CN104429971A - Tissue culture seedling-raising method of millettia fordii dunn - Google Patents
Tissue culture seedling-raising method of millettia fordii dunn Download PDFInfo
- Publication number
- CN104429971A CN104429971A CN201410798739.1A CN201410798739A CN104429971A CN 104429971 A CN104429971 A CN 104429971A CN 201410798739 A CN201410798739 A CN 201410798739A CN 104429971 A CN104429971 A CN 104429971A
- Authority
- CN
- China
- Prior art keywords
- precipice
- days
- guangdong
- seedling
- tissue culture
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 241000521581 Millettia Species 0.000 title claims abstract description 11
- 238000000034 method Methods 0.000 title abstract description 11
- 230000035755 proliferation Effects 0.000 claims abstract 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 claims abstract description 11
- 229930006000 Sucrose Natural products 0.000 claims abstract description 11
- NWBJYWHLCVSVIJ-UHFFFAOYSA-N N-benzyladenine Chemical compound N=1C=NC=2NC=NC=2C=1NCC1=CC=CC=C1 NWBJYWHLCVSVIJ-UHFFFAOYSA-N 0.000 claims abstract description 8
- 230000000249 desinfective effect Effects 0.000 claims abstract description 3
- 241000345998 Calamus manan Species 0.000 claims description 39
- 235000010627 Phaseolus vulgaris Nutrition 0.000 claims description 39
- 244000046052 Phaseolus vulgaris Species 0.000 claims description 39
- 235000012950 rattan cane Nutrition 0.000 claims description 39
- 239000002609 medium Substances 0.000 claims description 24
- 238000004659 sterilization and disinfection Methods 0.000 claims description 24
- 230000001954 sterilising effect Effects 0.000 claims description 21
- 238000012136 culture method Methods 0.000 claims description 13
- 238000005406 washing Methods 0.000 claims description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 13
- 239000002689 soil Substances 0.000 claims description 11
- 241000206575 Chondrus crispus Species 0.000 claims description 10
- 239000005720 sucrose Substances 0.000 claims description 10
- 238000005286 illumination Methods 0.000 claims description 9
- 239000006870 ms-medium Substances 0.000 claims description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- RCTYPNKXASFOBE-UHFFFAOYSA-M chloromercury Chemical compound [Hg]Cl RCTYPNKXASFOBE-UHFFFAOYSA-M 0.000 claims description 6
- 230000000844 anti-bacterial effect Effects 0.000 claims description 5
- 239000003899 bactericide agent Substances 0.000 claims description 5
- 239000013039 cover film Substances 0.000 claims description 5
- 239000010408 film Substances 0.000 claims description 5
- 238000011081 inoculation Methods 0.000 claims description 5
- 239000010409 thin film Substances 0.000 claims description 5
- 230000006698 induction Effects 0.000 claims description 4
- 239000007921 spray Substances 0.000 claims description 4
- 239000008399 tap water Substances 0.000 claims description 2
- 235000020679 tap water Nutrition 0.000 claims description 2
- 230000008569 process Effects 0.000 abstract description 3
- 239000001963 growth medium Substances 0.000 abstract 2
- 229940113118 carrageenan Drugs 0.000 abstract 1
- 235000010418 carrageenan Nutrition 0.000 abstract 1
- 229920001525 carrageenan Polymers 0.000 abstract 1
- 239000000679 carrageenan Substances 0.000 abstract 1
- 210000001161 mammalian embryo Anatomy 0.000 abstract 1
- 229960004793 sucrose Drugs 0.000 abstract 1
- 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 abstract 1
- 238000010586 diagram Methods 0.000 description 18
- 238000012360 testing method Methods 0.000 description 9
- 210000001519 tissue Anatomy 0.000 description 8
- JTEDVYBZBROSJT-UHFFFAOYSA-N indole-3-butyric acid Chemical compound C1=CC=C2C(CCCC(=O)O)=CNC2=C1 JTEDVYBZBROSJT-UHFFFAOYSA-N 0.000 description 7
- 239000011159 matrix material Substances 0.000 description 6
- 241000196324 Embryophyta Species 0.000 description 5
- 230000018109 developmental process Effects 0.000 description 5
- 230000008901 benefit Effects 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- HFCYZXMHUIHAQI-UHFFFAOYSA-N Thidiazuron Chemical compound C=1C=CC=CC=1NC(=O)NC1=CN=NS1 HFCYZXMHUIHAQI-UHFFFAOYSA-N 0.000 description 3
- 238000004140 cleaning Methods 0.000 description 3
- 238000011109 contamination Methods 0.000 description 3
- 238000007796 conventional method Methods 0.000 description 3
- 238000005520 cutting process Methods 0.000 description 3
- 210000002615 epidermis Anatomy 0.000 description 3
- 230000035784 germination Effects 0.000 description 3
- 238000012549 training Methods 0.000 description 3
- 239000003814 drug Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000001902 propagating effect Effects 0.000 description 2
- 230000008929 regeneration Effects 0.000 description 2
- 238000011069 regeneration method Methods 0.000 description 2
- 239000005972 6-Benzyladenine Substances 0.000 description 1
- 206010011224 Cough Diseases 0.000 description 1
- 238000010923 batch production Methods 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- UORPMTWHYAUNDH-UHFFFAOYSA-N phenylurea thiadiazole Chemical compound C1(=CC=CC=C1)NC(=O)N.S1N=NC=C1 UORPMTWHYAUNDH-UHFFFAOYSA-N 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
- 238000005728 strengthening Methods 0.000 description 1
- 238000012090 tissue culture technique Methods 0.000 description 1
Landscapes
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a tissue culture seedling-raising method of millettia fordii dunn. The method comprises the following steps: disinfecting and sprouting seeds, selecting germinal sterile embryo axes as explants, carrying out adventitious bud proliferation, carrying out rooting culture, hardening seedlings and transplanting the seedlings to obtain millettia fordii dunn seedlings, wherein a multiplicative culture medium comprises an MS culture medium, 0.3-0.5mg/L of 6-BA, 0.06-0.1mg/L of TDZ, 0.15-0.3mg/L of NAA, 30g/L of cane sugar and 5.8g/L of carrageenan; the pH value is 5.5-5.8. The millettia fordii dunn seedlings cultured by the method has the characteristics of good hereditary stability, high reproduction speed, short seedling raising period, low cost and simplicity in operation; the experimental facility requirements are low; the operation process is simple; the millettia fordii dunn seedlings are bred in scales; the result is stable and reliable.
Description
Technical field
The invention belongs to applied biology, agricultural biological technical field, particularly the tissue culture method of precipice, a kind of Guangdong beans rattan.
Background technology
Precipice, Guangdong beans rattan (Millettia fordii Dunn) originates in Guangdong, Guangxi, is born in the mountain valley sparse woods of height above sea level about 500 meters.Liana; Winglike compound leaf; The usual armpit of raceme is raw; Pod is linear; Seed brown, avette, be about 7 millimeters, wide about 6 millimeters, smooth; 9 ~ October of florescence, really November phase.
Among the people think precipice, Guangdong beans rattan have moisten the lung and relieve the cough, the effect such as invigorating the spleen qi-restoratives, strengthening yang and invigorating kidney.Therefore, be with in area, Wuzhou, Guangxi and forest in Maofengshan, Guangzhou one, the root of precipice, Guangdong prevailing beans rattan and pig bone compatibility Baoshang.Excavate due to manually unordered, cause resource day by day to reduce, the market price raises year by year.Guangzhou great You Yuxuan agricultural development Co., Ltd is the demand in short supply alleviating market, determines the scaled artificial planting base setting up precipice, Guangdong beans rattan.But because precipice, Guangdong beans rattan still belongs to wild state at present, seed source is few, and cottage propagation speed is slow, be badly in need of the supply problem solving seedling.
The tissue culture rapid propagating technology of plant has and takes up room little, and the advantages such as by area, season limits, small investment, and economic benefit is high, by sterile working, short time amount reproduction target plant under manual control condition, obtains the whole plant of regeneration.In the urgent need to the tissue culture technique of research and development precipice, Guangdong beans rattan, realized the biological control of precipice, Guangdong beans rattan seedling by tissue culture technology, solve the difficult problem that precipice, Guangdong beans rattan seed is few, restrict production development.
Summary of the invention
In order to overcome the shortcoming of existing propagation technique with not enough, the object of the present invention is to provide the tissue culture method of precipice, a kind of Guangdong beans rattan.Utilize the method to cultivate precipice, Guangdong beans rattan seedling and there is the feature that genetic stability is good, reproduction speed is fast, growing-seedling period is short, with low cost, easy and simple to handle; And achieving precipice, biological control Guangdong beans rattan seedling, result is reliable and stable.
Object of the present invention is achieved through the following technical solutions: the tissue culture method of precipice, a kind of Guangdong beans rattan, comprise seed disinfection and vernalization, adventitious bud proliferation, culture of rootage and test-tube seedling transplanting step: be, after the alcohol of 75% and the mercury chloride of 1g/L are sterilized, be sown in aseptic moistening gauze or MS medium by evenly full precipice, Guangdong beans rattan seed volumetric concentration; With the plumular axis of germinating seed for explant, MS+6BA+TDZ+NAA medium carries out differentiation adventitious buds, propagation, 1/2MS+IBA medium carries out culture of rootage, transplant after seedling hardening of taking root, the plant regeneration system formed.
The tissue culture method of described precipice, Guangdong beans rattan, comprises the steps:
(1) seed disinfection and vernalization: select evenly full precipice, Guangdong beans rattan (Millettia fordii Dunn) seed, first use tap water, then after desinfection chamber sterilization, carry out vernalization;
(2) explant is selected: the aseptic plumular axis sprouted after selecting step (1) vernalization is explant;
(3) adventitious bud proliferation: be inoculated in subculture multiplication medium by the explant of step (2), carry out Multiplying culture, obtains indefinite bud;
(4) culture of rootage: indefinite bud step (3) obtained, is inoculated in root induction on root media, obtains seedling of taking root;
(5) hardening and transplanting: the seedling of taking root step (4) obtained, transplants after hardening, obtains precipice, Guangdong beans rattan seedling.
The condition of the sterilization described in step (1) is preferably with 75% (v/v) alcohol disinfecting, 8 ~ 15min, with aseptic water washing 2 ~ 3 times; Again with 1g/L mercury chloride sterilization 4 ~ 8min, with aseptic water washing 4 ~ 6 times;
The condition of the vernalization described in step (1) is preferably the seed after by sterilization and is inoculated in aseptic moistening gauze or MS medium, vernalization 20 ~ 30 days, temperature 20 ~ 25 DEG C, light application time 12h/d, intensity of illumination 2,000 ~ 3,000lux;
The composition of the subculture multiplication medium described in step (3) is preferably MS medium+6-BA 0.3 ~ 0.5mg/L+TDZ 0.06 ~ 0.1mg/L+NAA 0.15 ~ 0.3mg/L+ sucrose 30g/L+ carragheen 5.8g/L, pH value 5.5 ~ 5.8; Described 6-BA refers to 6-benzyladenine; Described NAA, refers to a-methyl α-naphthyl acetate; Described TDZ refers to thiadiazole phenylurea, trade name Thidiazuron (Thidiazuron, N-Phenyl-N '-1,2,3-thiadiazol-5-ylurea, N-phenyl-N-1,2,3-thiadiazoles-5-urea, TDZ);
The condition of the cultivation described in step (3) is preferably temperature 24 ~ 30 DEG C, cultivate 24 ~ 30 days, relative moisture 60% ~ 70%, light application time 12h/d, intensity of illumination 2,000 ~ 3,000lux;
The composition of the root media described in step (4) is preferably 1/2MS medium+IBA 0.3 ~ 0.4mg/L+ sucrose 15g/L+ carragheen 5.8g/L, pH value 5.5 ~ 5.8; Described IBA, refers to indolebutyric acid; Described 1/2MS medium refers to that the macroelement of MS medium reduces by half;
The condition of the root induction described in step (4) is preferably temperature 28 ~ 32 DEG C, relative moisture 75% ~ 90%, and illumination condition is the rear first light culture of inoculation 5 days, cultivates 25 ~ 30 days with natural scattering light subsequently, light application time 12h/d;
The condition of the hardening described in step (5) preferably will be taken root after seedling proceeds to film booth, hardening 25 ~ 30 days;
Transplant in yellow soil after the condition of the transplanting described in step (5) is preferably hardening, cover film, adds a cover sunshade net, and transplant after 7 ~ 10 days, the bactericide spraying 0.1% (m/m) once, lifts thin film and sunshade net after 30 ~ 40 days;
The present invention has following advantage and effect relative to prior art:
(1) the seedling genetic character obtained by method of the present invention is good, and result is reliable and stable.The material that the present invention adopts is elite seed, and according to the totipotency of plant cell, the descendant inheritting of each seed growing is very consistent, can keep the merit of original kind.
(2) Experimental Establishment is less demanding, and operating process is simple.The present invention is adopted to cultivate precipice, Guangdong beans rattan seedling, required instrument and medicine are group training conventional equipment and common drug, the operating process such as involved explant selection and sterilization, adventitious bud proliferation, culture of rootage and test-tube seedling transplanting, general personnel all can reach technical requirement through training, and general Zu Pei factory all can carry out the group training production of precipice, Guangdong beans rattan according to the present invention.
(3) method of the present invention has the advantages that reproduction speed is fast, growing-seedling period is short, with low cost.
(4) reproduction coefficient is high, and be produced on a large scale precipice, Guangdong beans rattan seedling.Precipice, Guangdong of the present invention beans rattan tissue culture technology is that the carrying out under manual control condition produces, less by affecting in natural environment; Only need intercept seed plumular axis as propagating materials, once, proliferation times reaches 4.2 to 25 days propagation, and within 30 days, can complete culture of rootage, rooting rate reaches more than 60%; Utilize that the present invention can carry out in the anniversary, precipice, batch production amount reproduction Guangdong beans rattan seedling.
Accompanying drawing explanation
Fig. 1 is the schematic diagram being inoculated into subculture multiplication medium adventitious bud proliferation after 10 days in embodiment 1.
Fig. 2 is the schematic diagram being inoculated into subculture multiplication medium adventitious bud proliferation after 25 days in embodiment 1.
Fig. 3 is inoculated into root media to take root after 20 days the schematic diagram of seedling in embodiment 1.
Fig. 4 is inoculated into root media to take root after 30 days the schematic diagram of seedling in embodiment 1.
Fig. 5 be transplant in embodiment 1 with yellow soil be matrix Seedling bag in take root after 15 days the schematic diagram of seedling.
Fig. 6 be transplant in embodiment 1 with yellow soil be matrix Seedling bag in take root after 30 days the schematic diagram of seedling.
Fig. 7 is the schematic diagram being inoculated into subculture multiplication medium adventitious bud proliferation after 13 days in embodiment 2.
Fig. 8 is the schematic diagram being inoculated into subculture multiplication medium adventitious bud proliferation after 28 days in embodiment 2.
Fig. 9 is inoculated into root media to take root after 20 days the schematic diagram of seedling in embodiment 2.
Figure 10 is inoculated into root media to take root after 30 days the schematic diagram of seedling in embodiment 2.
Figure 11 be transplant in embodiment 2 with yellow soil be matrix Seedling bag in take root after 10 days the schematic diagram of seedling.
Figure 12 be transplant in embodiment 2 with yellow soil be matrix Seedling bag in take root after 25 days the schematic diagram of seedling.
Figure 13 is the schematic diagram being inoculated into subculture multiplication medium adventitious bud proliferation after 15 days in embodiment 3.
Figure 14 is the schematic diagram being inoculated into subculture multiplication medium adventitious bud proliferation after 25 days in embodiment 3.
Figure 15 is inoculated into root media to take root after 15 days the schematic diagram of seedling in embodiment 3.
Figure 16 is inoculated into root media to take root after 30 days the schematic diagram of seedling in embodiment 3.
Figure 17 be transplant in embodiment 3 with yellow soil be matrix Seedling bag in take root after 12 days the schematic diagram of seedling.
Figure 18 be transplant in embodiment 3 with yellow soil be matrix Seedling bag in take root after 30 days the schematic diagram of seedling.
Embodiment
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited thereto.
Embodiment 1
(1) seed disinfection and vernalization: precipice, Guangdong beans rattan (Millettia fordii Dunn) seed is purchased from Guangzhou great You Yuxuan agricultural development Co., Ltd; Select ripe, full, uniform seed, after carefully cleaning epidermis with running water, be positioned on superclean bench; In sterilizing bottle, pour the alcohol of 75% into, double swerve, sterilization 8min, with aseptic water washing 2 times; Pour the mercury chloride sterilization 4min of 1g/L again into, finally use aseptic water washing 4 times; Be placed in aseptic moistening gauze, with gauze planting subcovering.After sterilization, the pollution-free rate of seed can reach 70.6%.
(2) select explant: after seed inoculates 15 days, start rudiment; When 30 days, it is high that 3cm is grown in rudiment, selects the rudiment bar of free of contamination stalwartness, after cutting away radicle and cotyledon, gets plumular axis as explant.Total germination rate reaches 85.0%.
(3) adventitious bud proliferation: the explant that step (2) obtains is inoculated in subculture multiplication medium (MS medium+6-BA 0.3mg/L+TDZ 0.1mg/L+NAA 0.15mg/L+ sucrose 30g/L+ carragheen 5.8g/L, pH value 5.5) on, carry out Multiplying culture, obtain indefinite bud; Condition of culture: temperature 24 DEG C, relative moisture 60%, light application time 12h/d, light intensity 3,000lux, within every 25 days, by the switching of the indefinite bud of propagation once result of the test is shown in Fig. 1 and Fig. 2; Shoot proliferation multiple can reach 3.2.
(4) culture of rootage: the indefinite bud that step (3) is obtained, superclean bench is cut into 2 ~ 2.5cm long, be inoculated in the upper cultivation of root media (1/2MS medium+IBA 0.3mg/L+ sucrose 15g/L+ carragheen 5.8g/L, pH value 5.5), obtain seedling of taking root; Condition of culture: temperature 28 DEG C, relative moisture 75%, illumination condition are the rear first light culture of inoculation 5 days, cultivate 25 days with natural scattering light subsequently, light application time 12h/d, and result of the test is shown in Fig. 3 and Fig. 4; Rooting rate reaches 62%.
(5) hardening and transplanting: after the seedling of taking root that step (4) obtains is proceeded to film booth, hardening 30 days, grow to after 1 ~ 2cm until root system, take out bottle seedling, transplant after washing medium off in yellow soil, cover film, add a cover sunshade net, transplant after 7 days, the bactericide of spray 0.1% (m/m) once, thin film and sunshade net is lifted after 30 days, obtain precipice, Guangdong beans rattan seedling, manage by the conventional method in nursery, result of the test is shown in Fig. 5 and Fig. 6.
Embodiment 2
(1) seed disinfection and vernalization: precipice, Guangdong beans rattan (Millettia fordii Dunn) seed is purchased from Guangzhou great You Yuxuan agricultural development Co., Ltd; Select ripe, full, uniform seed, after carefully cleaning epidermis with running water, be positioned on superclean bench; In sterilizing bottle, pour the alcohol of 75% into, double swerve, sterilization 12min, with aseptic water washing 2 times; Pour the mercury chloride sterilization 6min of 1g/L again into, finally use aseptic water washing 4 times; Be placed in aseptic moistening gauze, with gauze planting subcovering.After sterilization, the pollution-free rate of seed can reach 73%.
(2) select explant: after seed inoculates 15 days, start rudiment; When 30 days, it is high that 3cm is grown in rudiment, selects the rudiment bar of free of contamination stalwartness, after cutting away radicle and cotyledon, gets plumular axis as explant.Total germination rate reaches 85.2%.
(3) adventitious bud proliferation: the explant that step (2) obtains is inoculated in subculture multiplication medium (MS medium+6-BA 0.4mg/L+TDZ 0.08mg/L+NAA 0.2mg/L+ sucrose 30g/L+ carragheen 5.8g/L, pH value 5.6) on, carry out Multiplying culture, obtain indefinite bud; Condition of culture: temperature 28 DEG C, relative moisture 65%, light application time 12h/d, light intensity 2,500lux, within every 25 days, by the switching of the indefinite bud of propagation once result of the test is shown in Fig. 7 and Fig. 8; Shoot proliferation multiple can reach 3.8.
(4) culture of rootage: indefinite bud step (3) obtained, is inoculated in the upper cultivation of root media (1/2MS medium+IBA 0.35mg/L+ sucrose 15g/L+ carragheen 5.8g/L, pH value 5.7), obtains seedling of taking root; Condition of culture: temperature 30 DEG C, relative moisture 80%, illumination condition are the rear first light culture of inoculation 5 days, cultivate 25 days with natural scattering light subsequently, light application time 12h/d, and result of the test is shown in Fig. 9 and Figure 10; Rooting rate reaches 64%.
(5) hardening and transplanting: after the seedling of taking root that step (4) obtains is proceeded to film booth, hardening 30 days, grow to after 1 ~ 2cm until root system, take out bottle seedling, transplant after washing medium off in yellow soil, cover film, add a cover sunshade net, transplant after 8 days, the bactericide of spray 0.1% (m/m) once, thin film and sunshade net is lifted after 35 days, obtain precipice, Guangdong beans rattan seedling, manage by the conventional method in nursery, result of the test is shown in Figure 11 and Figure 12.
Embodiment 3
(1) seed disinfection and vernalization: precipice, Guangdong beans rattan (Millettia fordii Dunn) seed is purchased from Guangzhou great You Yuxuan agricultural development Co., Ltd; Select ripe, full, uniform seed, after carefully cleaning epidermis with running water, be positioned on superclean bench; In sterilizing bottle, pour the alcohol of 75% into, double swerve, sterilization 15min, with aseptic water washing 2 times; Pour the mercury chloride sterilization 8min of 1g/L again into, finally use aseptic water washing 4 times; Be placed in aseptic moistening gauze, with gauze planting subcovering.After sterilization, the pollution-free rate of seed can reach 90%.
(2) select explant: after seed inoculates 15 days, start rudiment; When 30 days, it is high that 3cm is grown in rudiment, selects the rudiment bar of free of contamination stalwartness, after cutting away radicle and cotyledon, gets plumular axis as explant.Total germination rate reaches 86%.
(3) adventitious bud proliferation: the explant that step (2) obtains is inoculated in subculture multiplication medium (MS medium+6-BA 0.5mg/L+TDZ 0.1mg/L+NAA 0.3mg/L+ sucrose 30g/L+ carragheen 5.8g/L, pH value 5.8) on, carry out Multiplying culture, obtain indefinite bud; Condition of culture: temperature 30 DEG C, relative moisture 70%, light application time 12h/d, light intensity 2,000lux, within every 25 days, by the switching of the indefinite bud of propagation once result of the test is shown in Figure 13 and Figure 14; Shoot proliferation multiple can reach 4.2.
(4) culture of rootage: indefinite bud step (3) obtained, is inoculated in the upper cultivation of root media (1/2MS medium+IBA 0.4mg/L+ sucrose 15g/L+ carragheen 5.8g/L, pH value 5.8), obtains seedling of taking root; Condition of culture: temperature 32 DEG C, relative moisture 90%, illumination condition are the rear first light culture of inoculation 5 days, cultivate 25 days with natural scattering light subsequently, light application time 12h/d, and result of the test is shown in Figure 15 and Figure 16; Rooting rate reaches 64.6%.
(5) hardening and transplanting: after the seedling of taking root that step (4) obtains is proceeded to film booth, hardening 30 days, grow to after 1 ~ 2cm until root system, take out bottle seedling, transplant after washing medium off in yellow soil, cover film, add a cover sunshade net, transplant after 10 days, the bactericide of spray 0.1% (m/m) once, thin film and sunshade net is lifted after 40 days, obtain precipice, Guangdong beans rattan seedling, manage by the conventional method in nursery, result of the test is shown in Figure 17 and Figure 18.
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from Spirit Essence of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (8)
1. a tissue culture method for precipice, Guangdong beans rattan, is characterized in that comprising the steps:
(1) seed disinfection and vernalization: select evenly full precipice, Guangdong beans rattan (Millettia fordii Dunn) seed, first use tap water, then after desinfection chamber sterilization, carry out vernalization;
(2) explant is selected: the aseptic plumular axis sprouted after selecting step (1) vernalization is explant;
(3) adventitious bud proliferation: be inoculated in subculture multiplication medium by the explant of step (2), carry out Multiplying culture, obtains indefinite bud;
(4) culture of rootage: indefinite bud step (3) obtained, is inoculated in root induction on root media, obtains seedling of taking root;
(5) hardening and transplanting: the seedling of taking root step (4) obtained, transplants after hardening, obtains precipice, Guangdong beans rattan seedling;
Subculture multiplication medium described in step (3) consist of MS medium+6-BA 0.3 ~ 0.5mg/L+TDZ 0.06 ~ 0.1mg/L+NAA 0.15 ~ 0.3mg/L+ sucrose 30g/L+ carragheen 5.8g/L, pH value 5.5 ~ 5.8.
2. the tissue culture method of precipice, Guangdong according to claim 1 beans rattan, is characterized in that:
The condition of the sterilization described in step (1) for volume fraction 75% alcohol disinfecting 8 ~ 15min, with aseptic water washing 2 ~ 3 times; Again with 1g/L mercury chloride sterilization 4 ~ 8min, with aseptic water washing 4 ~ 6 times.
3. the tissue culture method of precipice, Guangdong according to claim 1 beans rattan, is characterized in that:
The condition of the vernalization described in step (1) be by sterilization after seed be inoculated in aseptic moistening gauze or MS medium, vernalization 20 ~ 30 days, temperature 20 ~ 25 DEG C, light application time 12h/d, intensity of illumination 2,000 ~ 3,000lux.
4. the tissue culture method of precipice, Guangdong according to claim 1 beans rattan, is characterized in that:
The condition of the cultivation described in step (3) is temperature 24 ~ 30 DEG C, cultivate 24 ~ 30 days, relative moisture 60% ~ 70%, light application time 12h/d, intensity of illumination 2,000 ~ 3,000lux.
5. the tissue culture method of precipice, Guangdong according to claim 1 beans rattan, is characterized in that:
Root media described in step (4) consist of 1/2MS medium+IBA 0.3 ~ 0.4mg/L+ sucrose 15g/L+ carragheen 5.8g/L, pH value 5.5 ~ 5.8.
6. the tissue culture method of precipice, Guangdong according to claim 1 beans rattan, is characterized in that:
The condition of the root induction described in step (4) is temperature 28 ~ 32 DEG C, relative moisture 75% ~ 90%, and illumination condition is the rear first light culture of inoculation 5 days, cultivates 25 ~ 30 days with natural scattering light subsequently, light application time 12h/d.
7. the tissue culture method of precipice, Guangdong according to claim 1 beans rattan, is characterized in that:
The condition of the hardening described in step (5) for after seedling of taking root proceeds to film booth, hardening 25 ~ 30 days.
8. the tissue culture method of precipice, Guangdong according to claim 1 beans rattan, is characterized in that:
The condition of the transplanting described in step (5) is transplant in yellow soil after hardening, and cover film, adds a cover sunshade net, transplants after 7 ~ 10 days, sprays the bactericide of mass percent 0.1% once, lift thin film and sunshade net after 30 ~ 40 days.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410798739.1A CN104429971B (en) | 2014-12-18 | 2014-12-18 | Tissue culture seedling-raising method of millettia fordii dunn |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410798739.1A CN104429971B (en) | 2014-12-18 | 2014-12-18 | Tissue culture seedling-raising method of millettia fordii dunn |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104429971A true CN104429971A (en) | 2015-03-25 |
| CN104429971B CN104429971B (en) | 2017-01-11 |
Family
ID=52877334
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410798739.1A Expired - Fee Related CN104429971B (en) | 2014-12-18 | 2014-12-18 | Tissue culture seedling-raising method of millettia fordii dunn |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104429971B (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105935020A (en) * | 2016-04-26 | 2016-09-14 | 广西智宝科技有限公司 | Plantation method of millettia speciosa champ |
| CN106550872A (en) * | 2016-11-02 | 2017-04-05 | 甘肃省治沙研究所 | A kind of Ramulus et Folium Tamariciss Rapid Rooting culture medium and Ramulus et Folium Tamariciss method for tissue culture |
| CN106688893A (en) * | 2017-01-15 | 2017-05-24 | 黄振忠 | Beautiful millettia root tissue culture seedling raising method |
| CN106962188A (en) * | 2017-03-01 | 2017-07-21 | 防城港市防城区蚕种场 | Beautiful millettia root seed directly induces the method from bud and fast breeding seedling |
| CN107125134A (en) * | 2017-05-04 | 2017-09-05 | 南宁市辉雄中药材农民专业合作社 | A kind of beautiful millettia root high yield cultivating method |
| CN108739387A (en) * | 2018-06-07 | 2018-11-06 | 金寨县金绿源特色中药材种植农场 | A kind of method for culturing seedlings of wild bletilla striata |
| CN109548650A (en) * | 2018-10-10 | 2019-04-02 | 广西医科大学 | A kind of breeding method of beautiful millettia root |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101124891A (en) * | 2006-08-15 | 2008-02-20 | 钦州市中医药研究所 | Method for tissue culture propagation of beautiful millettia root seedlings |
| CN102893870A (en) * | 2012-10-09 | 2013-01-30 | 冯志祥 | Rapid propagation and seedling raising method for beautiful millettia root seedling tissue culture |
| CN103039361A (en) * | 2013-01-11 | 2013-04-17 | 广西壮族自治区药用植物园 | Method for directly inducing cluster buds of milletia speciosa champ seeds and quickly breeding seedlings |
-
2014
- 2014-12-18 CN CN201410798739.1A patent/CN104429971B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101124891A (en) * | 2006-08-15 | 2008-02-20 | 钦州市中医药研究所 | Method for tissue culture propagation of beautiful millettia root seedlings |
| CN102893870A (en) * | 2012-10-09 | 2013-01-30 | 冯志祥 | Rapid propagation and seedling raising method for beautiful millettia root seedling tissue culture |
| CN103039361A (en) * | 2013-01-11 | 2013-04-17 | 广西壮族自治区药用植物园 | Method for directly inducing cluster buds of milletia speciosa champ seeds and quickly breeding seedlings |
Non-Patent Citations (2)
| Title |
|---|
| 韦玉燕等: "牛大力研究概况", 《广西科学院学报》 * |
| 黄艳宁等: "香花崖豆藤的组织培养与快速繁殖", 《植物生理学通讯》 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105935020A (en) * | 2016-04-26 | 2016-09-14 | 广西智宝科技有限公司 | Plantation method of millettia speciosa champ |
| CN106550872A (en) * | 2016-11-02 | 2017-04-05 | 甘肃省治沙研究所 | A kind of Ramulus et Folium Tamariciss Rapid Rooting culture medium and Ramulus et Folium Tamariciss method for tissue culture |
| CN106550872B (en) * | 2016-11-02 | 2018-10-30 | 甘肃省治沙研究所 | A kind of Chinese tamarisk Rapid Rooting culture medium and Chinese tamarisk method for tissue culture |
| CN106688893A (en) * | 2017-01-15 | 2017-05-24 | 黄振忠 | Beautiful millettia root tissue culture seedling raising method |
| CN106962188A (en) * | 2017-03-01 | 2017-07-21 | 防城港市防城区蚕种场 | Beautiful millettia root seed directly induces the method from bud and fast breeding seedling |
| CN107125134A (en) * | 2017-05-04 | 2017-09-05 | 南宁市辉雄中药材农民专业合作社 | A kind of beautiful millettia root high yield cultivating method |
| CN108739387A (en) * | 2018-06-07 | 2018-11-06 | 金寨县金绿源特色中药材种植农场 | A kind of method for culturing seedlings of wild bletilla striata |
| CN109548650A (en) * | 2018-10-10 | 2019-04-02 | 广西医科大学 | A kind of breeding method of beautiful millettia root |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104429971B (en) | 2017-01-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104429971B (en) | Tissue culture seedling-raising method of millettia fordii dunn | |
| CN102960245B (en) | Artificial breeding and cultivating method of Dendrobium officinale | |
| CN102422816A (en) | Method for quickly culturing shoot tips in vitro | |
| CN103734014B (en) | A kind of quick breeding method for tissue culture of anisetree bark | |
| CN103931492A (en) | Tissue-culture rapid seedling growing method for apple rootstock M9 | |
| CN102067783B (en) | Miniature lotus root seed breeding technique for lotus root | |
| CN105706694B (en) | Cultural method in strong winter habit wheat room | |
| CN107018896B (en) | A kind of method of facility cuttage tilia miqueliana | |
| CN103380729B (en) | A kind of method of biplate lettuce tongue tissue cultures and Fast-propagation | |
| CN105993956A (en) | Fast propagating method for atractylis lancea | |
| CN106376328A (en) | Wild bletila striata seed quick breeding method | |
| CN106069543B (en) | A kind of Kiwi berry detoxication and tissue culture method for transplanting | |
| CN103548692A (en) | Tissue culture rapid propagation method for asplenium antiquum osaka germchit | |
| CN103168690B (en) | Breeding method of Qi dioscorea opposita virus-free miniature seed beans | |
| CN105145363B (en) | It is a kind of to significantly improve the method that China fir tissue culture produces emergence rate | |
| CN104542307A (en) | Culturing method of momordica cochinchinensis | |
| CN105010142B (en) | The method of the strange nanmu agalloch eaglewood tissue cultures of Vietnam | |
| CN103548695B (en) | A kind of meadowrueleaf corydalis root quick breeding method for tissue culture | |
| CN103155868A (en) | Rapid seeding raising method of cherry rootstock ZY-1 tissue culture | |
| CN104365484B (en) | The tissue culture propagation method of butterfly flower cuckoo | |
| CN106489503A (en) | Oil tree peony Clonal regeneration method for culturing seedlings | |
| CN110278874A (en) | A kind of narrow crown black poplar 11 tissue culture and rapid propagation methods | |
| CN108077067B (en) | Tissue culture and rapid propagation method of cotton rose | |
| CN101248758B (en) | Tissue culture method for fine stalk double butterflies | |
| CN112106664B (en) | Sterile germination and rapid propagation method for michelia spectabilis seeds |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C41 | Transfer of patent application or patent right or utility model | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20161026 Address after: My Xin Zhen He Yi Cun quassiawood Zengcheng District in Guangzhou city of Guangdong province 511365 Applicant after: Guangzhou Jingli Biotechnology Co.,Ltd. Address before: 510520, No. 233, Guang Shan Road, Tianhe District, Guangdong, Guangzhou Applicant before: Zeng Lei Applicant before: Xie Jianneng |
|
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20170111 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |