CN107295970B - A method of induction castor-oil plant anther explant forms callus - Google Patents
A method of induction castor-oil plant anther explant forms callus Download PDFInfo
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- CN107295970B CN107295970B CN201710571480.0A CN201710571480A CN107295970B CN 107295970 B CN107295970 B CN 107295970B CN 201710571480 A CN201710571480 A CN 201710571480A CN 107295970 B CN107295970 B CN 107295970B
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- 206010020649 Hyperkeratosis Diseases 0.000 title claims abstract description 90
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- 240000000528 Ricinus communis Species 0.000 title claims abstract description 81
- 230000006698 induction Effects 0.000 title claims abstract description 46
- 238000000034 method Methods 0.000 title claims abstract description 30
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- 239000002609 medium Substances 0.000 claims abstract description 20
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- 229910001431 copper ion Inorganic materials 0.000 claims abstract description 13
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 claims abstract description 11
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- 238000007654 immersion Methods 0.000 claims description 12
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- 230000014509 gene expression Effects 0.000 description 6
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- 230000015572 biosynthetic process Effects 0.000 description 3
- NLKNQRATVPKPDG-UHFFFAOYSA-M potassium iodide Chemical compound [K+].[I-] NLKNQRATVPKPDG-UHFFFAOYSA-M 0.000 description 3
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- 241000238631 Hexapoda Species 0.000 description 2
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- NWBJYWHLCVSVIJ-UHFFFAOYSA-N N-benzyladenine Chemical compound N=1C=NC=2NC=NC=2C=1NCC1=CC=CC=C1 NWBJYWHLCVSVIJ-UHFFFAOYSA-N 0.000 description 2
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- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 description 2
- 238000011160 research Methods 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
- LFDGRWDETVOGDT-UHFFFAOYSA-N 1h-pyrrole;hydrochloride Chemical compound Cl.C=1C=CNC=1 LFDGRWDETVOGDT-UHFFFAOYSA-N 0.000 description 1
- 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
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 240000008853 Datura stramonium Species 0.000 description 1
- 240000001008 Dimocarpus longan Species 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
- SQUHHTBVTRBESD-UHFFFAOYSA-N Hexa-Ac-myo-Inositol Natural products CC(=O)OC1C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C1OC(C)=O SQUHHTBVTRBESD-UHFFFAOYSA-N 0.000 description 1
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 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
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 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
- 241000219000 Populus Species 0.000 description 1
- 235000003846 Ricinus Nutrition 0.000 description 1
- 241000322381 Ricinus <louse> 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
- LTZCTXYLWIRYJM-UHFFFAOYSA-H [Na+].[Na+].[Mo+4].O.O.S(=O)(=O)([O-])[O-].S(=O)(=O)([O-])[O-].S(=O)(=O)([O-])[O-] Chemical compound [Na+].[Na+].[Mo+4].O.O.S(=O)(=O)([O-])[O-].S(=O)(=O)([O-])[O-].S(=O)(=O)([O-])[O-] LTZCTXYLWIRYJM-UHFFFAOYSA-H 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- -1 aminoacyl amine Chemical class 0.000 description 1
- 230000009286 beneficial effect Effects 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
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 1
- 229910000366 copper(II) sulfate Inorganic materials 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
- 239000012153 distilled water Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 235000008434 ginseng Nutrition 0.000 description 1
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 description 1
- 229960000367 inositol Drugs 0.000 description 1
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Substances [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 1
- 229910052742 iron 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
- 229910000402 monopotassium phosphate Inorganic materials 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
- FWFGVMYFCODZRD-UHFFFAOYSA-N oxidanium;hydrogen sulfate Chemical compound O.OS(O)(=O)=O FWFGVMYFCODZRD-UHFFFAOYSA-N 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 238000003976 plant breeding Methods 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
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 description 1
- 238000005204 segregation Methods 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 238000004659 sterilization and disinfection Methods 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
- 229960000344 thiamine hydrochloride Drugs 0.000 description 1
- 235000019190 thiamine hydrochloride Nutrition 0.000 description 1
- 239000011747 thiamine hydrochloride Substances 0.000 description 1
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 description 1
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 description 1
- 229910000368 zinc sulfate Inorganic materials 0.000 description 1
- 239000011686 zinc sulphate Substances 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 kind of method that induction castor-oil plant anther explant forms callus, includes the following steps: that castor-oil plant anther explant is placed in the BA solution of 15~30 mg/L by S1. and impregnate 10~20min；S2. it will be inoculated on callus inducing medium and cultivate by the castor-oil plant anther explant of S1 processing, obtain callus；The callus inducing medium is the MS culture medium for being added to the SNP of 4~16mg/L, or is added to the MS culture medium of 1~2mg/L copper ion, or is added to the MS culture medium of 3~6mg/L glutamine.The present invention can promote anther explant, and dedifferentiation forms more callus with higher efficiency, while improving the quality of obtained callus, haves laid a good foundation for the work of castor-oil plant associated biomolecule technology Breeding.
Description
Technical field
The present invention relates to field of plant cell engineering technology, and in particular, to a kind of induction castor-oil plant anther explant is formed
The method of callus.
Background technique
Since Guhas reports that hair Thorn-Apple Anther Culture obtains haplobiont for the first time, about grinding for Anther Culture
Study carefully rapid development, many countries including China carry out the research work of this respect in succession, have about more than 200 kinds works so far
Object obtains haplobiont by anther cultivation.
In breeding field, Breeding by anther culture and conventional cross-breeding, distant hybridization breeding, mutation breeding and transgenosis
Technology combines, and has become biotechnology one of relatively broad and effective method in crop breeding, be crop improvement and
The important method of science of heredity and physiology, Biochemical Research.Up to the present, China has 40 various plants and is obtained with anther culture method
Pollen plant was obtained, mainly has wheat, corn, rice, grape, citrus, apple, longan, rubber, poplar and ginseng etc. some
Medicinal plant.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, is one of ten big oil crops in the world, tool
Have the features such as widely used, economic value is high, be have special industry purposes essential industry oil, seed, blade, stem,
Shell comprehensive utilization value is high.Currently, also lacking competitive excellent variety.
For a long time due to castor-oil plant mainly with hybridization, traditional mode such as select, introduce a fine variety carry out breeding, largely
Limit its development.Haplobiont can be obtained using Anther Culture, then after carrying out chromosome doubling to it, can be quickly obtained
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, the breeding time limit can be significantly shortened.But currently, castor-oil plant Anther Culture is still in infancy, in relation to castor-oil plant
The report of Anther Culture is also seldom, asks there is also the inductivity of castor-oil plant anther callus is low and callus quality is poor etc.
Topic, seriously constrains the progress of castor-oil plant vitro anther culture.Therefore, efficient induction castor-oil plant anther explant is established to be formed
The tissue culture system of callus all has the haploid breeding for accelerating castor-oil plant and the further castor-oil plant industry for developing China important
Meaning.
Summary of the invention
The purpose of the invention is to overcome the deficiencies of the prior art and provide a kind of formation of induction castor-oil plant anther explant to be cured
The method of injured tissue.
To achieve the goals above, the present invention is achieved by the following technical programs:
A method of induction castor-oil plant anther explant forms callus, includes the following steps:
S1. castor-oil plant anther 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 cultivate by the castor-oil plant anther explant of S1 processing, obtain
Obtain callus；The callus inducing medium is the MS culture medium for being added to the SNP of 4~16mg/L, or it is added to 1~
The MS culture medium of 2mg/L copper ion, or it is added to the MS culture medium of 3~6mg/L glutamine.
The method that tradition induction castor-oil plant anther explant forms callus is all to select for anther explant to be seeded in contain
It is directly induced on the culture medium for having various hormones, creativeness of the invention is first to carry out an early period to anther explant
Immersion treatment, then explant is seeded in on the culture medium of copper ions, SNP or glutamine progress callus induction group again
It knits.Carrying out immersion treatment early period to anther explant is advantageous in that the BA of high concentration can promote anther explant with higher
Efficiency dedifferentiation forms more callus, while improving the quality of obtained callus.
Preferably, castor-oil plant anther explant is placed in the BA solution of 15mg/L and impregnates 10min.
Although identical explant generating process of evoked callus on different culture mediums be substantially it is consistent,
But its callus quality and inductivity are then different and different because of culture medium prescription；In addition, different explant types is in phase
The quality and inductivity of the callus induced on same culture medium are also different.
Preferably, the callus inducing medium is the MS culture medium for being added to the SNP of 8mg/L.
Preferably, the callus inducing medium is the MS culture medium for being added to 2mg/L copper ion.
Preferably, the callus inducing medium is the MS culture medium for being added to 6mg/L glutamine.
Preferably, the acquisition methods of the castor-oil plant anther explant are as follows: obtain without insect food take, full and perianth not yet
The male flower of opening carries out surface sterilizing to castor-oil plant male flower using 2% sodium hypochlorite, and removes the perianth on male flower surface, careful to obtain
Castor-oil plant anther explant therein.
The configuration method of the BA solution of various concentration of the present invention are as follows: accurately weigh a certain amount of 6-benzyl aminopurine (6-BA,
Abbreviation BA) (Sigma, USA) powder, is sufficiently dissolved, then with deionized water constant volume, be configured to dense with the NaOH solution of 1 mol/L
Degree is respectively the BA processing solution of 0,7.5,15,30,60,120 mg/L.Then with the HCl solution adjustment BA processing of 1 mol/L
The pH to 5.8~6.0 of solution, using it is preceding with 0.22 micron of water system filter membrane (Millipore, USA) to prepared BA at
Reason solution is filtered sterilizing.
The MS culture medium: inorganic constituents (the 16.5 g/L ammonium nitrate, 19 g/L potassium nitrate, 1.7 of MS culture medium prescription
G/L potassium dihydrogen phosphate, 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 vitriol, 6.2 mg/L boric acid, 0.83 mg/L potassium iodide, 0.25 mg/L sulfate dihydrate molybdenum disodium, 0.025 mg/L five
Brochanite, 0.025 mg/L CoCL2 6H2O, 37.3 mg/L, bis- water sodium ethylene diamine tetracetate, 27.8 mg/L, seven water sulfuric acid are sub-
Iron) (2 mg/L glycine, 0.1 mg/L thiamine hydrochloride, 0.5 mg/L hydrochloric acid pyrrole are trembled for the organic principle of+MS culture medium prescription
Alcohol, 0.5 mg/L niacin)+7 g/L agar of+30 g/L sucrose of+100 mg/L inositol, extremely with 1 mg/L NaOH solution adjustment pH
5.8~6.0.Culture medium sterilizes 15 minutes under conditions of 121 DEG C, 0.1 MPa.
Condition of culture described in step S2 are as follows: room temperature is 25 DEG C, intensity of illumination is 2000 lx, light application time is daily 12
Hour.
Compared with prior art, the invention has the following beneficial effects:
The method for forming callus the novelty of the present invention is changing tradition induction castor-oil plant anther explant, i.e., it is sharp
Short-term immersion treatment is carried out to anther explant with high concentration BA solution, thus promotes anther explant to take off with higher efficiency and divides
Change forms more callus.The quality of obtained callus is improved simultaneously, is castor-oil plant associated biomolecule technology Breeding work
It haves laid a good foundation.
Specific embodiment
The present invention is made combined with specific embodiments below and further being elaborated, the embodiment is served only for explaining this
Invention, is not intended to limit the scope of the present invention.Test method as used in the following examples is normal unless otherwise specified
Rule method；Used material, reagent etc., unless otherwise specified, for the reagent and material commercially obtained.
Embodiment 1
A method of induction castor-oil plant anther explant forms callus, includes the following steps:
(1) obtain anther explant: selection takes without insect food, full and perianth is not yet opened in spherical castor-oil plant male flower.
Taking the time that should be selected in, temperature is low, and when the weak fine day morning 10 of illumination or so, the bud on castor-oil plant can reduce dirt without fog at this time
Dye, illumination is weaker and can prevent the forfeiture of anther moisture.The male flower bud of acquisition is lived with tissue-wrapped, is first moistened with distilled water
It is wet, then extra moisture is gently squeezed out, it then puts into vial, is handled 3 days in 4 DEG C of refrigerator.On superclean bench
1 min first first sterilized to castor-oil plant male flower with 75% ethanol solution, then with 2% NaClO solution disinfection 20min, rinsed with sterile water castor
Numb male flower 5 times.Carefully the perianth of male flower outer layer is removed with sterilized dissecting needle later, then anther therein is carefully divided
From can be obtained castor-oil plant anther explant.
(2) by castor-oil plant anther explant be seeded in addition various concentration BA MS minimal medium on, room temperature be 25 DEG C,
Intensity of illumination is 2000 lx, light application time is evoked callus under conditions of daily 12 hours.The results are shown in Table 1, addition
Suitable BA has facilitation to the formation of castor-oil plant anther callus, and when the concentration of BA is 1 mg/L, castor-oil plant anther is cured
The inductivity of injured tissue reaches maximum value, is 10.67%；And continuing growing with the concentration of BA, castor-oil plant anther callus lure
Conductance gradually decreases.Meanwhile experimental result is also shown that and forms callus using conventional method induction castor-oil plant anther explant
In the process, the induced efficiency of obtained callus is lower and second-rate, small volume.
Table 1 is influence 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 software of SPSS Statistics
(P≤0.05), significant difference between the different expressions processing of letter after data.
Embodiment 2
A method of induction castor-oil plant anther explant forms callus, includes the following steps:
(1) anther explant is obtained: with embodiment 1.
(2) castor-oil plant anther explant is placed in immersion different time (0~80 min) in the BA solution of 15 mg/L；
(3) then castor-oil plant anther explant is placed on after sucking surplus liquid on sterile blotting paper, careful is seeded to
It is cultivated 30 days on no hormone MS culture medium.
The experimental results showed that when the time of immersion treatment explant is 10 min, explant callus induction efficiency effect
Fruit is best, reaches 41.25%(table 2).Continue to extend the processing time, castor-oil plant anther explant callus induction rate will gradually decrease.
Table 2 is the influence for handling the 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 software of SPSS Statistics
(P≤0.05), significant difference between the different expressions processing of letter after data.
Embodiment 3
A method of induction castor-oil plant anther explant forms callus, includes the following steps:
(1) anther explant is obtained: with embodiment 1.
(2) castor-oil plant anther explant is placed in the BA solution of various concentration (0~120 mg/L) and impregnates 10min.
(3) then castor-oil plant anther explant is placed on after sucking surplus liquid on sterile blotting paper, careful is seeded to
It is cultivated 30 days on no hormone MS culture medium.
The experimental results showed that with the increase of BA solution concentration, castor-oil plant anther explant callus induction efficiency will be by
Step improves, and when the concentration of immersion treatment explant is 15 mg/L, explant callus induction effect is best, callus
Inductivity reaches maximum value, is 41.25%(table 3).And continue to improve BA treatment fluid concentration, castor-oil plant anther explant callus lures
Leading efficiency will gradually reduce.Meanwhile castor-oil plant anther explant is induced in method using short-term high concentration BA solution immersion treatment
During body forms callus, it was found that obtained callus quality is preferable, and volume is larger.
Table 3 is influence of the BA solution concentration 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 software of SPSS Statistics
(P≤0.05), significant difference between the different expressions processing of letter after data.
By above-mentioned experimental result it is found that compared with forming callus using conventional method induction castor-oil plant anther explant,
(callus is significantly improved using the callus induction rate of high concentration BA solution immersion treatment castor-oil plant anther explant institute induced synthesis
30.58%) tissue inductivity improves, and the better quality of callus obtained, volume are bigger.Therefore, using 15
Mg/L BA solution carries out the immersion treatment of 10 min to castor-oil plant anther explant, can be used to reach to obtain more, quality
The purpose of better callus.
Embodiment 4
A method of induction castor-oil plant anther explant forms callus, includes the following steps:
(1) anther explant is obtained: with embodiment 1.
(2) castor-oil plant anther explant is placed in the BA solution of 15mg/L and impregnates 10min.
(3) then castor-oil plant anther explant is placed on after sucking surplus liquid on sterile blotting paper, careful is seeded to
It is added on the MS culture medium of the sodium nitroprussiate (SNP) (0,2,4,8,16 and 32mg/L) of various concentration and cultivates 30 days.
The experimental results showed that castor-oil plant induction of anther callus rate works as SNP also with raising with the raising of SNP concentration
When concentration is 8 mg/L, callus induction rate reaches maximum value, is 60.37%, improves 49.70% than conventional method, than passing through
It is seeded on no hormone MS culture medium after the processing of high concentration BA solution and improves 19.12%；But when being to continue with the concentration for improving SNP,
Callus induction will be suppressed, and induced efficiency will gradually reduce.
Table 4 is influence 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 software of SPSS Statistics
(P≤0.05), significant difference between the different expressions processing of letter after data.
Embodiment 5
A method of induction castor-oil plant anther explant forms callus, includes the following steps:
(1) anther explant is obtained: with embodiment 1.
(2) castor-oil plant anther explant is placed in the BA solution of 15mg/L and impregnates 10min.
(3) then castor-oil plant anther explant is placed on after sucking surplus liquid on sterile blotting paper, careful is seeded to
It is added on the MS culture medium of the copper ion (anhydrous cupric sulfate) (0,0.5,1,2,4 and 8 mg/L) of various concentration and cultivates 30 days.
The experimental results showed that with the raising of copper ion concentration, castor-oil plant induction of anther callus rate also with raising, when
When copper ion concentration is 2 mg/L, callus induction rate reaches maximum value, is 68.36%, improves 57.69% than conventional method,
27.11% is improved than being seeded on no hormone MS culture medium after the processing of excessive concentrations BA solution；；But be to continue with improve copper from
When the concentration of son, 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 software of SPSS Statistics
(P≤0.05), significant difference between the different expressions processing of letter after data.
Embodiment 6
A method of induction castor-oil plant anther explant forms callus, includes the following steps:
(1) anther explant is obtained: with embodiment 1.
(2) castor-oil plant anther explant is placed in the BA solution of 15mg/L and impregnates 10min.
(3) then castor-oil plant anther explant is placed on after sucking surplus liquid on sterile blotting paper, careful is seeded to
It is added on the MS culture medium of the glutamine (0,1.5,3,6,12 and 24 mg/L) of various concentration and cultivates 30 days.
The experimental results showed that castor-oil plant induction of anther callus rate works as paddy also with raising with the raising of Gln concentration
When aminoacyl amine concentration is 6 mg/L, callus induction rate reaches maximum value, is 54.63%, improves 43.96% than conventional method,
13.38% is improved than being seeded on no hormone MS culture medium after the processing of excessive concentrations BA solution；But it is to continue with raising glutamy
When the concentration of amine, 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 software of SPSS Statistics
(P≤0.05), significant difference between the different expressions processing of letter after data.
Embodiment 7
A method of induction castor-oil plant anther explant forms callus, and step (1) and (3) are with embodiment 2, with implementation
The unique different place of example 2 is that step (2) are impregnated using different hormones, and the present embodiment includes following 4 different
And column processing: processing 1 is that the TDZ solution of 15mg/L is used to impregnate anther explant 10min；Processing 2 is to use 15mg/L's
NAA solution impregnates anther explant 10min；Processing 3 is that the IBA solution of 15mg/L is used to impregnate anther explant 10min；Place
Reason 4 is that the IAA solution of 15mg/L is used to impregnate anther explant 10min.Callus induction result after four kinds of different disposals
It is shown in Table 7.
Table 7 is influence of the hormon immersion to castor-oil plant induction of anther callus efficiency
Embodiment 8
A method of induction castor-oil plant anther explant forms callus, includes the following steps:
(1) anther explant is obtained: with embodiment 1.
(2) castor-oil plant anther explant is seeded on the MS minimal medium of addition hormon, is 25 DEG C, light in room temperature
It is 2000 lx according to intensity, evoked callus under conditions of light application time is daily 12 hours.The present embodiment includes following 2
Different and column processing: processing 1 uses the MS culture medium of the NAA of the BA+0.2mg/L of addition 3mg/L as callus induction
Culture medium；Processing 2 uses the MS culture medium of the 2,4-D of the BA+2mg/L of addition 2mg/L as callus inducing medium；Knot
Fruit is 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) anther explant is obtained: with embodiment 1.
(2) castor-oil plant anther explant is placed in the BA solution of 15mg/L and impregnates 10min.
(3) then castor-oil plant anther explant is placed on after sucking surplus liquid on sterile blotting paper, careful is seeded to
On the MS minimal medium for adding hormon, room temperature is 25 DEG C, intensity of illumination is 2000 lx, light application time is daily 12
Evoked callus under conditions of hour.The present embodiment includes following 4 different and column processing: processing 1 is added to use
The MS culture medium of the SNP+2mg/L copper ion of 8mg/L is as callus inducing medium；Processing 2 adds 2mg/L to use
The MS culture medium of copper ion+6mg/L glutamine is as callus inducing medium；Processing 3 is using addition 8mg/L
The MS culture medium of SNP+6mg/L glutamine is as callus inducing medium；Processing 4 is the SNP using addition 8mg/L
+ add the MS culture medium of 2mg/L copper ion+6mg/L glutamine 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 embodiments are served only for explaining the present invention, are not intended to limit the scope of the present invention.
Claims (5)
1. a kind of method that induction castor-oil plant anther explant forms callus, which comprises the steps of:
S1. castor-oil plant anther 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 cultivate by the castor-oil plant anther explant of S1 processing, be cured
Injured tissue；The callus inducing medium is the MS culture medium for being added to the sodium nitroprussiate of 4~16mg/L, or it is added to 1~
The MS culture medium of 2mg/L copper ion, or it is added to the MS culture medium of 3~6mg/L glutamine.
2. the method according to claim 1, wherein castor-oil plant anther explant is placed in the BA solution of 15mg/L
Impregnate 10min.
3. the method according to claim 1, wherein the callus inducing medium is to be added to 8mg/L
Sodium nitroprussiate MS culture medium.
4. the method according to claim 1, wherein the callus inducing medium is to be added to 2mg/L
The MS culture medium of copper ion.
5. the method according to claim 1, wherein the callus inducing medium is to be added to 6mg/L
The MS culture medium of glutamine.
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