CN111616053B - Curcuma wenyujin embryonic callus induction and plant regeneration method - Google Patents
Curcuma wenyujin embryonic callus induction and plant regeneration method Download PDFInfo
- Publication number
- CN111616053B CN111616053B CN202010522942.1A CN202010522942A CN111616053B CN 111616053 B CN111616053 B CN 111616053B CN 202010522942 A CN202010522942 A CN 202010522942A CN 111616053 B CN111616053 B CN 111616053B
- Authority
- CN
- China
- Prior art keywords
- culture medium
- curcuma wenyujin
- callus
- culture
- sterile
- 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
- 206010020649 Hyperkeratosis Diseases 0.000 title claims abstract description 76
- 241000963390 Curcuma wenyujin Species 0.000 title claims abstract description 72
- 235000003394 Curcuma wenyujin Nutrition 0.000 title claims abstract description 72
- 230000006698 induction Effects 0.000 title claims abstract description 49
- 241000196324 Embryophyta Species 0.000 title claims abstract description 21
- 238000011069 regeneration method Methods 0.000 title abstract description 24
- 239000001963 growth medium Substances 0.000 claims abstract description 44
- 230000004069 differentiation Effects 0.000 claims abstract description 29
- 230000000408 embryogenic effect Effects 0.000 claims abstract description 19
- 238000000034 method Methods 0.000 claims abstract description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 15
- 238000005406 washing Methods 0.000 claims abstract description 14
- 238000004659 sterilization and disinfection Methods 0.000 claims abstract description 12
- 230000001939 inductive effect Effects 0.000 claims abstract description 7
- 239000008223 sterile water Substances 0.000 claims abstract description 5
- 230000001172 regenerating effect Effects 0.000 claims abstract description 4
- 238000005286 illumination Methods 0.000 claims description 24
- 238000012258 culturing Methods 0.000 claims description 16
- 239000007864 aqueous solution Substances 0.000 claims description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 8
- 238000002791 soaking Methods 0.000 claims description 8
- 230000001954 sterilising effect Effects 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 5
- 239000005708 Sodium hypochlorite Substances 0.000 claims description 4
- 239000011521 glass Substances 0.000 claims description 4
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 claims description 4
- 230000008929 regeneration Effects 0.000 abstract description 22
- 238000001784 detoxification Methods 0.000 abstract description 7
- 238000004161 plant tissue culture Methods 0.000 abstract description 3
- 239000002609 medium Substances 0.000 description 24
- 244000163122 Curcuma domestica Species 0.000 description 22
- 235000003392 Curcuma domestica Nutrition 0.000 description 15
- 235000003373 curcuma longa Nutrition 0.000 description 11
- VFLDPWHFBUODDF-FCXRPNKRSA-N curcumin Chemical compound C1=C(O)C(OC)=CC(\C=C\C(=O)CC(=O)\C=C\C=2C=C(OC)C(O)=CC=2)=C1 VFLDPWHFBUODDF-FCXRPNKRSA-N 0.000 description 8
- 235000013976 turmeric Nutrition 0.000 description 8
- 230000000052 comparative effect Effects 0.000 description 7
- 240000009138 Curcuma zedoaria Species 0.000 description 6
- 235000003405 Curcuma zedoaria Nutrition 0.000 description 6
- 239000001812 curcuma zedoaria berg. rosc. Substances 0.000 description 6
- 239000012153 distilled water Substances 0.000 description 6
- 239000006870 ms-medium Substances 0.000 description 6
- 235000019509 white turmeric Nutrition 0.000 description 6
- 235000014375 Curcuma Nutrition 0.000 description 5
- 230000009286 beneficial effect Effects 0.000 description 5
- 239000003864 humus Substances 0.000 description 5
- 229920001817 Agar Polymers 0.000 description 4
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 4
- 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 4
- 229930006000 Sucrose Natural products 0.000 description 4
- 239000008272 agar Substances 0.000 description 4
- 238000009395 breeding Methods 0.000 description 4
- 230000001488 breeding effect Effects 0.000 description 4
- 235000020415 coconut juice Nutrition 0.000 description 4
- 230000002068 genetic effect Effects 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 239000005720 sucrose Substances 0.000 description 4
- 229930003231 vitamin Natural products 0.000 description 4
- 239000011782 vitamin Substances 0.000 description 4
- 229940088594 vitamin Drugs 0.000 description 4
- 235000013343 vitamin Nutrition 0.000 description 4
- 230000012010 growth Effects 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 230000009466 transformation Effects 0.000 description 3
- 239000000341 volatile oil Substances 0.000 description 3
- 235000003398 Curcuma aromatica Nutrition 0.000 description 2
- 244000008991 Curcuma longa Species 0.000 description 2
- 241000234299 Zingiberaceae Species 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 238000012136 culture method Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 230000035699 permeability Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 241000589158 Agrobacterium Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 206010008342 Cervix carcinoma Diseases 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 208000025865 Ulcer Diseases 0.000 description 1
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 201000010881 cervical cancer Diseases 0.000 description 1
- 239000001215 curcuma longa l. root Substances 0.000 description 1
- 235000012754 curcumin Nutrition 0.000 description 1
- 229940109262 curcumin Drugs 0.000 description 1
- 239000004148 curcumin Substances 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- VFLDPWHFBUODDF-UHFFFAOYSA-N diferuloylmethane Natural products C1=C(O)C(OC)=CC(C=CC(=O)CC(=O)C=CC=2C=C(OC)C(O)=CC=2)=C1 VFLDPWHFBUODDF-UHFFFAOYSA-N 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000003337 fertilizer Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000012737 fresh medium Substances 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 229910001385 heavy metal Inorganic materials 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000002398 materia medica Substances 0.000 description 1
- 230000007721 medicinal effect Effects 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 230000008635 plant growth Effects 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 229930000044 secondary metabolite Natural products 0.000 description 1
- 229930004725 sesquiterpene Natural products 0.000 description 1
- -1 sesquiterpene compound Chemical class 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000002352 surface water Substances 0.000 description 1
- 239000008399 tap water Substances 0.000 description 1
- 235000020679 tap water Nutrition 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 239000010681 turmeric oil Substances 0.000 description 1
- 231100000397 ulcer Toxicity 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
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
- A01H4/008—Methods for regeneration to complete plants
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a method for inducing embryonic callus of curcuma wenyujin and regenerating plants, belonging to the technical field of plant tissue culture. The method comprises the following steps: (1) taking sprouts on the tubers of the curcuma wenyujin, washing the sprouts with sterile water after disinfection, and then inoculating the sprouts into a sprout culture medium to culture so as to obtain sterile seedlings of the curcuma wenyujin; (2) transferring the sterile seedlings to a cluster bud induction culture medium for culture to obtain sterile cluster seedlings; (3) taking the root base part of the sterile cluster seedling as an explant, inoculating the explant into a callus induction culture medium, and carrying out dark culture to obtain Curcuma wenyujin embryonic callus; (4) inoculating the Curcuma wenyujin embryonic callus to a differentiation culture medium for culture to obtain a Curcuma wenyujin regeneration plant. The method adopts multi-step disinfection on the curcuma wenyujin sprouts to realize quick and effective detoxification; the embryogenic callus with good state is a key step of differentiation and regeneration, and the generation rate of the embryogenic callus induced by the callus induction culture medium provided by the invention reaches about 95%.
Description
Technical Field
The invention belongs to the technical field of plant tissue culture, and particularly relates to a method for inducing Curcuma wenyujin embryonic callus and regenerating a plant.
Background
The curcuma wenyujin (wen zedoary) is a perennial herb of curcuma genus of zingiberaceae family, has a long medicinal history, and is recorded in tang "medical property treatise" and li shizhen "compendium of materia medica: curcumae rhizoma has the actions of moving qi and breaking blood, resolving food stagnation and alleviating pain, and is called qi-flowing herb in blood. In modern medical research and clinical application in China, the curcuma zedoary is an effective anticancer traditional Chinese medicine, and the main components of the curcuma zedoary are volatile oil, curcumin, polysaccharide and the like. Has better curative effect on preventing and treating malignant tumors, particularly early cervical cancer. The volatile oil is mainly sesquiterpene compound, and belongs to secondary metabolite of plant. The zedoary volatile oil has the capacity of resisting tumor, ulcer and virus in clinical treatment.
The production of the common turmeric (common turmeric) basically follows the traditional conventional breeding, seed reproduction and cultivation technical measures, and besides having obvious resource advantages, the differences between the breeding and reproduction technology, the standardized and standardized cultivation technology and the control aspects of heavy metals, pesticides, fertilizers and the like and the international advanced level still exist. The most outstanding problem is that asexual propagation is adopted, about one fifth of the root tuber of the curcuma wenyujin dug every year is reserved as the stock for plant growth in the second year, which not only affects the yield, but also causes obvious phenomenon of variety degradation, slow seed reproduction speed, and simultaneously can not effectively detoxify, the ingredient and content of the produced zedoary turmeric are unstable, and the safety and effectiveness of clinical medication are affected to a certain extent.
In order to solve the problems of low yield, serious variety degeneration and ineffective detoxification of common turmeric breeding, a plurality of researchers in China realize detoxification, rapid propagation and excellent variety screening of common turmeric through a tissue culture method. The detoxification and rapid propagation of the curcuma wenyujin are realized by respectively utilizing a tissue culture method in a Maobizeng professor of Zhejiang university and a laboratory of Fuyun Xin professor; meanwhile, Wenzhou medical college Limin and the like realize high yield of the Curcuma wenyujin by improving each link in the traditional Curcuma wenyujin field planting mode. Although the above means realizes the rapid propagation of the curcuma wenyujin and the screening of the excellent variety, the content of the effective component zedoary turmeric oil of the curcuma wenyujin is low, and the market demand can not be met.
At present, there are few reports about the establishment of a Curcuma wenyujin tissue differentiation and regeneration system. The Wenzhou medical college Wang Xiaohui et al report the callus induction, differentiation and regeneration system of the Curcuma wenyujin, but the callus induction rate of the system is low, and the differentiation and regeneration are difficult to repeat.
Curcuma wenyujin is a plant of the genus Curcuma of the family Zingiberaceae, and a plurality of reports are provided on the establishment of a tissue differentiation, regeneration and genetic transformation system of Curcuma longa at present, for example, Zhang Shijun et al establish a tissue differentiation and regeneration system of Curcuma longa; he Ruifeng and the like realize the agrobacterium transfection and differentiation regeneration of turmeric callus and establish a genetic transformation system of turmeric; phan Van Tan realizes the tissue differentiation and regeneration of the turmeric by adjusting the formula of the MS basic culture medium and adding the conditions of active carbon, humus and the like; mohanty, S reported the tissue differentiation regeneration system of curcuma aromatica. Because the Chinese medicinal materials have obvious difference in genuine property, even if the turmeric is used together, the culture medium conditions for realizing a tissue differentiation and regeneration system are also greatly different due to different material production places.
The radix curcumae is a famous medicinal material in Zhejiang, and research shows that the effective component content of the radix curcumae from the production area of the Renan Yunjiang in Wenzhou is the highest. The establishment of a high-efficiency technical system for induction, multiplication and plant regeneration of Curcuma wenyujin embryonic callus is a problem to be solved by the technical personnel in the field.
Disclosure of Invention
The invention aims to provide a technical method for induction of embryogenic callus of common turmeric and plant regeneration, which meets the breeding production requirement of common turmeric and lays a foundation for establishment and genetic improvement of a common turmeric genetic transformation system.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for inducing Curcuma wenyujin embryonic callus and regenerating plant comprises the following steps:
(1) taking buds on the Curcuma wenyujin tubers, washing the buds with sterile water after disinfection, and then inoculating the buds into a bud culture medium to culture to obtain Curcuma wenyujin aseptic seedlings, wherein the bud culture medium is an MS culture medium added with PPM (0.1-0.2% by volume) and 6-BA (1.0-4.0 mg/L);
(2) transferring the sterile seedlings to a cluster bud induction culture medium for culture to obtain sterile cluster seedlings, wherein the cluster bud induction culture medium is an MS culture medium added with 1.0-4.0 mg/L6-BA;
(3) taking a root base part of the sterile clump seedling as an explant, inoculating the root base part into a callus induction culture medium, and performing dark culture to obtain Curcuma wenyujin embryonic callus, wherein the callus induction culture medium is obtained by adding 0.2-1 mg/L of 6-BA and 0.5-1 mg/L of 2 and 4-D into an MS culture medium;
(4) and inoculating the Curcuma wenyujin embryonic callus into a differentiation culture medium for culturing to obtain a Curcuma wenyujin regeneration plant, wherein the differentiation culture medium is an MS culture medium added with 1-3 mg/L of 6-BA and 0.5-2 mg/L of NAA.
Specifically, the Curcuma wenyujin comes from GAP demonstration planting base of Curcuma wenyujin in Reian, Wenzhou, Zhejiang.
In the step (1), a rapid and effective Curcuma wenyujin sprout detoxification method is provided.
The tuber of Curcuma wenyujin Y.H.Chen et C.Ling naturally sprouts at 25 deg.C, and the sprouts about 1.5cm in length are cut off, and the sprouts are slightly provided with tuber tissue, which is beneficial to inoculation and survival of sprouts.
The buds are washed by running water and then disinfected by 0.1g/L of Yipeiling aqueous solution, 50% of plant tissue culture bacteriostat PPM aqueous solution, 75% of ethanol and 3.78% of sodium hypochlorite aqueous solution in sequence. Specifically, soaking the buds in 0.1g/L YIBEILING aqueous solution, shaking for 5min, repeating for 2-3 times, and shaking overnight; soaking the buds in PPM aqueous solution with volume ratio of 50% for disinfection for 10 min; soaking the buds in 75% ethanol, and sterilizing for 2-3 min; soaking the sprouts in 3.78% sodium hypochlorite water solution for disinfection for 15 min. After each step of disinfection treatment, shake washing with sterile water for 2-4 times, 5min each time.
After the disinfection is finished, the surface water of the sprouts is sucked dry, and then the sprouts are inoculated in a sprout culture medium for aseptic culture, preferably, the sprout culture medium is MS culture medium added with PPM with the volume ratio of 0.15 percent and 6-BA with the volume ratio of 3.0 mg/L.
The culture of Curcuma wenyujin aseptic seedling is carried out in two steps, firstly, culturing under weak light condition which is not beneficial to bacterial growth, but because of insufficient light, Curcuma wenyujin bud and growth speed are fastThe temperature is slow; after two weeks, the determined pollution-free sprouts are transferred to normal illumination conditions for culture, and a batch of strong turmeric root tuber sterile seedlings can be obtained. Specifically, the method comprises the following steps: firstly, placing the mixture in a place with the temperature of 25-28 ℃, the illumination time of 6 h/dark for 18h and the illumination intensity of 30-40 mu mol-2.s-1Culturing for 10-14 days under the condition; the second step, the mixture is placed at a temperature of between 25 and 28 ℃, and the illumination is carried out for 12 hours/dark for 12 hours, and the illumination intensity is 50 to 60 mu mol-2.s-1Culturing for 10-14 days under the condition.
In the step (2), a method for inducing clustered shoots of curcuma aromatica is provided.
Cutting off the root and leaf parts of sterile seedlings, inoculating the sterile seedlings in a cluster bud induction culture medium, and culturing and inducing for 4 weeks under normal illumination to obtain sterile cluster seedlings of Curcuma wenyujin.
Preferably, the cluster bud induction medium is MS medium supplemented with 3.0 mg/L6-BA.
The culture conditions of the sterile cluster seedlings are as follows: 25-28 ℃, 12h of illumination/12 h of darkness and 50-60 mu mol.m of illumination intensity-2.s-1Culturing for 25-30 days under the condition.
In the step (3), an induction method of Curcuma wenyujin embryonic callus is provided.
Cutting off the leaf, root and rhizome combination part of the Curcuma wenyujin cluster seedling, reserving about 1.5cm of root base part, inoculating the root base part into a callus induction culture medium, and carrying out dark culture to obtain Curcuma wenyujin embryonic callus.
Preferably, the callus induction medium is MS medium supplemented with 0.5mg/L of 6-BA and 0.5mg/L of 2, 4-D.
The container of the callus induction culture medium is a glass culture dish. The glass culture dish has certain air permeability and is beneficial to the generation of embryogenic callus.
The culture conditions of the Curcuma wenyujin embryonic callus are as follows: culturing in dark at 25-28 ℃ for 45-60 days.
In the step (4), a method for differentiation and regeneration of Curcuma wenyujin embryonic callus is provided.
Selecting light yellow granular Curcuma wenyujin embryonic callus, inoculating in a differentiation culture medium, and culturing under normal illumination to obtain a regeneration plant.
Preferably, the differentiation medium is MS medium supplemented with 2 mg/L6-BA and 1mg/L NAA.
The culture conditions of the Curcuma wenyujin regenerated plant are 25-28 ℃, the illumination is 12 h/dark 12h, and the illumination intensity is 50-60 mu mol-2.s-1Culturing for 60-90 days under the condition.
The invention has the following beneficial effects:
(1) the method adopts multi-step disinfection on the curcuma wenyujin sprouts to realize quick and effective detoxification.
(2) The embryogenic callus with good state is a key step of differentiation and regeneration, and the generation rate of the embryogenic callus induced by the callus induction culture medium provided by the invention reaches about 95%.
(3) Under the differentiation and regeneration conditions provided by the invention, the Curcuma wenyujin regenerated plant is successfully obtained.
Drawings
FIG. 1 shows that sprouts growing from tubers of Curcuma wenyujin Y.H. Cheng et C.Ling, which are naturally placed at about 25 deg.C in 5, 6, and 7 months, grow to about 1.5cm, and are cut off for detoxification treatment.
FIG. 2 shows sterilized shoots of Curcuma wenyujin Y.H.Chen et C.Ling obtained by multi-step sterilization, inoculated into MS medium containing 0.15% PPM (v/v) +3.0 mg/L6-BA, and cultured at 25 deg.C under low light for two weeks.
FIG. 3 shows the sterile seedlings of Curcuma wenyujin Y.H.Chen et C.Ling obtained by transferring the sterile seedlings of Curcuma wenyujin Y.H.Chen et C.Ling to cluster bud induction medium (MS +3.0 mg/L6-BA) and performing induction culture in a normal light incubator at 25 deg.C for 4 weeks.
FIG. 4 shows the isolation of individual sterile clumped seedlings, which were cut to remove the leaf, root, and rhizome junctions, and then inoculated with approximately 1.5cm long root roots into callus induction medium (MS +0.5 mg/L6-BA +0.5 mg/L2, 4-D).
FIG. 5 shows the induction of callus after about 50 days of induction of Curcuma wenyujin root explant in callus induction medium, the pale yellow granular callus pointed by the arrow is embryogenic callus.
FIG. 6 shows that the callus of Curcuma wenyujin Y.H.Chen et C.Ling is induced in MS +0.55mg/L TDZ +0.45 mg/L6-BA +0.27 mg/L2, 4-D callus induction medium for about 50 days to obtain non-embryogenic callus.
FIG. 7 shows non-embryogenic callus obtained by inducing Curcuma wenyujin Y.H.Chen et C.Ling explant in MS +2 mg/L2, 4-D +0.5mg/L KT callus induction medium for about 50 days.
FIG. 8 shows that Curcuma wenyujin Y.H.Chen et H.Chen et H.Chan explant is induced in 1/4MS macroelement + MS microelement + vitamins + 10% coconut juice +25g/L sucrose +1ml/L humus +7.5g/L agar +1mg/L TDZ +1 mg/L6-BA +0.5 mg/L2, 4-D callus induction medium for about 50 days to obtain non-embryogenic callus.
FIG. 9 shows the regeneration plant obtained after the Curcuma wenyujin embryonic callus of the present invention is placed in the differentiation medium for about 70 days of differentiation regeneration.
FIG. 10 shows the callus state of Curcuma wenyujin embryonic callus that was not successfully differentiated into regenerated plants in differentiation medium (1/4MS macroelements + MS microelements + vitamins + 10% coconut juice +25g/L sucrose +2ml/L humus +7.5g/L agar +1mg/L TDZ +1 mg/L6-BA +0.5 mg/L2, 4-D +2mg/L KT +2mg/L NAA +2.0g/L activated carbon).
Detailed Description
The present invention will be further described with reference to the following examples, but the present invention is not limited thereto.
In the following cases, the tuber of Curcuma wenyujin Y.H.Cheng an Curcuma wenyujin G.G. GAP demonstration plantation base in Wen Jiang.
MS medium was purchased by Qingdao Haibo Biotechnology, Inc.
Example 1
(1) The Curcuma wenyujin tubers are naturally placed in a laboratory at the room temperature of about 25 ℃, and the months of 5, 6 and 7 per year are the seasons with the tubers sprouting most.
Sprouts of about 1.5cm in length are cut with a scalpel, taking care that the cut is slightly accompanied by part of tuber tissue, which is more beneficial to the inoculation and survival of the sprouts thereafter.
(2) Washing the cut buds with tap water to remove soil and dirt, placing the buds in a 50ml sterile centrifuge tube, shaking and washing the buds with sterile distilled water on a horizontal shaking table for 2 times, then shaking and washing the buds with sterile distilled water containing 0.1g/L of Yipeiling for 2 times, and shaking and washing the buds overnight for the third time; shaking and washing with sterile distilled water for 3 times the next day, then sterilizing with sterile water containing 50% PPM (v/v) for 10min, and shaking and washing with sterile distilled water for 3 times; sterilizing with 75% ethanol for 2-3min, and washing with sterile distilled water for 4 times; and finally, sterilizing in a 3.78% sodium hypochlorite aqueous solution for 15min, and washing with sterile distilled water for 4 times, wherein the washing with shaking is 5 min/time.
Finally, the surface of the sprouts was blotted up with sterile filter paper, inoculated into MS medium containing 0.15% PPM (v/v) +3.0 mg/L6-BA, and placed at 25 deg.C, light/dark (6h/18h), light intensity of 30 μmol-2.s-1The artificial climate incubator is used for culturing under the condition of weak illumination.
After two weeks, a batch of Curcuma wenyujin aseptic sprouts (figure 2) can be obtained, the weak light culture condition is not favorable for the growth of bacteria, but the Curcuma wenyujin sprouts are yellow and grow slowly due to insufficient light; then, the illumination conditions of the incubator were adjusted to light/dark (12h/12h) and the illumination intensity was 60. mu. mol.m-2.s-1And culturing for two weeks to obtain a batch of strong Curcuma wenyujin aseptic seedlings.
(3) Transferring the obtained sterile seedlings to fresh cluster bud induction medium (MS +3.0 mg/L6-BA), and standing at 25 deg.C, light/dark (2h/12h), and illumination intensity of 60 μmol.m-2.s-1The cultivation box (2) is used for about 4 weeks, and a batch of sterile clumpy seedlings can be obtained (figure 3).
(4) The sterile seedling individual obtained above was isolated, then the leaf, root and rhizome junctions of the seedlings were cut off, the root base approximately 1.5cm long was used as an explant, inoculated on callus induction medium (MS +0.5 mg/L6-BA +0.5 mg/L2, 4-D) (FIG. 4), placed in a dark incubator at 25 ℃ for approximately 50 days to obtain Curcuma wenyujin embryonic callus (FIG. 5). According to our experience, the callus induction medium is preferably poured into a glass culture dish, and a disposable plastic culture dish is likely to cause water drops to gather on the surface due to poor air permeability caused by the fact that the upper surface and the lower surface are sealed, and finally the moisture on the surface of an explant is high, so that the embryogenic callus is not generated.
(5) The obtained pale yellow granular embryogenic callus is placed in a differentiation medium (MS +2 mg/L6-BA +1mg/L NAA) with 25 deg, light/dark (12h/12h) and illumination intensity of 60 μmol-2.s-1After the culture box is differentiated and regenerated for about 70 days, the regenerated plant of the curcuma wenyujin is obtained (figure 9).
Comparative example 1
In the callus induction medium search, referring to the turmeric callus induction medium formulation published by Zhang Shijun et al (MS +0.55mg/L TDZ +0.45 mg/L6-BA +0.27 mg/L2, 4-D), under the same culture conditions as in example 1, after about 50 days, a large number of milky, finely divided, snowflake-like (FIG. 6) non-embryogenic calli were formed around the explants.
Comparative example 2
In the callus induction medium search, a batch of vitrified, brownish non-embryogenic calli was obtained after about 50 days under the same culture conditions as in example 1, with reference to the turmeric callus induction medium formulation (MS +2 mg/L2, 4-D +0.5mg/L KT) reported by Mohanty, S (FIG. 7).
The calli of the two types obtained in comparative example 1 and comparative example 2 were severely hydrated, browned and gradually died during the subsequent subculture.
Comparative example 3
In the callus induction culture medium search, the callus induction amount was small and the callus was browned after about 50 days under the same culture conditions as in example 1 with reference to the modified Curcuma rhizome callus induction culture medium reported by Phan Van Tan (1/4MS macroelement + MS microelement + vitamin + 10% coconut juice +25g/L sucrose +1ml/L humus +7.5g/L agar +1mg/L TDZ +1 mg/L6-BA +0.5 mg/L2, 4-D) (FIG. 8).
Comparative example 4
In the process of searching for the callus differentiation medium, referring to a Curcuma wenyujin callus differentiation medium formula MS +5mg/L KT +0.3mg/L NAA published by Wangxiao Hui et al of Wenzhou medical college, embryonic callus is differentiated on the medium for about 70 days and even is prolonged to 100 days, no regeneration plant is generated.
Comparative example 5
In the process of searching for a callus differentiation medium, referring to a formula of a Phan Van Tan improved turmeric differentiation medium (1/4MS macroelements + MS microelements + vitamins + 10% coconut juice +25g/L sucrose +2ml/L humus +7.5g/L agar +1mg/L TDZ +1 mg/L6-BA +0.5 mg/L2, 4-D +2mg/L KT +2mg/L NAA +2.0g/L active carbon), embryogenic callus is differentiated and regenerated for about 70 days, a large amount of green tissues are generated on the surface (figure 10), the embryogenic callus is transferred to a fresh medium to be continuously differentiated and regenerated for about 150 days, and no regenerated plant is generated all the time.
The research result of the invention shows that the embryogenic callus with good state is the key step of differentiation and regeneration, which is the basis of success or failure, and compared with the reported Curcuma callus induction culture medium, in the induction aspect of Curcuma wenyujin embryonic callus, the induction rate of the obtained induction formula embryogenic callus reaches about 95%, and the induction culture medium has obvious advantages.
Claims (1)
1. A method for inducing embryogenic callus and regenerating plants of Curcuma wenyujin from GAP demonstration plantation of Curcuma wenyujin in Renaan, Wenzhou, is characterized by comprising the following steps:
(1) picking buds on tubers of the Curcuma wenyujin, wherein the picked buds have tuber tissues, washing the picked buds by flowing water, sequentially soaking the buds in 0.1g/L Yipeiling aqueous solution, shaking for 5min, repeating for 2-3 times, and then shaking overnight; soaking the buds in PPM aqueous solution with volume ratio of 50% for disinfection for 10 min; soaking the buds in 75% ethanol, and sterilizing for 2-3 min; soaking the buds in 3.78% sodium hypochlorite aqueous solution for disinfection for 15min, and washing the buds with sterile water for 5min each time for 2-4 times after each step of disinfection treatment; then inoculating the curcuma wenyujin sterile seedlings in a bud culture medium to be cultured, wherein the bud culture medium is MS culture medium added with PPM (PPM) with the volume ratio of 0.15% and 6-BA with the volume ratio of 3.0 mg/L;
the culture of the Curcuma wenyujin aseptic seedlings is carried out in two steps, wherein in the first step, the Curcuma wenyujin aseptic seedlings are placed at the temperature of 25-28 ℃, the illumination time is 6 h/dark 18h, and the illumination intensity is 30-40 mu mol.m-2.s-1Culturing for 10-14 days under the condition; in the second step, the first step is that,placing the mixture in a place with the temperature of 25-28 ℃, the illumination time of 12 h/dark time of 12h and the illumination intensity of 50-60 mu mol-2.s-1Culturing for 10-14 days under the condition;
(2) after cutting off root and leaf parts, transferring the sterile seedlings to a cluster bud induction culture medium for culture, wherein the culture conditions are as follows: 25-28 ℃, 12h of illumination/12 h of darkness and 50-60 mu mol.m of illumination intensity-2.s-1Culturing for 25-30 days under the condition to obtain sterile cluster seedlings, wherein a cluster bud induction culture medium is an MS culture medium added with 3.0 mg/L6-BA;
(3) taking a root base part of the sterile clump seedlings as an explant, inoculating the explant into a callus induction culture medium, wherein a container of the callus induction culture medium is a glass culture dish, and carrying out dark culture at the temperature of 25-28 ℃ for 45-60 days to obtain Curcuma wenyujin embryonic callus, and the callus induction culture medium is an MS culture medium added with 0.5mg/L of 6-BA and 0.5mg/L of 2 and 4-D;
(4) inoculating Curcuma wenyujin embryonic callus into differentiation culture medium for culturing under the conditions of 25-28 ℃, 12h of illumination/12 h of darkness and 50-60 mu mol.m of illumination intensity-2.s-1Culturing for 60-90 days under the condition to obtain a Curcuma wenyujin regenerated plant, wherein the differentiation culture medium is MS culture medium added with 2 mg/L6-BA and 1mg/L NAA.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010522942.1A CN111616053B (en) | 2020-06-10 | 2020-06-10 | Curcuma wenyujin embryonic callus induction and plant regeneration method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010522942.1A CN111616053B (en) | 2020-06-10 | 2020-06-10 | Curcuma wenyujin embryonic callus induction and plant regeneration method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111616053A CN111616053A (en) | 2020-09-04 |
| CN111616053B true CN111616053B (en) | 2022-03-25 |
Family
ID=72267405
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010522942.1A Active CN111616053B (en) | 2020-06-10 | 2020-06-10 | Curcuma wenyujin embryonic callus induction and plant regeneration method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111616053B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107410032A (en) * | 2017-09-26 | 2017-12-01 | 杭州师范大学 | A kind of method of RADIX CURCUMAE Regenerated Plantlets |
| CN109463279A (en) * | 2018-12-10 | 2019-03-15 | 绍兴文理学院 | A kind of RADIX CURCUMAE takes off the fast breeding technique of vaccine |
-
2020
- 2020-06-10 CN CN202010522942.1A patent/CN111616053B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107410032A (en) * | 2017-09-26 | 2017-12-01 | 杭州师范大学 | A kind of method of RADIX CURCUMAE Regenerated Plantlets |
| CN109463279A (en) * | 2018-12-10 | 2019-03-15 | 绍兴文理学院 | A kind of RADIX CURCUMAE takes off the fast breeding technique of vaccine |
Non-Patent Citations (6)
| Title |
|---|
| 3种抑菌剂在黄槐组培中的抑菌效果研究;安冰;《现代农业科技》;20181231(第2期);第147-148页 * |
| Characterization of Secondary Metabolites of an Endophytic Fungus from Curcuma wenyujin;Jvfen Yan等;《Current Microbiology》;20141231;第69卷;第740-744页 * |
| PPM抗菌剂—广谱型植物组培抗微生物剂Plant Preservative Mixture;aini3311;《每日生物评论》;20140724;www.bio-review.com/ppm-plant-preservative-mixture/ * |
| 温郁金愈伤组织培养及快速繁殖;王晓慧等;《北方园艺》;20081231(第10期);第143-146页 * |
| 温郁金愈伤组织的培养与繁殖;瞿丽蔚;《医学信息》;20151231;第28卷(第20期);第331-332页 * |
| 温郁金的组织培养与快速繁殖;汪洪等;《植物生理学通讯》;20070630(第3期);第509-510页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111616053A (en) | 2020-09-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105638458B (en) | A kind of method for tissue culture of bulbus fritillariae cirrhosae | |
| CN103270952B (en) | Sinkiang drug mulberry tissue culture and rapid propagation culture medium | |
| CN105010146B (en) | The rapid propagation method of Dysosma versipellis | |
| CN105532448A (en) | Polygonatum kingianum tissue culture method | |
| CN110278870A (en) | Utilize the tissue culture method with leaf petiole forming seedling through one step culture of Jing Banxia tissue culture tufted seedling | |
| CN114342810A (en) | Tissue culture method of rhizoma atractylodis with high disease resistance | |
| CN109220790A (en) | A kind of in vitro outer breeding method of red fruit ginseng | |
| CN106538382B (en) | Method for establishing efficient eremochloa ophiuroides regeneration system by taking young ears as explants | |
| CN105830918A (en) | Method for improving survival rate of transplanting of Huperzia serrata spores | |
| CN105340750A (en) | Honeysuckle tissue-culture-seedling culture medium and honeysuckle tissue-culture rapid propagation method | |
| CN105724257A (en) | Tobacco microspore induction culture medium and tobacco microspore induction culture method | |
| CN104737906A (en) | Building method of rosemarinus officinalis tissue culture system | |
| CN104813931A (en) | Tissue culture and rapid propagation method for Dendrobium officinale | |
| CN105432466B (en) | Method with plant regeneration occurs for a kind of pittosporum tobira somatic embryo | |
| CN111616053B (en) | Curcuma wenyujin embryonic callus induction and plant regeneration method | |
| CN109618924B (en) | Method suitable for reversing vitrified test tube plantlets of various plants | |
| CN106879468A (en) | It is a kind of to improve the method that sugarcane stem apex is trained motility rate | |
| CN110199876A (en) | A kind of method of sonchus oleraceus tissue cultures | |
| CN103621399A (en) | Peony tissue culture method | |
| CN102090336B (en) | Propagation method for Asarum forbesiiMaxim tissue culture | |
| CN107242136A (en) | A kind of method for tissue culture of raspberry | |
| CN104255532B (en) | A kind of tissue cultures forming seedling through one step culture method for quickly breeding of the dish that nourishes heart | |
| CN107535354A (en) | A kind of scientific and effective river monkshood propagation method | |
| CN106577286B (en) | A kind of method of pipex kadsura tissue-culturing rapid propagation | |
| CN112616663A (en) | Method for greatly shortening planting period of lilium davidii var davidii and rapidly propagating seedlings |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |