tissue culture rapid propagation method for alpine rhododendron | |
| CN115918534B (en) | Method for establishing soybean fir embryo rapid propagation system | |
| CN107548991B (en) | A kind of inoculation method of lily test tube bulbs | |
| CN102405834B (en) | Rapid propagation method of Tianjiao |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant |