CN107295970A9 - Method for inducing castor anther explant to form callus - Google Patents
Method for inducing castor anther explant to form callus Download PDFInfo
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- CN107295970A9 CN107295970A9 CN201710571480.0A CN201710571480A CN107295970A9 CN 107295970 A9 CN107295970 A9 CN 107295970A9 CN 201710571480 A CN201710571480 A CN 201710571480A CN 107295970 A9 CN107295970 A9 CN 107295970A9
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- castor
- explant
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- oil plant
- flower pesticide
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- 206010020649 Hyperkeratosis Diseases 0.000 title claims abstract description 90
- 235000004443 Ricinus communis Nutrition 0.000 title claims abstract description 81
- 230000001939 inductive effect Effects 0.000 title claims abstract description 34
- 238000000034 method Methods 0.000 title claims abstract description 27
- JPVYNHNXODAKFH-UHFFFAOYSA-N Cu2+ Chemical compound [Cu+2] JPVYNHNXODAKFH-UHFFFAOYSA-N 0.000 claims abstract description 14
- 229910001431 copper ion Inorganic materials 0.000 claims abstract description 14
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 claims abstract description 13
- 240000000528 Ricinus communis Species 0.000 claims description 77
- 239000000575 pesticide Substances 0.000 claims description 68
- 238000012545 processing Methods 0.000 claims description 26
- 238000007654 immersion Methods 0.000 claims description 9
- 230000006698 induction Effects 0.000 abstract description 34
- 239000001963 growth medium Substances 0.000 abstract description 12
- 238000002791 soaking Methods 0.000 abstract 1
- 238000011282 treatment Methods 0.000 description 12
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- 238000007796 conventional method Methods 0.000 description 6
- 230000014509 gene expression Effects 0.000 description 6
- 238000005286 illumination Methods 0.000 description 6
- 239000007788 liquid Substances 0.000 description 6
- 238000007619 statistical method Methods 0.000 description 6
- 230000000763 evoking effect Effects 0.000 description 5
- 241000196324 Embryophyta Species 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- NLKNQRATVPKPDG-UHFFFAOYSA-M potassium iodide Chemical compound [K+].[I-] NLKNQRATVPKPDG-UHFFFAOYSA-M 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 241000238631 Hexapoda Species 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical class [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- NWBJYWHLCVSVIJ-UHFFFAOYSA-N N-benzyladenine Chemical compound N=1C=NC=2NC=NC=2C=1NCC1=CC=CC=C1 NWBJYWHLCVSVIJ-UHFFFAOYSA-N 0.000 description 2
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 description 2
- 235000021307 Triticum Nutrition 0.000 description 2
- 244000098338 Triticum aestivum Species 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 2
- 229910000366 copper(II) sulfate Inorganic materials 0.000 description 2
- 230000032459 dedifferentiation Effects 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000003921 oil Substances 0.000 description 2
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 description 2
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 239000005631 2,4-Dichlorophenoxyacetic acid Substances 0.000 description 1
- PAWQVTBBRAZDMG-UHFFFAOYSA-N 2-(3-bromo-2-fluorophenyl)acetic acid Chemical compound OC(=O)CC1=CC=CC(Br)=C1F PAWQVTBBRAZDMG-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- LGNCPYIUMKDLCM-UHFFFAOYSA-N C=C.[Na].O.O Chemical group C=C.[Na].O.O LGNCPYIUMKDLCM-UHFFFAOYSA-N 0.000 description 1
- 241000207199 Citrus Species 0.000 description 1
- 240000008853 Datura stramonium Species 0.000 description 1
- 240000001008 Dimocarpus longan Species 0.000 description 1
- QXNVGIXVLWOKEQ-UHFFFAOYSA-N Disodium Chemical compound [Na][Na] QXNVGIXVLWOKEQ-UHFFFAOYSA-N 0.000 description 1
- 241000221017 Euphorbiaceae Species 0.000 description 1
- 235000000235 Euphoria longan Nutrition 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 235000011430 Malus pumila Nutrition 0.000 description 1
- 244000070406 Malus silvestris Species 0.000 description 1
- 235000015103 Malus silvestris Nutrition 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 244000131316 Panax pseudoginseng Species 0.000 description 1
- 235000005035 Panax pseudoginseng ssp. pseudoginseng Nutrition 0.000 description 1
- 235000003140 Panax quinquefolius Nutrition 0.000 description 1
- 235000003846 Ricinus Nutrition 0.000 description 1
- 241000322381 Ricinus <louse> Species 0.000 description 1
- 241000124033 Salix Species 0.000 description 1
- 239000005708 Sodium hypochlorite Substances 0.000 description 1
- 235000000208 Solanum incanum Nutrition 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 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 1
- 235000009754 Vitis X bourquina Nutrition 0.000 description 1
- 235000012333 Vitis X labruscana Nutrition 0.000 description 1
- 240000006365 Vitis vinifera Species 0.000 description 1
- 235000014787 Vitis vinifera Nutrition 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 1
- 239000004327 boric acid Substances 0.000 description 1
- LLSDKQJKOVVTOJ-UHFFFAOYSA-L calcium chloride dihydrate Chemical class O.O.[Cl-].[Cl-].[Ca+2] LLSDKQJKOVVTOJ-UHFFFAOYSA-L 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 235000020971 citrus fruits Nutrition 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 238000009402 cross-breeding Methods 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- VCSZKSHWUBFOOE-UHFFFAOYSA-N dioxidanium;sulfate Chemical compound O.O.OS(O)(=O)=O VCSZKSHWUBFOOE-UHFFFAOYSA-N 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 239000011790 ferrous sulphate Substances 0.000 description 1
- 235000003891 ferrous sulphate Nutrition 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 235000008434 ginseng Nutrition 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 150000003840 hydrochlorides Chemical class 0.000 description 1
- 150000004001 inositols Chemical class 0.000 description 1
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 1
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- ISPYRSDWRDQNSW-UHFFFAOYSA-L manganese(II) sulfate monohydrate Chemical class O.[Mn+2].[O-]S([O-])(=O)=O ISPYRSDWRDQNSW-UHFFFAOYSA-L 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 235000016768 molybdenum Nutrition 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 235000001968 nicotinic acid Nutrition 0.000 description 1
- 229960003512 nicotinic acid Drugs 0.000 description 1
- 239000011664 nicotinic acid Substances 0.000 description 1
- 238000009400 out breeding Methods 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 238000003976 plant breeding Methods 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical class [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 239000004323 potassium nitrate Substances 0.000 description 1
- 235000010333 potassium nitrate Nutrition 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 238000005204 segregation Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- BQMKAHQKDSZAIQ-UHFFFAOYSA-N tetrasodium;iron(3+);nitroxyl anion;pentacyanide Chemical compound [Na+].[Na+].[Na+].[Na+].[Fe+3].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].O=[N-] BQMKAHQKDSZAIQ-UHFFFAOYSA-N 0.000 description 1
- 235000019190 thiamine hydrochloride Nutrition 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 230000009261 transgenic effect Effects 0.000 description 1
- 235000009529 zinc sulphate Nutrition 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
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
-
- 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Developmental Biology & Embryology (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 a castor anther explant to form callus, which comprises the following steps of S1, soaking the castor anther explant in 15-30 mg/L BA solution for 10-20 min, and S2, inoculating the castor anther explant processed by S1 to a callus induction culture medium for culture to obtain callus, wherein the callus induction culture medium is an MS culture medium added with 4-16 mg/L SNP, or an MS culture medium added with 1-2 mg/L copper ions, or an MS culture medium added with 3-6 mg/L glutamine.
Description
Technical field
The present invention relates to field of plant cell engineering technology, and in particular, to a kind of method for inducing castor-oil plant flower pesticide explant to form callus.
Background technology
Since Guhas reports that mao Thorn-Apple Anther Culture obtains haplobiont first, research on Anther Culture develops rapidly, many countries including China carry out the research work of this respect in succession, and about 200 various crops are cultivated by flower pesticide and obtain haplobiont so far.
In breeding field, Breeding by anther culture is combined with conventional cross-breeding, distant hybridization breeding, mutation breeding and transgenic technology, have become biotechnology one of relatively broad and effective method in crop breeding, be crop improvement and science of heredity and physiology, the important method of Biochemical Research.Up to the present, China has 40 various plants and obtains pollen plant with anther culture method, mainly there is some medicinal plants such as wheat, corn, rice, grape, citrus, apple, longan, rubber, willow and ginseng.A paddy pollen new lines and new varieties more than 80 are turned out, a Wheat Pollen new varieties and new lines more than 20.
Castor-oil plant is that Euphorbiaceae Ricinus is annual or herbaceos perennial, it is one of ten big oil crops in the world, there is the features such as widely used, economic value is high, it is the essential industry oil with special industry purposes, its seed, blade, cane, shell comprehensive utilization value are high.At present, competitive improved seeds are also lacked.
For a long time due to castor-oil plant mainly by hybridize, select, introduce a fine variety etc. it is traditional in a manner of carry out breeding, greatly limit its development.Haplobiont can be obtained using Anther Culture, then after carrying out chromosome doubling to it, can quickly obtain widely applied pure line cultivar in castor-oil plant breeding；And trait segregation will not occur for the offspring for the excellent homozygous line therefrom selected, neat and consistent is showed, can significantly shorten the breeding time limit.But at present, castor-oil plant Anther Culture is still in infancy, and the report in relation to castor-oil plant Anther Culture is also seldom, also there are castor-oil plant anther callus inductivity it is low poor with callus quality the problems such as, seriously constrain the progress of castor-oil plant vitro anther culture.Therefore, the tissue culture system that efficiently induction castor-oil plant flower pesticide explant forms callus is established, is all of great significance to the haploid breeding for accelerating castor-oil plant and the castor-oil plant industry for further developing China.
The content of the invention
The purpose of the invention is to overcome the deficiencies of the prior art and provide a kind of method for inducing castor-oil plant flower pesticide explant to form callus.
To achieve these goals, the present invention is achieved by the following technical programs：
A kind of method for inducing castor-oil plant flower pesticide explant to form callus, includes the following steps：
S1. castor-oil plant flower pesticide explant is placed in 15~30
10~20min is soaked in the BA solution of mg/L；
S2. it will be inoculated on callus inducing medium and cultivated by the castor-oil plant flower pesticide explant of S1 processing, obtain callus；The MS culture mediums that the callus inducing medium is the SNP that with the addition of 4~16mg/L, or the MS culture mediums of 1~2mg/L copper ions are with the addition of, or with the addition of the MS culture mediums of 3~6mg/L glutamine.
The method that tradition induction castor-oil plant flower pesticide explant forms callus is all to select flower pesticide explant being seeded on the culture medium containing various hormones directly to be induced, the creativeness of the present invention is the immersion treatment that an early period is first carried out to flower pesticide explant, and then explant is seeded on the culture medium of copper ions, SNP or glutamine again and carries out evoked callus.Carrying out immersion treatment early period to flower pesticide explant is advantageous in that the BA of high concentration can promote flower pesticide explant to form more callus with the efficiency dedifferentiation of higher, while improves the quality of obtained callus.
Preferably, castor-oil plant flower pesticide explant is placed in the BA solution of 15mg/L and soaks 10min.
Although identical explant generating process of evoked callus on different culture mediums is substantially consistent, its callus quality and inductivity are then different and different because of culture medium prescription；In addition, the quality and inductivity of the callus that different explant types induces on identical culture medium are also different.
Preferably, the MS culture mediums that the callus inducing medium is the SNP that with the addition of 8mg/L.
Preferably, the callus inducing medium is the MS culture mediums that with the addition of 2mg/L copper ions.
Preferably, the callus inducing medium is the MS culture mediums that with the addition of 6mg/L glutamine.
Preferably, the acquisition methods of the castor-oil plant flower pesticide explant are：Acquisition takes without insect food, the male flower that full and perianth is not yet opened, and surface sterilizing is carried out to castor-oil plant male flower using 2% sodium hypochlorite, and peels off the perianth on male flower surface, carefully obtains castor-oil plant flower pesticide explant therein.
The collocation method of the BA solution of various concentrations of the present invention is：Accurately weigh a certain amount of 6-benzyl aminopurine（6-BA, abbreviation BA）（Sigma, USA）Powder, is fully dissolved with the NaOH solution of 1 mol/L, then with deionized water constant volume, is configured to the BA processing solutions that concentration is respectively 0,7.5,15,30,60,120 mg/L.Then with the pH to 5.8~6.0 of the HCl solution of 1 mol/L adjustment BA processing solutions, the preceding water system filter membrane with 0.22 micron is used（Millipore, USA）Filtration sterilization is carried out to prepared BA processing solutions.
The MS culture mediums：The inorganic constituents of MS culture medium prescriptions（16.5 g/L ammonium nitrate, 19
G/L potassium nitrate, 1.7 g/L potassium dihydrogen phosphates, 3.7
G/L epsom salts, 4.4 g/L calcium chloride dihydrates, 16.9
Mg/L manganese sulfate monohydrates, 8.6 mg/L white vitriols, 6.2
Mg/L boric acid, 0.83 mg/L potassium iodide, 0.25
Mg/L sulfate dihydrate molybdenums disodium, 0.025 mg/L cupric sulfate pentahydrates, 0.025
Mg/L CoCL2 6H2Os, 37.3 mg/L, bis- water sodium ethylene diamine tetracetates, 27.8
Mg/L ferrous sulfate heptahydrates）The organic principle of+MS culture medium prescriptions（2
Mg/L glycine, 0.1 mg/L thiamine hydrochlorides, 0.5
Mg/L puridoxine hydrochlorides, 0.5 mg/L nicotinic acid）+100
+ 30 g/L sucrose+7 of mg/L inositols
G/L agar, pH to 5.8~6.0 is adjusted with 1 mg/L NaOH solutions.Culture medium sterilizes 15 minutes under conditions of 121 DEG C, 0.1 MPa.
Condition of culture described in step S2 is：When room temperature is 25 DEG C, intensity of illumination is 2000 lx, light application time is daily 12 small.
Compared with prior art, the present invention has the advantages that：
The novelty of the present invention is to change the method that tradition induction castor-oil plant flower pesticide explant forms callus, short-term immersion treatment is carried out to flower pesticide explant using high concentration BA solution, thus promotes flower pesticide explant to form more callus with the efficiency dedifferentiation of higher.The quality of obtained callus is improved at the same time, is had laid a good foundation for the work of castor-oil plant associated biomolecule technology Breeding.
Embodiment
The present invention is made with reference to specific embodiment and further being elaborated, the embodiment is served only for explaining the present invention, is not intended to limit the scope of the present invention.Test method used in following embodiments is conventional method unless otherwise specified；Used material, reagent etc., unless otherwise specified, for the reagent and material commercially obtained.
Embodiment
1
A kind of method for inducing castor-oil plant flower pesticide explant to form callus, includes the following steps：
(1) flower pesticide explant is obtained：Choose without insect food takes, full and perianth is not yet opened in spherical castor-oil plant male flower.Taking the time to be selected in, temperature is low, during the weak fine day morning 10 of illumination or so, the bud on castor-oil plant can reduce pollution without fog at this time, and illumination is weaker and can prevent the forfeiture of flower pesticide moisture.The male flower bud of collection is lived with tissue-wrapped, is first soaked with distilled water, then gently squeezes out unnecessary moisture, then puts into vial, is handled 3 days in 4 DEG C of refrigerator.First first sterilize 1 min to castor-oil plant male flower with 75% ethanol solution on superclean bench, then with 2% NaClO solution disinfection 20min, rinsed with sterile water castor-oil plant male flower 5 times.Carefully the perianth of male flower outer layer is removed with sterilized dissecting needle afterwards, then by flower pesticide careful separation therein, you can obtain castor-oil plant flower pesticide explant.
(2) castor-oil plant flower pesticide explant is seeded on the MS minimal mediums of addition various concentrations BA, room temperature be 25 DEG C, intensity of illumination is 2000 lx, light application time is daily 12 small when under conditions of evoked callus.The results are shown in Table 1, and adding formation of suitable BA to castor-oil plant anther callus has facilitation, and when the concentration of BA is 1 mg/L, the inductivity of castor-oil plant anther callus reaches maximum, is 10.67%；And continue to increase with the concentration of BA, the inductivity of castor-oil plant anther callus gradually reduces.Meanwhile experimental result is also shown that during forming callus using conventional method induction castor-oil plant flower pesticide explant, it is relatively low and second-rate to obtain the induced efficiency of callus, small volume.
Table 1 is influences of the BA to castor-oil plant induction of anther callus efficiency
Note：Data carry out variance analysis and Duncan Multiple range test using 17.0 statistical analysis softwares of SPSS Statistics（P ≦ 0.05）, significant difference between the different expressions processing of letter after data.
Embodiment
2
A kind of method for inducing castor-oil plant flower pesticide explant to form callus, includes the following steps：
(1) flower pesticide explant is obtained：With embodiment 1.
(2) castor-oil plant flower pesticide explant is placed in the BA solution of 15 mg/L and soaks different time（0~80 min）；
(3) and then by castor-oil plant flower pesticide explant it is placed on after sucking surplus liquid on sterile blotting paper, careful being seeded on no hormone MS culture mediums is cultivated 30 days.
Test result indicates that when the time of immersion treatment explant is 10 min, explant callus induction efficiency best results, up to 41.25%（Table 2）.Continue to extend processing time, castor-oil plant flower pesticide explant callus induction rate will be reduced gradually.
Table 2 is influence of the processing time to castor-oil plant induction of anther callus efficiency
Note：Data carry out variance analysis and Duncan Multiple range test using 17.0 statistical analysis softwares of SPSS Statistics（P ≦ 0.05）, significant difference between the different expressions processing of letter after data.
Embodiment
3
A kind of method for inducing castor-oil plant flower pesticide explant to form callus, includes the following steps：
(1) flower pesticide explant is obtained：With embodiment 1.
(2) castor-oil plant flower pesticide explant is placed in the BA solution of various concentrations (0~120 mg/L) and soaks 10min.
(3) and then by castor-oil plant flower pesticide explant it is placed on after sucking surplus liquid on sterile blotting paper, careful being seeded on no hormone MS culture mediums is cultivated 30 days.
Test result indicates that with the increase of BA solution concentrations, castor-oil plant flower pesticide explant callus induction efficiency will step up, when the concentration of immersion treatment explant is 15
During mg/L, explant callus induction best results, callus induction rate reaches maximum, is 41.25%（Table 3）.And continue to improve BA treatment fluid concentration, castor-oil plant flower pesticide explant callus induction efficiency will gradually reduce.Meanwhile during inducing castor-oil plant flower pesticide explant to form callus using the method for short-term high concentration BA solution immersion treatments, it was found that obtain that callus quality is preferable, and volume is larger.
Table 3 is influence of the BA solution concentrations to castor-oil plant induction of anther callus efficiency
Note：Data carry out variance analysis and Duncan Multiple range test using 17.0 statistical analysis softwares of SPSS Statistics（P ≦ 0.05）, significant difference between the different expressions processing of letter after data.
From above-mentioned experimental result, compared with forming callus using conventional method induction castor-oil plant flower pesticide explant, significantly improved using the callus induction rate of high concentration BA solution immersion treatment castor-oil plant flower pesticide explants institute induced synthesis（Callus induction rate improves 30.58%）, and the better quality of the callus obtained, volume bigger.Therefore, the immersion treatment of 10 min is carried out to castor-oil plant flower pesticide explant using 15 mg/L BA solution, can be used for achieving the purpose that to obtain more, better quality callus.
Embodiment
4
A kind of method for inducing castor-oil plant flower pesticide explant to form callus, includes the following steps：
(1) flower pesticide explant is obtained：With embodiment 1.
(2) castor-oil plant flower pesticide explant is placed in the BA solution of 15mg/L and soaks 10min.
(3) and then by castor-oil plant flower pesticide explant it is placed on after sucking surplus liquid on sterile blotting paper, careful is seeded to the sodium nitroprussiate that with the addition of various concentrations（SNP）（0th, 2,4,8,16 and 32mg/L）MS culture mediums on cultivate 30 days.
Test result indicates that, with the raising of SNP concentration, castor-oil plant induction of anther callus rate is also with improve, when SNP concentration is 8 mg/L, callus induction rate reaches maximum, for 60.37%, 49.70% is improved than conventional method, 19.12% is improved than being seeded to after excessive concentrations BA solution treatments on no hormone MS culture mediums；But when being to continue with improving the concentration of SNP, callus induction will be suppressed, and induced efficiency will gradually reduce.
Table 4 is influences of the SNP to castor-oil plant induction of anther callus efficiency
Note：Data carry out variance analysis and Duncan Multiple range test using 17.0 statistical analysis softwares of SPSS Statistics（P ≦ 0.05）, significant difference between the different expressions processing of letter after data.
Embodiment
5
A kind of method for inducing castor-oil plant flower pesticide explant to form callus, includes the following steps：
(1) flower pesticide explant is obtained：With embodiment 1.
(2) castor-oil plant flower pesticide explant is placed in the BA solution of 15mg/L and soaks 10min.
(3) and then by castor-oil plant flower pesticide explant it is placed on after sucking surplus liquid on sterile blotting paper, careful is seeded to the copper ion that with the addition of various concentrations（Anhydrous cupric sulfate）（0th, 0.5,1,2,4 and 8 mg/L）MS culture mediums on cultivate 30 days.
Test result indicates that, with the raising of copper ion concentration, castor-oil plant induction of anther callus rate is also with improve, when copper ion concentration is 2 mg/L, callus induction rate reaches maximum, for 68.36%, 57.69% is improved than conventional method, 27.11% is improved than being seeded to after excessive concentrations BA solution treatments on no hormone MS culture mediums；；But when being to continue with improving the concentration of copper ion, callus induction will be suppressed, and induced efficiency will gradually reduce.
Table 5 is influence of the copper ion to castor-oil plant induction of anther callus efficiency
Note：Data carry out variance analysis and Duncan Multiple range test using 17.0 statistical analysis softwares of SPSS Statistics（P ≦ 0.05）, significant difference between the different expressions processing of letter after data.
Embodiment
6
A kind of method for inducing castor-oil plant flower pesticide explant to form callus, includes the following steps：
(1) flower pesticide explant is obtained：With embodiment 1.
(2) castor-oil plant flower pesticide explant is placed in the BA solution of 15mg/L and soaks 10min.
(3) and then by castor-oil plant flower pesticide explant it is placed on after sucking surplus liquid on sterile blotting paper, careful is seeded to the glutamine that with the addition of various concentrations（0th, 1.5,3,6,12 and 24 mg/L）MS culture mediums on cultivate 30 days.
Test result indicates that with the raising of Gln concentration, castor-oil plant induction of anther callus rate is also with improve, when glutamine concentration is 6
During mg/L, callus induction rate reaches maximum, is 54.63%, improves 43.96% than conventional method, 13.38% is improved than being seeded to after excessive concentrations BA solution treatments on no hormone MS culture mediums；But when being to continue with improving the concentration of glutamine, callus induction will be suppressed, and induced efficiency will gradually reduce.
Table 6 is influence of the glutamine to castor-oil plant induction of anther callus efficiency
Note：Data carry out variance analysis and Duncan Multiple range test using 17.0 statistical analysis softwares of SPSS Statistics（P ≦ 0.05）, significant difference between the different expressions processing of letter after data.
Embodiment
7
A kind of method for inducing castor-oil plant flower pesticide explant to form callus, step (1) and (3) are the same as embodiment 2, it is that step (2) is soaked using different hormones from the unique different place of embodiment 2, the present embodiment includes following 4 different and column processing：Processing 1 is to soak flower pesticide explant 10min using the TDZ solution of 15mg/L；Processing 2 is to soak flower pesticide explant 10min using the NAA solution of 15mg/L；Processing 3 is to soak flower pesticide explant 10min using the IBA solution of 15mg/L；Processing 4 is to soak flower pesticide explant 10min using the IAA solution of 15mg/L.Callus induction after four kinds of different disposals the results are shown in Table 7.
Table 7 soaks the influence to castor-oil plant induction of anther callus efficiency for hormon
Embodiment
8
A kind of method for inducing castor-oil plant flower pesticide explant to form callus, includes the following steps：
(1) flower pesticide explant is obtained：With embodiment 1.
(2) by castor-oil plant flower pesticide explant be seeded in addition hormon MS minimal mediums on, room temperature be 25 DEG C, intensity of illumination is 2000 lx, light application time is daily 12 small when under conditions of evoked callus.The present embodiment includes following 2 different and column processing：Processing 1 uses the MS culture mediums of the NAA of the BA+0.2mg/L of addition 3mg/L as callus inducing medium；Processing 2 uses the MS culture mediums of the 2,4-D of the BA+2mg/L of addition 2mg/L as callus inducing medium；It the results are shown in Table 8.
Table 8 is influence of the different callus inducing mediums to castor-oil plant induction of anther callus efficiency
Embodiment
9
(1) flower pesticide explant is obtained：With embodiment 1.
(2) castor-oil plant flower pesticide explant is placed in the BA solution of 15mg/L and soaks 10min.
(3) and then by castor-oil plant flower pesticide explant it is placed on after sucking surplus liquid on sterile blotting paper, careful is seeded on the MS minimal mediums of addition hormon, room temperature be 25 DEG C, intensity of illumination is 2000 lx, light application time is daily 12 small when under conditions of evoked callus.The present embodiment includes following 4 different and column processing：Processing 1 is the MS culture mediums for the SNP+2mg/L copper ions for using addition 8mg/L as callus inducing medium；Processing 2 is using addition 2mg/L
The MS culture mediums of copper ion+6mg/L glutamine are as callus inducing medium；Processing 3 is the MS culture mediums for the SNP+6mg/L glutamine for using addition 8mg/L as callus inducing medium；Processing 4 adds the SNP+of 8mg/L plus the MS culture mediums of 2mg/L copper ion+6mg/L glutamine to use as callus inducing medium；It the results are shown in Table 9.
Table 9 is influence of the different callus inducing mediums to castor-oil plant induction of anther callus efficiency
Above example is served only for explaining the present invention, is not intended to limit the scope of the present invention.
Claims (5)
1..
A kind of method for inducing castor-oil plant flower pesticide explant to form callus, it is characterised in that include the following steps：
S1. castor-oil plant flower pesticide explant is placed in 10~20min of immersion in the BA solution of 15~30 mg/L；
S2. it will be inoculated on callus inducing medium and cultivated by the castor-oil plant flower pesticide explant of S1 processing, obtain callus；The MS culture mediums that the callus inducing medium is the SNP that with the addition of 4~16mg/L, or the MS culture mediums of 1~2mg/L copper ions are with the addition of, or with the addition of the MS culture mediums of 3~6mg/L glutamine.
2. according to the method described in claim 1, soak 10min it is characterized in that, castor-oil plant flower pesticide explant is placed in the BA solution of 15mg/L.
3. the according to the method described in claim 1, it is characterized in that, MS culture mediums that the callus inducing medium is the SNP that with the addition of 8mg/L.
4. according to the method described in claim 1, it is characterized in that, the callus inducing medium is the MS culture mediums that with the addition of 2mg/L copper ions.
5. according to the method described in claim 1, it is characterized in that, the callus inducing medium is the MS culture mediums that with the addition of 6mg/L glutamine.
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