CN116616179A - Tissue culture rapid propagation method of excellent clone of Gardenia jasminoides ellis with high content of geniposide - Google Patents
Tissue culture rapid propagation method of excellent clone of Gardenia jasminoides ellis with high content of geniposide Download PDFInfo
- Publication number
- CN116616179A CN116616179A CN202310648728.4A CN202310648728A CN116616179A CN 116616179 A CN116616179 A CN 116616179A CN 202310648728 A CN202310648728 A CN 202310648728A CN 116616179 A CN116616179 A CN 116616179A
- Authority
- CN
- China
- Prior art keywords
- culture medium
- seedling
- content
- geniposide
- gardenia
- 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.)
- Granted
Links
- IBFYXTRXDNAPMM-BVTMAQQCSA-N Geniposide Chemical compound O([C@@H]1OC=C([C@@H]2[C@H]1C(=CC2)CO)C(=O)OC)[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O IBFYXTRXDNAPMM-BVTMAQQCSA-N 0.000 title claims abstract description 34
- IBFYXTRXDNAPMM-FZEIBHLUSA-N Geniposide Natural products COC(=O)C1=CO[C@@H](O[C@H]2O[C@@H](CO)[C@H](O)[C@@H](O)[C@@H]2O)[C@H]2[C@@H]1CC=C2CO IBFYXTRXDNAPMM-FZEIBHLUSA-N 0.000 title claims abstract description 32
- VGLLGNISLBPZNL-RBUKDIBWSA-N arborescoside Natural products O=C(OC)C=1[C@@H]2C([C@H](O[C@H]3[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O3)OC=1)=C(CO)CC2 VGLLGNISLBPZNL-RBUKDIBWSA-N 0.000 title claims abstract description 32
- 240000001972 Gardenia jasminoides Species 0.000 title claims abstract description 20
- 238000000034 method Methods 0.000 title claims abstract description 18
- 235000018958 Gardenia augusta Nutrition 0.000 title claims abstract description 17
- 239000001963 growth medium Substances 0.000 claims abstract description 44
- 230000035755 proliferation Effects 0.000 claims abstract description 40
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 16
- 230000006698 induction Effects 0.000 claims abstract description 14
- 239000011159 matrix material Substances 0.000 claims abstract description 14
- 241000196324 Embryophyta Species 0.000 claims abstract description 13
- 238000012258 culturing Methods 0.000 claims abstract description 10
- 239000012883 rooting culture medium Substances 0.000 claims abstract description 9
- 230000001954 sterilising effect Effects 0.000 claims abstract description 7
- 239000007640 basal medium Substances 0.000 claims abstract description 5
- 238000005286 illumination Methods 0.000 claims description 36
- 241000157835 Gardenia Species 0.000 claims description 23
- 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 claims description 16
- 229930006000 Sucrose Natural products 0.000 claims description 16
- 229940113118 carrageenan Drugs 0.000 claims description 16
- 235000010418 carrageenan Nutrition 0.000 claims description 16
- 229920001525 carrageenan Polymers 0.000 claims description 16
- 239000000679 carrageenan Substances 0.000 claims description 16
- 239000005720 sucrose Substances 0.000 claims description 16
- UHVMMEOXYDMDKI-JKYCWFKZSA-L zinc;1-(5-cyanopyridin-2-yl)-3-[(1s,2s)-2-(6-fluoro-2-hydroxy-3-propanoylphenyl)cyclopropyl]urea;diacetate Chemical compound [Zn+2].CC([O-])=O.CC([O-])=O.CCC(=O)C1=CC=C(F)C([C@H]2[C@H](C2)NC(=O)NC=2N=CC(=CC=2)C#N)=C1O UHVMMEOXYDMDKI-JKYCWFKZSA-L 0.000 claims description 16
- 206010020649 Hyperkeratosis Diseases 0.000 claims description 13
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 8
- 229910052799 carbon Inorganic materials 0.000 claims description 8
- 230000001502 supplementing effect Effects 0.000 claims description 5
- 235000019362 perlite Nutrition 0.000 claims description 4
- 239000010451 perlite Substances 0.000 claims description 4
- 239000002689 soil Substances 0.000 claims description 4
- 239000003337 fertilizer Substances 0.000 claims description 3
- 239000002609 medium Substances 0.000 claims 3
- 230000028446 budding cell bud growth Effects 0.000 claims 1
- 239000000463 material Substances 0.000 abstract description 6
- 238000004659 sterilization and disinfection Methods 0.000 abstract description 5
- 238000009395 breeding Methods 0.000 abstract description 4
- 230000001488 breeding effect Effects 0.000 abstract description 4
- 230000008569 process Effects 0.000 description 5
- 235000016709 nutrition Nutrition 0.000 description 4
- 230000035764 nutrition Effects 0.000 description 4
- 230000008929 regeneration Effects 0.000 description 4
- 238000011069 regeneration method Methods 0.000 description 4
- 230000002068 genetic effect Effects 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 230000002087 whitening effect Effects 0.000 description 3
- 239000008280 blood Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000003306 harvesting Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000003595 mist Substances 0.000 description 2
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 2
- 239000008223 sterile water Substances 0.000 description 2
- 238000004383 yellowing Methods 0.000 description 2
- 206010001557 Albinism Diseases 0.000 description 1
- 208000004880 Polyuria Diseases 0.000 description 1
- 208000001431 Psychomotor Agitation Diseases 0.000 description 1
- 206010038743 Restlessness Diseases 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- -1 anti-tumor Substances 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000035619 diuresis Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 229930182470 glycoside Natural products 0.000 description 1
- 150000002338 glycosides Chemical class 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 239000009427 jiangzhi Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000010899 nucleation Methods 0.000 description 1
- 238000004161 plant tissue culture Methods 0.000 description 1
- 230000010152 pollination Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000010926 purge Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000004083 survival effect Effects 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
- A01H4/001—Culture apparatus for tissue culture
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/20—Reduction of greenhouse gas [GHG] emissions in agriculture, e.g. CO2
- Y02P60/21—Dinitrogen oxide [N2O], e.g. using aquaponics, hydroponics or efficiency measures
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)
Abstract
The invention discloses a tissue culture rapid propagation method of a high-jasminoidin-content excellent clone of gardenia jasminoides ellis, which takes current annual branches of a high-yield high-jasminoidin-content Jiang Zhizi mother plant as explants, and the current annual branches are inoculated into an induction culture medium after sterilization to alternately culture in light and dark to grow primary buds; inoculating the primary buds to a first proliferation culture medium, and alternately performing subculture in light and dark; inoculating the strain to a second proliferation culture medium to obtain proliferation seedlings; inoculating the proliferation seedling to a rooting culture medium, and alternately culturing in light and darkness to obtain a rooting seedling; and (3) transplanting the root Miao Xiqu basal medium after seedling hardening into a matrix, and watering the root fixing water, and performing conventional seedling growing management to obtain the gardenia seedling with high jasminoidin content. The invention selects excellent clone of gardenia with high yield and high geniposide content as a material, and constructs a high-efficiency asexual tissue culture and breeding technical system to reserve the characteristics of the parent to the maximum extent, thereby rapidly and efficiently obtaining high-quality high-geniposide content gardenia seedling.
Description
Technical Field
The invention belongs to the technical field of plant tissue culture, and particularly relates to a tissue culture rapid propagation method of a high-gardenoside-content excellent clone of gardenia jasminoides ellis.
Background
The gardenia belongs to a first medicine and food dual-purpose resource issued by the ministry of health, has the effects of purging fire and relieving restlessness, clearing heat and promoting diuresis, cooling blood and detoxifying, has the application history of over 2000 years as a traditional bulk medicinal material in China, mainly produces Jiangxi, hunan, chongqing, sichuan, hubei, zhejiang, anhui, fujian, guangxi and other provinces and cities, is cultivated mostly in Jiangxi, has large production quantity, is a well-known genuine medicinal material, has the characteristics of round body, thin skin, red and full color, and is known as "small red gardenia", "Jiangzhi". The gardenia glycoside is used as a main active ingredient of the gardenia jasminoides ellis and has various biological effects of anti-inflammatory, antioxidant, anti-tumor, blood sugar reducing and the like.
The gardenia in the Jiangxi indigenous areas mainly adopts a seeding mode to grow seedlings, cutting seedlings are few, along with large-scale introduction of foreign gardenia varieties by growers, the types of gardenia in the Jiangxi indigenous areas are extremely complex, different types of resources are used for hybrid cultivation, products of the same area are different in form and maturity, quality of gardenia medicinal materials in the current market is uneven, and even harvest and processing of planting farmers and enterprises are extremely inconvenient, so that the raising cost and harvesting cost in intangible states are improved.
Jiang Zhizi progeny of free pollination can generate serious differentiation in the aspects of characters, yield, quality and the like, and can obtain excellent single plants with high yield and high quality through breeding, and the large-scale asexual propagation of the excellent single plants is a key problem of high-efficiency and rapid application to production. Tissue culture propagation is one of the ways of high-efficiency rapid propagation of excellent single gardenia plants with high geniposide content.
Currently, reports on gardenia tissue culture fall into three general categories: the first is that stem tissues are used as explants to directly carry out plant regeneration of fructus gardeniae; the second category is that seeds are used as explants, and plant regeneration of gardenia is carried out without differentiation; the third category is to use gardenia leaves, seeds and young buds as explants, and realize plant regeneration by inducing callus. Although aseptic Miao Zaisheng report of gardenia is reported, the explant adopts seeds, and only the seeds are subjected to aseptic culture, so that most of cultured gardenia tissue culture seedlings are low in quality, have different phenotypes and have serious yellowing and browning phenomena. Therefore, how to clone and scale the bred excellent single gardenia plant with high geniposide content through tissue culture, optimize and improve the tissue culture mode, the seedling hardening mode and the transplanting matrix to improve the transplanting survival rate of the tissue culture seedling of the gardenia becomes a key problem of the current research.
Disclosure of Invention
Aiming at the defects and the problems in the prior art, the invention aims to provide a tissue culture rapid propagation method of excellent clone of Gardenia jasminoides Ellis with high content of geniposide, and the method can be used for rapidly and efficiently obtaining high-quality seedlings of Gardenia jasminoides Ellis with high content of geniposide.
The invention is realized by the following technical scheme:
the invention provides a tissue culture rapid propagation method of a high-gardenoside-content excellent clone of gardenia jasminoides ellis, which comprises the following steps:
(1) Establishment of a sterile line: taking the current annual branch of a parent strain with high yield and high geniposide content (more than or equal to 5.00g/100 g) and Jiang Zhizi as an explant, sterilizing, inoculating into an induction culture medium, and culturing under alternating conditions of illumination intensity of 2500 Lux-3000 Lux, illumination and darkness at 24+/-1 ℃, wherein the illumination time is 12 hours and the darkness time is 12 hours in each period until primary buds grow out, wherein each liter of the induction culture medium comprises: 0.5mg of 6-BA, 0.3mg of NAA, 30.0g of sucrose, 5.0g of carrageenan and the balance of MS culture medium, and the pH is 5.5-5.8; in the early stages of sterile line establishment, in order to maintain the genetic characteristics of the parent to the maximum extent, it is necessary to impair the ability to differentiate buds from callus, and to induce primary buds in the form of buds or cluster buds.
(2) Proliferation culture: inoculating primary buds to a first proliferation culture medium, and performing secondary culture under the conditions of illumination intensity of 2500 Lux-3000 Lux, illumination and darkness at 24+/-1 ℃, wherein the illumination time is 12 hours and the darkness time is 12 hours in each period, and each liter of proliferation culture medium contains: 1.0mg of 6-BA, 0.3mg to 0.5mg of NAA, 30.0g of sucrose, 7.5g of carrageenan and the balance of MS culture medium, and the pH is 5.5 to 5.8; if the leaves of the proliferation seedling are brown, whitened or white callus appears at the basal part, inoculating the leaves on a second proliferation culture medium to obtain the proliferation seedling, wherein each liter of proliferation culture medium contains: 1.0mg of 6-BA, 0.3mg to 0.5mg of NAA, 5.0g of active carbon, 30.0g of sucrose, 7.5g of carrageenan and the balance of MS culture medium. The proliferation culture medium can lead the number of adventitious buds and the number of effective seedlings (3 cm-4 cm in height, 3 leaves and more) generated in the proliferation culture process to be evenly developed, thereby reducing the occurrence of browning and albinism of the leaves and slowing down the generation of callus.
(3) Rooting culture: inoculating healthy rooting-free seedlings with 3 or more leaves to a rooting culture medium, and culturing under alternating conditions of illumination intensity of 2500 Lux-3000 Lux and illumination and darkness at 24+/-1 ℃ to obtain rooting seedlings, wherein the illumination time is 12 hours and the darkness time is 12 hours in each period, and each liter of rooting culture medium contains: 1.0mg IBA, 0.1 mg-0.5 mg NAA, 5.0g active carbon, 30.0g sucrose, 7.5g carrageenan and the balance of MS culture medium, and the pH is 5.5-5.8.
(4) Transplanting tissue culture seedlings: transplanting the rooting Miao Xiqu basal medium after seedling hardening into a matrix, pouring root fixing water after transplanting, performing conventional seedling cultivation management, and growing Jiang Zhizi seedlings with high geniposide content, wherein the seedling hardening mode is as follows: the indoor incubator is used for hardening off 5d and then is used for outdoor hardening off 5d, and the matrix is prepared by uniformly mixing and stirring red loam, turfy soil and perlite according to the volume ratio of 1:1:1.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention selects excellent clone of gardenia with high yield and high geniposide content from Jiangxi dao medicinal material Jiang Zhizi as a material, constructs a high-efficiency asexual tissue culture breeding technical system thereof, so as to keep the characteristics of the parent stock to the maximum extent, quickly and efficiently obtain high-quality Jiang Zhizi seedlings with high geniposide content, and overcomes the defects that most of cultured gardenia tissue culture seedlings are not high in quality, have different phenotypes, are serious in yellowing and browning phenomena and the like when seeds are used as explants for aseptic culture in the prior art.
2. The invention solves the technical problems that in the subculture, the leaves of the proliferation seedling are easy to brown and whiten, white callus is easy to appear at the base, and the like.
3. The invention screens out proper formulas and culture conditions of all links of the tissue culture propagation of the gardenia with high content of the geniposide, and successfully obtains the regeneration plant of the gardenia with high content of the geniposide. Therefore, the invention effectively solves the technical problems of browning and whitening in the tissue culture and breeding of the gardenia with the content of the geniposide of more than 5.00g/100g and white callus at the basal part, can rapidly and efficiently obtain high-quality Jiang Zhizi seedlings with high content of the geniposide, and provides high-quality seedlings for the gardenia industry.
Drawings
FIG. 1 shows the initial buds of Gardenia jasminoides Ellis with high geniposide content.
FIG. 2 shows a bottle seedling of Gardenia jasminoides Ellis proliferation with high content of geniposide.
Figure 3 is Jiang Zhizi tissue culture root seedling with high geniposide content.
Fig. 4 is a Jiang Zhizi tissue culture transplanted seedling with high geniposide content.
Detailed Description
The invention will be further described with reference to the accompanying drawings.
Example 1:
(1) Establishment of a sterile line: the new-born disease-free tender branches of the parent plant of the geniposide Jiang Zhizi with high yield and high geniposide content (5.13 g/100 g) are used as explants, the branches are sheared off neatly by scissors, the broken parts of the branches are wrapped by toilet paper which is fully soaked with water, sterile water is sprayed to the branches every 1h in the transportation process to ensure that the branches are always in a moist state, and the branches are brought back to laboratory for research and use. Treating the explant with 3% NaClO solution for 10min for sterilization; after sterilization, inoculating the culture medium into an induction culture medium, and culturing under the conditions of illumination intensity of 2500 Lux-3000 Lux, illumination and darkness at 24+/-1 ℃, wherein the illumination time is 12 hours and the darkness time is 12 hours in each period until primary buds grow, and the induction culture medium contains the following components per liter as shown in figure 1: 0.5mg of 6-BA, 0.3mg of NAA, 30.0g of sucrose, 7.5g of carrageenan and the balance of MS culture medium, and the pH is 5.5-5.8; in the initial stage of sterile line establishment, in order to maximally maintain the genetic characteristics of parent, the capability of differentiating buds from callus needs to be weakened, and primary bud induction is performed in a bud or cluster bud mode, wherein the primary bud induction rate is more than 70.0%.
(2) Proliferation culture: inoculating primary buds to a first proliferation culture medium, and carrying out subculture under the conditions of illumination intensity of 2500-3000 Lux, illumination and darkness at 24+/-1 ℃ alternately, wherein the illumination time is 12 hours and the darkness time is 12 hours in each period, and each liter of proliferation culture medium comprises: 1.0mg of 6-BA, 0.5mg of NAA, 30.0g of sucrose, 7.5g of carrageenan and the balance of MS culture medium, and the pH is 5.5-5.8; if the leaves of the proliferation seedling are brown, whitened or white callus is generated at the basal part, the leaves are inoculated on a second proliferation culture medium to obtain the proliferation seedling, and as shown in figure 2, the proliferation culture medium contains the following components per liter: 1.0mg of 6-BA, 0.5mg of NAA, 5.0g of active carbon, 30.0g of sucrose, 7.5g of carrageenan and the balance of MS culture medium. The proliferation culture medium can lead the number of adventitious buds and the number of effective seedlings (3 cm-4 cm in height, 3 leaves and more) generated in the proliferation culture process to be evenly developed, thereby reducing the browning and whitening of the leaves, slowing down the generation of callus and leading the proliferation coefficient to be more than 4.83 per growth cycle.
(3) Rooting culture: inoculating healthy rooting-free seedlings with 3 or more leaves to a rooting culture medium, and culturing under alternating conditions of illumination intensity of 2500 Lux-3000 Lux, illumination and darkness at 24+/-1 ℃ for 12 hours and darkness for 12 hours in each period to obtain rooting seedlings, wherein each liter of rooting culture medium contains: 1.0mg IBA, 0.5mg NAA, 5.0g active carbon, 30.0g sucrose, 7.5g carrageenan and the balance of MS culture medium, and the pH is 5.5-5.8; after 30-35 d of general culture, the rooting rate is up to above 99%, the number of root systems is above 3, and the root-free seedlings grow into complete plants, as shown in figure 3.
(2) Transplanting tissue culture seedlings: transplanting the rooting Miao Xiqu basal medium after seedling hardening into a matrix, pouring root fixing water after transplanting, performing conventional seedling cultivation management, and growing Jiang Zhizi seedlings with high geniposide content, wherein the seedling hardening mode is as follows: the indoor incubator is used for hardening off 5d and then is used for outdoor hardening off 5d, and the matrix is prepared by uniformly mixing and stirring red loam, turfy soil and perlite according to the volume ratio of 1:1:1. The seedling is put into a hole tray or a nutrition bag with the length of 4.8cm multiplied by 4.8cm and the depth of 9cm, transplanted seedlings are put into the matrix of the nutrition bag, and after the seedling is planted, root fixing water is poured sufficiently to enable the matrix to be tightly fused with the root system of the seedling. Supplementing water to the leaf surface for 3-5 times a week by using a mist sprayer, and supplementing water for 1-2 times a day. After 1 month, when the leaves are smooth and flat, a proper amount of foliar fertilizer can be sprayed in time, and conventional seedling management is carried out, so that seedlings (shown in figure 4) of Gardenia jasminoides Ellis with high jasminoides Ellis content, which can be used for transplanting, are obtained.
Example 2:
(1) Establishment of a sterile line: the new-born disease-free tender branches of the parent plant of the geniposide Jiang Zhizi with high yield and high geniposide content (8.20 g/100 g) are used as explants, the branches are sheared off neatly by scissors, the broken parts of the branches are wrapped by toilet paper which is fully soaked with water, sterile water is sprayed to the branches every 1h in the transportation process to ensure that the branches are always in a moist state, and the branches are brought back to laboratory for research and use. Treating the explant with 3% NaClO solution for 10min for sterilization; after sterilization, inoculating the strain into an induction culture medium, and culturing under the conditions of illumination intensity of 2500-3000 Lux, illumination at 24+/-1 ℃ and darkness alternation, wherein the illumination time is 12 hours and the darkness time is 12 hours in each period until primary buds grow out, and each liter of the induction culture medium comprises: 0.5mg of 6-BA, 0.3mg of NAA, 30.0g of sucrose, 7.5g of carrageenan and the balance of MS culture medium, and the pH is 5.5-5.8; in the initial stage of sterile line establishment, in order to maximally maintain the genetic characteristics of parent, the capability of differentiating buds from callus needs to be weakened, and primary bud induction is performed in a bud or cluster bud mode, wherein the primary bud induction rate is more than 70.0%.
(2) Proliferation culture: inoculating primary buds to a first proliferation culture medium, and carrying out secondary culture under the conditions of illumination intensity of 2500-3000 Lux, illumination at 24+/-1 ℃ and darkness alternation, wherein the illumination time is 12 hours and the darkness time is 12 hours in each period, and each liter of proliferation culture medium contains: 1.0mg of 6-BA, 0.3mg of NAA, 30.0g of sucrose, 7.5g of carrageenan and the balance of MS culture medium, and the pH is 5.5-5.8; if the leaves of the proliferation seedling are brown, whitened or white callus appears at the basal part, inoculating the leaves on a second proliferation culture medium to obtain the proliferation seedling, wherein each liter of proliferation culture medium contains: 1.0mg of 6-BA, 0.3mg of NAA, 5.0g of active carbon, 30.0g of sucrose, 7.5g of carrageenan and the balance of MS culture medium. The proliferation culture medium can lead the number of adventitious buds and the number of effective seedlings (3 cm-4 cm in height, 3 leaves and more) generated in the proliferation culture process to be evenly developed, thereby reducing the browning and whitening of the leaves, slowing down the generation of callus and leading the proliferation coefficient to be more than 4.33 per growth cycle.
(3) Rooting culture: inoculating healthy rooting-free seedlings with 3 or more leaves to a rooting culture medium, and culturing under alternating conditions of illumination intensity of 2500 Lux-3000 Lux, illumination and darkness at 24+/-1 ℃ for 12 hours and darkness for 12 hours in each period to obtain rooting seedlings, wherein each liter of rooting culture medium contains: 1.0mg IBA, 0.1mg NAA, 5.0g active carbon, 30.0g sucrose, 7.5g carrageenan and the balance of MS culture medium, and the pH is 5.5-5.8; after the culture for 30-35 d, the rooting rate is up to 99% or more, the number of root systems is up to 4 or more, and the root-free seedlings grow into complete plants.
(4) Transplanting tissue culture seedlings: transplanting the rooting Miao Xiqu basal medium after seedling hardening into a matrix, pouring root fixing water after transplanting, performing conventional seedling cultivation management, and growing Jiang Zhizi seedlings with high geniposide content, wherein the seedling hardening mode is as follows: the indoor incubator is used for hardening off 5d and then is used for outdoor hardening off 5d, and the matrix is prepared by uniformly mixing and stirring red loam, turfy soil and perlite according to the volume ratio of 1:1:1. The seedling is put into a hole tray or a nutrition bag with the length of 4.8cm multiplied by 4.8cm and the depth of 9cm, transplanted seedlings are put into the matrix of the nutrition bag, and after the seedling is planted, root fixing water is poured sufficiently to enable the matrix to be tightly fused with the root system of the seedling. Supplementing water to the leaf surface for 3-5 times a week by using a mist sprayer, and supplementing water for 1-2 times a day. After 1 month, when the leaves are smooth and flat, a proper amount of foliar fertilizer can be sprayed in time, and conventional seedling management is carried out, so that seedlings of Gardenia jasminoides Ellis with high jasminoides Ellis content, which can be used for transplanting, are obtained.
The foregoing description of the preferred embodiments of the present invention has been presented only in terms of those specific and detailed descriptions, and is not, therefore, to be construed as limiting the scope of the invention. It should be noted that modifications, improvements and substitutions can be made by those skilled in the art without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (6)
1. A tissue culture rapid propagation method of a high-jasminoidin-content excellent clone of gardenia jasminoides ellis is characterized by comprising the following steps:
(1) Establishment of a sterile line: taking current annual branches of a parent plant with high yield and high geniposide content Jiang Zhizi as explants, sterilizing, inoculating the explants into an induction culture medium, and culturing under alternating conditions of illumination and darkness until primary buds grow;
(2) Proliferation culture: inoculating the primary buds to a first proliferation culture medium, and performing subculture under alternating light and dark conditions; inoculating the obtained multiplication seedling to a second multiplication medium to obtain multiplication seedlings when the multiplication seedlings have brown, whitened or white callus on the basal part;
(3) Rooting culture: inoculating healthy rooting-free plantlets with 3 or more leaves in the proliferation plantlets to a rooting culture medium, and culturing under alternating illumination and darkness to obtain rooting plantlets;
(4) Transplanting tissue culture seedlings: transplanting the rooting Miao Xiqu basal medium after seedling hardening into a matrix, and watering enough rooting water after transplanting and periodically supplementing water; when the leaves are smooth and flat, the foliar fertilizer is sprayed, the conventional seedling management is carried out, and the gardenia seedling with high jasminoidin content is grown.
2. The method for tissue culture and rapid propagation of excellent clone of gardenia jasminoides ellis with high content of geniposide as claimed in claim 1, wherein in the step (1), the light condition is: the illumination intensity is 2500 Lux-3000 Lux, 24+ -1 ℃; the illumination time is 12 hours and the darkness time is 12 hours in each period of alternating illumination and darkness; the induction culture medium contains per liter: 0.5mg of 6-BA, 0.3mg of NAA, 30.0g of sucrose, 5.0g of carrageenan and the balance of MS culture medium, and the pH is 5.5-5.8.
3. The method for tissue culture and rapid propagation of excellent clones of Gardenia jasminoides ellis with high geniposide content according to claim 1 or 2, wherein in the step (1), the geniposide content of the selected explant is not less than 5.00g/100g; the primary bud induction is carried out by adopting a bud growth mode or a cluster bud mode.
4. The method for tissue culture and rapid propagation of excellent clone of gardenia jasminoides ellis with high content of geniposide as claimed in claim 1, wherein in the step (2), the light condition is: the illumination intensity is 2500 Lux-3000 Lux, 24+ -1 ℃; the illumination time is 12 hours and the darkness time is 12 hours in each period of alternating illumination and darkness; the first proliferation medium contains per liter: 1.0mg of 6-BA, 0.3mg to 0.5mg of NAA, 30.0g of sucrose, 7.5g of carrageenan and the balance of MS culture medium, and the pH is 5.5 to 5.8; the second proliferation medium contains per liter: 1.0mg of 6-BA, 0.3mg to 0.5mg of NAA, 5.0g of active carbon, 30.0g of sucrose, 7.5g of carrageenan and the balance of MS culture medium.
5. The method for tissue culture and rapid propagation of excellent clone of gardenia jasminoides ellis with high content of geniposide as claimed in claim 1, wherein in the step (3), the light condition is: the illumination intensity is 2500 Lux-3000 Lux, 24+ -1 ℃; the illumination time is 12 hours and the darkness time is 12 hours in each period of alternating illumination and darkness; the rooting culture medium contains per liter: 1.0mg IBA, 0.1 mg-0.5 mg NAA, 5.0g active carbon, 30.0g sucrose, 7.5g carrageenan and the balance of MS culture medium, and the pH is 5.5-5.8; culturing for 30-35 d to obtain more than 3 root seedlings with root system number, so that the rootless seedlings grow into complete plants.
6. The method for tissue culture and rapid propagation of excellent clone of gardenia with high content of geniposide according to claim 1, wherein in the step (4), the seedling hardening mode is as follows: the indoor incubator acclimatizes 5d and then transfers to the outdoor acclimatizes 5d; the matrix is prepared from red loam, turfy soil and perlite according to the volume ratio of 1:1:1 are mixed and stirred uniformly.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310648728.4A CN116616179B (en) | 2023-06-02 | 2023-06-02 | Tissue culture rapid propagation method of excellent clone of Gardenia jasminoides ellis with high content of geniposide |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310648728.4A CN116616179B (en) | 2023-06-02 | 2023-06-02 | Tissue culture rapid propagation method of excellent clone of Gardenia jasminoides ellis with high content of geniposide |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116616179A true CN116616179A (en) | 2023-08-22 |
| CN116616179B CN116616179B (en) | 2024-02-13 |
Family
ID=87591814
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310648728.4A Active CN116616179B (en) | 2023-06-02 | 2023-06-02 | Tissue culture rapid propagation method of excellent clone of Gardenia jasminoides ellis with high content of geniposide |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116616179B (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103461127A (en) * | 2013-09-17 | 2013-12-25 | 南京通泽农业科技有限公司 | Rapid propagation method for gardenia jasminoides |
| CN110999782A (en) * | 2019-11-10 | 2020-04-14 | 六安市裕安区天缘中药材种植专业合作社 | Tissue culture and rapid propagation method for gardenia jasminoides |
| CN114793905A (en) * | 2022-05-23 | 2022-07-29 | 浙江省亚热带作物研究所(浙南林业科学研究院) | Rapid propagation method and culture medium for gardenia tissue culture seedlings |
| CN117158315A (en) * | 2023-05-19 | 2023-12-05 | 淮南师范学院 | Tissue culture method of gardenia lobule |
-
2023
- 2023-06-02 CN CN202310648728.4A patent/CN116616179B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103461127A (en) * | 2013-09-17 | 2013-12-25 | 南京通泽农业科技有限公司 | Rapid propagation method for gardenia jasminoides |
| CN110999782A (en) * | 2019-11-10 | 2020-04-14 | 六安市裕安区天缘中药材种植专业合作社 | Tissue culture and rapid propagation method for gardenia jasminoides |
| CN114793905A (en) * | 2022-05-23 | 2022-07-29 | 浙江省亚热带作物研究所(浙南林业科学研究院) | Rapid propagation method and culture medium for gardenia tissue culture seedlings |
| CN117158315A (en) * | 2023-05-19 | 2023-12-05 | 淮南师范学院 | Tissue culture method of gardenia lobule |
Non-Patent Citations (3)
| Title |
|---|
| 崔思然;陈彩霞;江艳华;李艾莲;: "栀子优良品种快繁体系的建立", 中国农学通报, no. 19 * |
| 赵长新, 廉美兰, 朴炫春: "栀子组织培养与快速繁殖", 延边大学农学学报, no. 03 * |
| 陆斐, 刘宝光, 刘玉波, 张弼弘, 王佰华: "栀子的组织培养快速繁殖技术", 北华大学学报(自然科学版), no. 06 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116616179B (en) | 2024-02-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN100444722C (en) | Cyrtopterin tissue culturing method and fast reproduction thereof | |
| CN105340747A (en) | Asexual rapid propagation method for radix glycyrrhizae | |
| CN110679482B (en) | Chrysanthemum multocida detoxification culture medium with high stem tip induction rate and tissue culture quality and method | |
| CN102124901A (en) | Artificial seedling method of Louisiana iris | |
| CN101822220A (en) | Method for culturing and rapidly propagating stem tip tissue of rare cymbidium goeringii | |
| CN101401549B (en) | Once-seedling forming quick propagating method for sugarcane tissue culture | |
| CN102845313A (en) | Method for quickly in-vitro actinidia kolomikta propagating | |
| CN115474546B (en) | Breeding method of columbin flowers | |
| CN101897297B (en) | Two-step tissue culture quick propagation method for hemerocallis | |
| CN103155862B (en) | Sinocalamus latiflorus flower pesticide inductor embryo the method obtaining regeneration plant | |
| CN101810144B (en) | Rapid breeding method of senecio cruentus | |
| CN106258960A (en) | A kind of orchid seed germination quick-breeding method | |
| CN103039360B (en) | Method for quickly propagating leeka through tissue culture | |
| CN117204342A (en) | Quick propagation method for cultivating capsicum seeds and cultivating seedlings | |
| CN109526745B (en) | Method for breeding seedlings by using paris polyphylla leaves | |
| CN106718910A (en) | The method that rapid induction breeds inclined fringe roegneria kamoji hypocotyledonery axis callus | |
| CN115119749B (en) | In-vitro culture method for young tomato embryo | |
| CN114600772B (en) | Tissue culture method and rapid propagation method of michelia figo in remote mountains | |
| CN116616179B (en) | Tissue culture rapid propagation method of excellent clone of Gardenia jasminoides ellis with high content of geniposide | |
| CN105532472A (en) | Direct induction seedling development method for hybrid cymbidium tortisepalum rhizomes | |
| CN101473792B (en) | Tissue culture of Ypsilandra thibetica and planting method | |
| CN110024688A (en) | A kind of method and its culture medium of caragana bud proliferation and plant regeneration | |
| CN101933457A (en) | Tissue culture and rapid propagation technology for wine bamboo | |
| CN110934066B (en) | Water culture method for regeneration of axillary buds of rice stem nodes, water culture solution and application thereof | |
| CN107568063B (en) | A kind of oilseed plant Asiatic sweet leaf tissue culture and rapid propagation method |
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 |