CN104770295B - A kind of japonica rice flower pesticide differentiation culture based formulas - Google Patents

A kind of japonica rice flower pesticide differentiation culture based formulas Download PDF

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CN104770295B
CN104770295B CN201510156933.4A CN201510156933A CN104770295B CN 104770295 B CN104770295 B CN 104770295B CN 201510156933 A CN201510156933 A CN 201510156933A CN 104770295 B CN104770295 B CN 104770295B
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culture medium
japonica rice
vitamin
culture
anther
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CN104770295A (en
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许伟
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Baoying Zhongbaode Garden Organic Farm
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Chongqing Yue Yue Machinery Manufacturing Co Ltd
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Abstract

The invention provides a kind of japonica rice flower pesticide division culture medium, the formula of this culture medium is by certain density KNO3、C8H7O2K、(NH4)2SO4、KH2PO4、CaCl2、MgSO4、Na2‑EDTA、FeSO4∙7H2O、MnSO4∙H2O、ZnSO4∙H2O、H3BO3、KI、CuSO4∙5H2O、Na2MoO4∙2H2O、CoCl2∙6H2O, glycerol, glycine, folic acid, methyl-nitroso-urea, alanine, lysine, vitamin B1, vitamin B6,3 pyridine carboxylic acids, sucrose, plant gel, NAA, 6 BA, Prohexadione calcium, sorbitol, L proline, hydrolyzed protein, colchicine and biotin are formed.Culture medium of the present invention has the advantage improving japonica rice anther callus plantlet differentiation rate expeditiously, reducing albefaction rate, can significantly improve japonica rice Anther Culture Efficiency.

Description

A kind of japonica rice flower pesticide differentiation culture based formulas
Technical field
The present invention relates to a kind of japonica rice flower pesticide division culture medium, be specifically related to one and improve japonica rice antherderived callus expeditiously Tissue plantlet differentiation rate, the differentiation culture based formulas of reduction albefaction rate, belong to technical field of agriculture science.
Background technology
Anther Culture is to utilize plant tissue culture technique, growing the flower pesticide to certain phase, by sterile working's skill Art, is seeded on synthetic medium to change the development program of pollen grain in flower pesticide, induces it to break up, and has been carried out continuously silk and divides Split, form cell mass, and then form a parenchyma-callus without differentiation, or be divided into embryoid, make wound healing subsequently Histo-differentiation becomes the process of complete plant, and therefore Breeding by anther culture is also haploid breeding.
Oryza sativa L. is one of the most most important cereal crops.Nineteen sixty-eight, Japan Xin Guan and big open country are trained by vitro anther Support and obtain Rice Pollen Plants, opened human use's flower pesticide and created the prelude of Oryza sativa L. new germ plasm, opened one for crop breeding The effective way that bar is new.Subsequently, utilize this technology breeding rice kind by the most attention of breeding man of various countries.Rice Anther is trained Bring up kind of a main feature to show as shortening breeding cycle, improve efficiency of selection, accelerate effective character transfer etc..Rice anther culture Breeding combines with conventional cross-breeding, distant hybridization breeding, mutagenic breeding and transgenic technology breeding, and development defines A set of breeding technique system, plays Link role at traditional agriculture in the conversion of high-tech agricultural.
China started Anther Culture technical research from 1970, and within 1975, the Tianjin institute of agricultural sciences is (before existing Tianjin crop institute Body) cooperated with Chinese Academy of Sciences heredity, bring out two flower training kind-flowers by haploid breeding method and educate No. 1 and No. 2,1977 Year in the international crop breeding method seminar that Japan holds, the big wild clear spring point out flower educate No. 1 and No. 2 be Anther Culture the earliest The kind being bred as.China achieves Anther culture breeding from theory to the breakthrough of application, also promotes Anther culture breeding technology both domestic and external Development.Along with researchs that deepens continuously such as anther cultural inherited character, Physiology and biochemistries, present Rice anther culture has become as New technology of breeding the most practical, effective in current biotechnology breeding, the applied research of China's Rice Anther Culture Breeding exists It is always maintained at leading position in the world.
The quantity of flower training regeneration plant is the most important thing of breed breeding.Due to by nutrient media components, the diversity of material And the impact of culture environment etc., current Efficiency is the most preferable, it is thus achieved that regeneration plant colony little, the table of anther culture descendant Existing type is inadequate, and this makes selection, utilization be limited by bigger.The growth promoter of Anther-culture is influenced by many factors, bag Including improvement of the improvement of culture medium, the increase and decrease of supplementary element, haploid doubling techniques, condition of culture etc., its purpose all exists In the inductivity and the differentiation rate of green Seedling that improve callus.Wherein culture medium is anther cultural material base, direct relation To growth and the differentiation of culture, nutrient media components is the critically important factor affecting Efficiency.The each component of culture medium Suitable amounts, and syntagmatic certain between them, may result in increasing substantially of Efficiency.Continue screening with excellent Change minimal medium, improve the specific aim that culture medium is selected, be the effective measures improving Anther Culture Ability；Substantial amounts of research finds Some Organic additives can significantly improve Anther Culture Ability, finds and optimum organization Anther Culture Organic additives matter, can enter One step improves Anther Culture Ability.Therefore, combination and Optimal Medium component, finding out efficient Rice anther culture based formulas is Improve the key of Anther Culture Efficiency.
Groping and put into practice japonica rice for many years is anther cultural, we continue to optimize culture medium prescription, and constantly taste Examination adds some additives improving japonica rice plantlet differentiation rate and combines the collocation of its kind and concentration, has finally found out a kind of energy Enough significantly improve japonica rice plantlet differentiation rate, the japonica rice anther callus differentiation culture based formulas of reduction albefaction rate.We grind Studying carefully result for further genralrlization and applies Rice Haploid Breeding and theoretical research without being suspected to have bigger realistic meaning and practicality It is worth.
Summary of the invention
The invention provides kind of a japonica rice flower pesticide division culture medium, the formula of this culture medium is as follows:
KNO32820～3080mg/L, C8H7O2K 1800～2000mg/L, (NH4)2SO4400～460mg/L, KH2PO4 320～380mg/L, CaCl2100～120mg/L, MgSO480～90mg/L, Na2-EDTA 30～35mg/L, FeSO4∙7H2O 22～26mg/L, MnSO4∙H2O 2.8～3.2mg/L, ZnSO4∙H2O 1.2～1.4mg/L, H3BO31.3～1.5mg/L, KI 0.6～0.7mg/L, CuSO4∙5H2O 0.04～0.06mg/L, Na2MoO4∙2H2O 0.14～0.18mg/L, CoCl2∙6H2O 0.14～0.18mg/L, glycerol 0.8～1.0mg/L, glycine 1.8～2.2mg/L, folic acid 0.35～0.45mg/L, first Base nitroso ureas 1.1～1.3mg/L, alanine 1.1～1.3mg/L, lysine 1.3～1.5mg/L, vitamin B1 4.5 ～5.5mg/L, vitamin B6 0.9～1.1mg/L, acidum nicotinicum 0.9～1.1mg/L, sucrose 23～27g/L, plant coagulates Glue 5～6g/L；
NAA 0.045～0.055mg/L, 6-BA 1.8～2.2mg/L, Prohexadione calcium 0.6～0.8mg/L, sorbitol 23 ～27g/L, L-PROLINE 100～140mg/L, hydrolyzed protein 0.9～1.1g/L, colchicine 0.18～0.22mg/L, raw Thing element 0.07～0.09mg/L, pH 5.8-6.0.
Culture medium of the present invention has raising japonica rice anther callus plantlet differentiation rate, reduction albefaction expeditiously The advantage of rate, can significantly improve japonica rice Anther Culture Efficiency.The following examples and contrast experiment can clearly be reflected The feature of culture medium of the present invention.
Detailed description of the invention
Embodiment 1
It is formulated as follows the culture medium of formula:
KNO32950mg/L, C8H7O2K 1900mg/L, (NH4)2SO4430mg/L, KH2PO4350mg/L, CaCl2 110mg/L, MgSO485mg/L, Na2-EDTA 32.5mg/L, FeSO4∙7H2O 24mg/L, MnSO4∙H2O 3.0mg/L, ZnSO4∙H2O 1.3mg/L, H3BO31.4mg/L, KI 0.65mg/L, CuSO4∙5H2O 0.05mg/L, Na2MoO4∙2H2O 0.16mg/L, CoCl2∙6H2O 0.16mg/L, glycerol 0.9mg/L, glycine 2.0mg/L, folic acid 0.4mg/L, methyl is sub- Nitrourea 1.2mg/L, alanine 1.2mg/L, lysine 1.4mg/L, vitamin B1 5.0mg/L, vitamin B6 1.0mg/ L, acidum nicotinicum 1.0mg/L, sucrose 25g/L, plant gel 5.5g/L；
NAA 0.05mg/L, 6-BA 2.0mg/L, Prohexadione calcium 0.7mg/L, sorbitol 25g/L, L-PROLINE 120mg/L, hydrolyzed protein 1.0g/L, colchicine 0.2mg/L, biotin 0.08mg/L, pH 5.9.
Choose japonica rice variety and lead to the anther callus of section's round-grained rice and black shell purple round-grained rice as donor, when the flower pesticide after inducing culture When callus grows to 2 mm in size, forwarding to induce green Seedling on division culture medium, condition of culture control is: temperature 26 DEG C, humidity 75%, the dark 10h of illumination 3000lx l4h/.(grow to about 2 mm) after Callus formation, calculate phenylacetic acid.
Wound healing block number × 100% of plantlet differentiation rate (%)=Differentiation From Calli number/transfer.
Embodiment 2
It is formulated as follows the culture medium (C of formula8H7O2K adds the preferred of concentration):
KNO32950mg/L, (NH4)2SO4430mg/L, KH2PO4350mg/L, CaCl2110mg/L, MgSO4 85mg/ L, Na2-EDTA 32.5mg/L, FeSO4∙7H2O 24mg/L, MnSO4∙H2O 3.0mg/L, ZnSO4∙H2O 1.3mg/L, H3BO3 1.4mg/L, KI 0.65mg/L, CuSO4∙5H2O 0.05mg/L, Na2MoO4∙2H2O 0.16mg/L, CoCl2∙6H2O 0.16mg/L, glycerol 0.9mg/L, glycine 2.0mg/L, folic acid 0.4mg/L, methyl-nitroso-urea 1.2mg/L, the third ammonia Acid 1.2mg/L, lysine 1.4mg/L, vitamin B1 5.0mg/L, vitamin B6 1.0mg/L, acidum nicotinicum 1.0mg/ L, sucrose 25g/L, plant gel 5.5g/L；
NAA 0.05mg/L, 6-BA 2.0mg/L, Prohexadione calcium 0.7mg/L, sorbitol 25g/L, L-PROLINE 120mg/L, hydrolyzed protein 1.0g/L, colchicine 0.2mg/L, biotin 0.08mg/L, pH 5.9.
C8H7O2The interpolation concentration of K arranges following 8 kinds: 900mg/L；1100mg/L；1300mg/L；1500mg/L； 1700mg/L；2100mg/L；2300mg/L；2500mg/L.
Choosing these two japonica rice variety antherderived callus equally as donor, operational approach, cultural method are with embodiment 1, meter Calculate plantlet differentiation rate.
Embodiment 3
It is formulated as follows the culture medium (glycerol add concentration preferred) of formula:
KNO32950mg/L, C8H7O2K 1900mg/L, (NH4)2SO4430mg/L, KH2PO4350mg/L, CaCl2 110mg/L, MgSO485mg/L, Na2-EDTA 32.5mg/L, FeSO4∙7H2O 24mg/L, MnSO4∙H2O 3.0mg/L, ZnSO4∙H2O 1.3mg/L, H3BO31.4mg/L, KI 0.65mg/L, CuSO4∙5H2O 0.05mg/L, Na2MoO4∙2H2O 0.16mg/L, CoCl2∙6H2O 0.16mg/L, glycine 2.0mg/L, folic acid 0.4mg/L, methyl-nitroso-urea 1.2mg/L, Alanine 1.2mg/L, lysine 1.4mg/L, vitamin B1 5.0mg/L, vitamin B6 1.0mg/L, acidum nicotinicum 1.0mg/L, sucrose 25g/L, plant gel 5.5g/L；
NAA 0.05mg/L, 6-BA 2.0mg/L, Prohexadione calcium 0.7mg/L, sorbitol 25g/L, L-PROLINE 120mg/L, hydrolyzed protein 1.0g/L, colchicine 0.2mg/L, biotin 0.08mg/L, pH 5.9.
The interpolation concentration of glycerol is following 6 kinds: 0；0.3mg/L；0.6mg/L；1.2mg/L；1.5mg/L；1.8mg/L.
Choosing these two japonica rice variety antherderived callus equally as donor, operational approach, cultural method are with embodiment 1, meter Calculate plantlet differentiation rate.
Embodiment 4
It is formulated as follows the culture medium (folic acid add concentration preferred) of formula:
KNO32950mg/L, C8H7O2K 1900mg/L, (NH4)2SO4430mg/L, KH2PO4350mg/L, CaCl2 110mg/L, MgSO485mg/L, Na2-EDTA 32.5mg/L, FeSO4∙7H2O 24mg/L, MnSO4∙H2O 3.0mg/L, ZnSO4∙H2O 1.3mg/L, H3BO31.4mg/L, KI 0.65mg/L, CuSO4∙5H2O 0.05mg/L, Na2MoO4∙2H2O 0.16mg/L, CoCl2∙6H2O 0.16mg/L, glycerol 0.9mg/L, glycine 2.0mg/L, methyl-nitroso-urea 1.2mg/ L, alanine 1.2mg/L, lysine 1.4mg/L, vitamin B1 5.0mg/L, vitamin B6 1.0mg/L, acidum nicotinicum 1.0mg/L, sucrose 25g/L, plant gel 5.5g/L；
NAA 0.05mg/L, 6-BA 2.0mg/L, Prohexadione calcium 0.7mg/L, sorbitol 25g/L, L-PROLINE 120mg/L, hydrolyzed protein 1.0g/L, colchicine 0.2mg/L, biotin 0.08mg/L, pH 5.9.
The interpolation concentration of folic acid is set to following 6 kinds: 0；0.1mg/L；0.2mg/L；0.3mg/L；0.5mg/L；0.6mg/ L。
Choosing these two japonica rice variety antherderived callus equally as donor, operational approach, cultural method are with embodiment 1, meter Calculate plantlet differentiation rate.
Embodiment 5
It is formulated as follows the culture medium (methyl-nitroso-urea add concentration preferred) of formula:
KNO32950mg/L, C8H7O2K 1900mg/L, (NH4)2SO4430mg/L, KH2PO4350mg/L, CaCl2 110mg/L, MgSO485mg/L, Na2-EDTA 32.5mg/L, FeSO4∙7H2O 24mg/L, MnSO4∙H2O 3.0mg/L, ZnSO4∙H2O 1.3mg/L, H3BO31.4mg/L, KI 0.65mg/L, CuSO4∙5H2O 0.05mg/L, Na2MoO4∙2H2O 0.16mg/L, CoCl2∙6H2O 0.16mg/L, glycerol 0.9mg/L, glycine 2.0mg/L, folic acid 0.4mg/L, alanine 1.2mg/L, lysine 1.4mg/L, vitamin B1 5.0mg/L, vitamin B6 1.0mg/L, acidum nicotinicum 1.0mg/L, Sucrose 25g/L, plant gel 5.5g/L；
NAA 0.05mg/L, 6-BA 2.0mg/L, Prohexadione calcium 0.7mg/L, sorbitol 25g/L, L-PROLINE 120mg/L, hydrolyzed protein 1.0g/L, colchicine 0.2mg/L, biotin 0.08mg/L, pH 5.9.
The interpolation concentration of methyl-nitroso-urea arranges following 6 kinds: 0；0.3mg/L；0.6mg/L；0.9mg/L；1.5mg/L； 1.8mg/L。
Choosing these two japonica rice variety antherderived callus equally as donor, operational approach, cultural method are with embodiment 1, meter Calculate plantlet differentiation rate.
Embodiment 6
It is formulated as follows the culture medium (alanine add concentration preferred) of formula:
KNO32950mg/L, C8H7O2K 1900mg/L, (NH4)2SO4430mg/L, KH2PO4350mg/L, CaCl2 110mg/L, MgSO485mg/L, Na2-EDTA 32.5mg/L, FeSO4∙7H2O 24mg/L, MnSO4∙H2O 3.0mg/L, ZnSO4∙H2O 1.3mg/L, H3BO31.4mg/L, KI 0.65mg/L, CuSO4∙5H2O 0.05mg/L, Na2MoO4∙2H2O 0.16mg/L, CoCl2∙6H2O 0.16mg/L, glycerol 0.9mg/L, glycine 2.0mg/L, folic acid 0.4mg/L, methyl is sub- Nitrourea 1.2mg/L, lysine 1.4mg/L, vitamin B1 5.0mg/L, vitamin B6 1.0mg/L, acidum nicotinicum 1.0mg/L, sucrose 25g/L, plant gel 5.5g/L；
NAA 0.05mg/L, 6-BA 2.0mg/L, Prohexadione calcium 0.7mg/L, sorbitol 25g/L, L-PROLINE 120mg/L, hydrolyzed protein 1.0g/L, colchicine 0.2mg/L, biotin 0.08mg/L, pH 5.9.
The interpolation concentration of alanine arranges following 6 kinds: 0；0.3mg/L；0.6mg/L；0.9mg/L；1.5mg/L；1.8mg/ L。
Choosing these two japonica rice variety antherderived callus equally as donor, operational approach, cultural method are with embodiment 1, meter Calculate plantlet differentiation rate.
Embodiment 7
It is formulated as follows the culture medium (lysine add concentration preferred) of formula:
KNO32950mg/L, C8H7O2K 1900mg/L, (NH4)2SO4430mg/L, KH2PO4350mg/L, CaCl2 110mg/L, MgSO485mg/L, Na2-EDTA 32.5mg/L, FeSO4∙7H2O 24mg/L, MnSO4∙H2O 3.0mg/L, ZnSO4∙H2O 1.3mg/L, H3BO31.4mg/L, KI 0.65mg/L, CuSO4∙5H2O 0.05mg/L, Na2MoO4∙2H2O 0.16mg/L, CoCl2∙6H2O 0.16mg/L, glycerol 0.9mg/L, glycine 2.0mg/L, folic acid 0.4mg/L, methyl is sub- Nitrourea 1.2mg/L, alanine 1.2mg/L, vitamin B1 5.0mg/L, vitamin B6 1.0mg/L, acidum nicotinicum 1.0mg/L, sucrose 25g/L, plant gel 5.5g/L；
NAA 0.05mg/L, 6-BA 2.0mg/L, Prohexadione calcium 0.7mg/L, sorbitol 25g/L, L-PROLINE 120mg/L, hydrolyzed protein 1.0g/L, colchicine 0.2mg/L, biotin 0.08mg/L, pH 5.9.
Lysine adds concentration and arranges following 10 kinds: 0；0.2mg/L；0.4mg/L；0.6mg/L；0.8mg/L；1.0mg/L； 1.2mg/L；1.6mg/L；1.8mg/L；2.0mg/L.
Choosing these two japonica rice variety antherderived callus equally as donor, operational approach, cultural method are with embodiment 1, meter Calculate plantlet differentiation rate.
Embodiment 8
It is formulated as follows the culture medium (Prohexadione calcium add concentration preferred) of formula:
KNO32950mg/L, C8H7O2K 1900mg/L, (NH4)2SO4430mg/L, KH2PO4350mg/L, CaCl2 110mg/L, MgSO485mg/L, Na2-EDTA 32.5mg/L, FeSO4∙7H2O 24mg/L, MnSO4∙H2O 3.0mg/L, ZnSO4∙H2O 1.3mg/L, H3BO31.4mg/L, KI 0.65mg/L, CuSO4∙5H2O 0.05mg/L, Na2MoO4∙2H2O 0.16mg/L, CoCl2∙6H2O 0.16mg/L, glycerol 0.9mg/L, glycine 2.0mg/L, folic acid 0.4mg/L, methyl is sub- Nitrourea 1.2mg/L, alanine 1.2mg/L, lysine 1.4mg/L, vitamin B1 5.0mg/L, vitamin B6 1.0mg/ L, acidum nicotinicum 1.0mg/L, sucrose 25g/L, plant gel 5.5g/L；
NAA 0.05mg/L, 6-BA 2.0mg/L, sorbitol 25g/L, L-PROLINE 120mg/L, hydrolyzed protein 1.0g/ L, colchicine 0.2mg/L, biotin 0.08mg/L, pH 5.9.
Prohexadione calcium adds concentration and arranges following 10 kinds: 0；0.1mg/L；0.2mg/L；0.3mg/L；0.4mg/L；0.5mg/ L；0.6mg/L；0.8mg/L；0.9mg/L；1.0mg/L.
Choosing these two japonica rice variety antherderived callus equally as donor, operational approach, cultural method are with embodiment 1, meter Calculate plantlet differentiation rate.
Embodiment 9
It is formulated as follows the culture medium (sorbitol add concentration preferred) of formula:
KNO32950mg/L, C8H7O2K 1900mg/L, (NH4)2SO4430mg/L, KH2PO4350mg/L, CaCl2 110mg/L, MgSO485mg/L, Na2-EDTA 32.5mg/L, FeSO4∙7H2O 24mg/L, MnSO4∙H2O 3.0mg/L, ZnSO4∙H2O 1.3mg/L, H3BO31.4mg/L, KI 0.65mg/L, CuSO4∙5H2O 0.05mg/L, Na2MoO4∙2H2O 0.16mg/L, CoCl2∙6H2O 0.16mg/L, glycerol 0.9mg/L, glycine 2.0mg/L, folic acid 0.4mg/L, methyl is sub- Nitrourea 1.2mg/L, alanine 1.2mg/L, lysine 1.4mg/L, vitamin B1 5.0mg/L, vitamin B6 1.0mg/ L, acidum nicotinicum 1.0mg/L, sucrose 25g/L, plant gel 5.5g/L；
NAA 0.05mg/L, 6-BA 2.0mg/L, Prohexadione calcium 0.7mg/L, L-PROLINE 120mg/L, hydrolyzed protein 1.0g/L, colchicine 0.2mg/L, biotin 0.08mg/L, pH 5.9.
Sorbitol adds concentration and arranges following 8 kinds: 0；5g/L；10g/L；15g/L；20g/L；30g/L；35g/L；40g/L.
Choosing these two japonica rice variety antherderived callus equally as donor, operational approach, cultural method are with embodiment 1, meter Calculate plantlet differentiation rate.
Embodiment 10
It is formulated as follows the culture medium (L-PROLINE add concentration preferred) of formula:
KNO32950mg/L, C8H7O2K 1900mg/L, (NH4)2SO4430mg/L, KH2PO4350mg/L, CaCl2 110mg/L, MgSO485mg/L, Na2-EDTA 32.5mg/L, FeSO4∙7H2O 24mg/L, MnSO4∙H2O 3.0mg/L, ZnSO4∙H2O 1.3mg/L, H3BO31.4mg/L, KI 0.65mg/L, CuSO4∙5H2O 0.05mg/L, Na2MoO4∙2H2O 0.16mg/L, CoCl2∙6H2O 0.16mg/L, glycerol 0.9mg/L, glycine 2.0mg/L, folic acid 0.4mg/L, methyl is sub- Nitrourea 1.2mg/L, alanine 1.2mg/L, lysine 1.4mg/L, vitamin B1 5.0mg/L, vitamin B6 1.0mg/ L, acidum nicotinicum 1.0mg/L, sucrose 25g/L, plant gel 5.5g/L；
NAA 0.05mg/L, 6-BA 2.0mg/L, Prohexadione calcium 0.7mg/L, sorbitol 25g/L, hydrolyzed protein 1.0g/ L, colchicine 0.2mg/L, biotin 0.08mg/L, pH 5.9.
L-PROLINE adds concentration and arranges following 6 kinds: 0；30mg/L；60mg/L；90mg/L；150mg/L；180mg/L.
Choosing these two japonica rice variety antherderived callus equally as donor, operational approach, cultural method are with embodiment 1, meter Calculate plantlet differentiation rate.
Embodiment 11
It is formulated as follows the culture medium (hydrolyzed protein add concentration preferred) of formula:
KNO32950mg/L, C8H7O2K 1900mg/L, (NH4)2SO4430mg/L, KH2PO4350mg/L, CaCl2 110mg/L, MgSO485mg/L, Na2-EDTA 32.5mg/L, FeSO4∙7H2O 24mg/L, MnSO4∙H2O 3.0mg/L, ZnSO4∙H2O 1.3mg/L, H3BO31.4mg/L, KI 0.65mg/L, CuSO4∙5H2O 0.05mg/L, Na2MoO4∙2H2O 0.16mg/L, CoCl2∙6H2O 0.16mg/L, glycerol 0.9mg/L, glycine 2.0mg/L, folic acid 0.4mg/L, methyl is sub- Nitrourea 1.2mg/L, alanine 1.2mg/L, lysine 1.4mg/L, vitamin B1 5.0mg/L, vitamin B6 1.0mg/ L, acidum nicotinicum 1.0mg/L, sucrose 25g/L, plant gel 5.5g/L；
NAA 0.05mg/L, 6-BA 2.0mg/L, Prohexadione calcium 0.7mg/L, sorbitol 25g/L, L-PROLINE 120mg/L, colchicine 0.2mg/L, biotin 0.08mg/L, pH 5.9.
Hydrolyzed protein adds concentration and arranges following 8 kinds: 0；0.2mg/L；0.4mg/L；0.6mg/L；0.8mg/L；1.2mg/ L；1.4mg/L；1.6 mg/L.
Choosing these two japonica rice variety antherderived callus equally as donor, operational approach, cultural method are with embodiment 1, meter Calculate plantlet differentiation rate.
Embodiment 12
It is formulated as follows the culture medium (colchicine add concentration preferred) of formula:
KNO32950mg/L, C8H7O2K 1900mg/L, (NH4)2SO4430mg/L, KH2PO4350mg/L, CaCl2 110mg/L, MgSO485mg/L, Na2-EDTA 32.5mg/L, FeSO4∙7H2O 24mg/L, MnSO4∙H2O 3.0mg/L, ZnSO4∙H2O 1.3mg/L, H3BO31.4mg/L, KI 0.65mg/L, CuSO4∙5H2O 0.05mg/L, Na2MoO4∙2H2O 0.16mg/L, CoCl2∙6H2O 0.16mg/L, glycerol 0.9mg/L, glycine 2.0mg/L, folic acid 0.4mg/L, methyl is sub- Nitrourea 1.2mg/L, alanine 1.2mg/L, lysine 1.4mg/L, vitamin B1 5.0mg/L, vitamin B6 1.0mg/ L, acidum nicotinicum 1.0mg/L, sucrose 25g/L, plant gel 5.5g/L；
NAA 0.05mg/L, 6-BA 2.0mg/L, Prohexadione calcium 0.7mg/L, sorbitol 25g/L, L-PROLINE 120mg/L, hydrolyzed protein 1.0g/L, biotin 0.08mg/L, pH 5.9.
Colchicine adds concentration and arranges following 6 kinds: 0；0.05mg/L；0.1mg/L；0.15mg/L；0.25mg/L； 0.3mg/L。
Choosing these two japonica rice variety antherderived callus equally as donor, operational approach, cultural method are with embodiment 1, meter Calculate plantlet differentiation rate.
Embodiment 13
It is formulated as follows the culture medium (biotin add concentration preferred) of formula:
KNO32950mg/L, C8H7O2K 1900mg/L, (NH4)2SO4430mg/L, KH2PO4350mg/L, CaCl2 110mg/L, MgSO485mg/L, Na2-EDTA 32.5mg/L, FeSO4∙7H2O 24mg/L, MnSO4∙H2O 3.0mg/L, ZnSO4∙H2O 1.3mg/L, H3BO31.4mg/L, KI 0.65mg/L, CuSO4∙5H2O 0.05mg/L, Na2MoO4∙2H2O 0.16mg/L, CoCl2∙6H2O 0.16mg/L, glycerol 0.9mg/L, glycine 2.0mg/L, folic acid 0.4mg/L, methyl is sub- Nitrourea 1.2mg/L, alanine 1.2mg/L, lysine 1.4mg/L, vitamin B1 5.0mg/L, vitamin B6 1.0mg/ L, acidum nicotinicum 1.0mg/L, sucrose 25g/L, plant gel 5.5g/L；
NAA 0.05mg/L, 6-BA 2.0mg/L, Prohexadione calcium 0.7mg/L, sorbitol 25g/L, L-PROLINE 120mg/L, hydrolyzed protein 1.0g/L, colchicine 0.2mg/L, pH 5.9.
Biotin adds concentration and arranges following 6 kinds: 0；0.02mg/L；0.04mg/L；0.06mg/L；0.1mg/L； 0.12mg/L。
Choosing these two japonica rice variety antherderived callus equally as donor, operational approach, cultural method are with embodiment 1, meter Calculate plantlet differentiation rate.
Culture medium contrast experiment
Embodiment 2-13 being contrasted with embodiment 1 respectively, the result of contrast is as shown in the table:
From table 1-12 it can be seen that use culture medium provided by the present invention than using other any contrast culture medium to round-grained rice The green plant regeneration of rice anther callus is in hgher efficiency, show the culture medium prescription that the present invention provides be through a large amount of single-factors, The optimum combination that multifactor experiment is screened, we have also done little scope single-factor around the combination matching of the present invention and have changed for many years Becoming optimum organization test, substantial amounts of experimentation also show that the component proportion of the present invention is optimum.Use provided by the present invention Culture medium japonica rice variety is led to section's round-grained rice, the callus plantlet differentiation rate of black shell purple round-grained rice has brought up to 56.4% and 65.5%, fill The culture medium that the clear present invention that defends oneself provides is the japonica rice flower pesticide division culture medium of a kind of notable improvement.
Embodiment 14
For the culture medium Seedling green to japonica rice anther callus that relatively culture medium provided by the present invention uses with forefathers The difference of differentiation effect, we have searched 3 japonica rice anther callus differentiation culture based formulas from pertinent literature, have been configured to flower Medicine wound healing division culture medium, chooses japonica rice variety equally and leads to section's round-grained rice and black shell purple round-grained rice is green as anther callus donor differentiated Seedling, operational approach, cultural method, with embodiment 1, calculate callus plantlet differentiation rate.
Other 3 division culture mediums are:
SK-culture medium: MS minimal medium+30g/L sucrose+6.5g/L agar+1mg/L NAA+1mg/L 6-BA+ 0.5mg/L KT, pH are that 5.9(documents comes from " efficiency of different culture media in hybrid paddy rice Anther Culture " such as Meng Xiangzhen)；
MS culture medium: MS minimal medium+30g/L sucrose+4.2g/L agar+0.5mg/L NAA+2mg/L 6-BA+ 0.5mg/L IAA+0.1mg/L IBA+100mg/L inositol+2mg/L MET+0.5g/L LH, pH be 5.8(documents from Yu Lixin etc. " North Japonica Rice Anther Culture Ability Genetic Analysis ")；
NB culture medium: NB minimal medium+30g/L sucrose+7g/L agar+3mg/L 6-BA+0.5mg/L NAA, pH is 5.8(documents comes from " screenings of high Anther Culture Efficiency japonica rice cross combination " such as Wang Qing).
Culture medium contrast experiment
Embodiment 14 being contrasted with embodiment 1, the result of contrast is as shown in the table:
From the point of view of the green plant regeneration effect of different culture media, culture medium of the present invention green Seedling average to japonica rice anther callus Differentiation rate is 61.0%, and SK-, MS, NB culture medium is then 34.9%, 37.0%, 31.4%, it appeared that the present invention is to japonica rice flower pesticide The plantlet differentiation rate of callus to be significantly higher than other 3 kinds of japonica rice flower pesticide division culture mediums.
Above 14 embodiments can illustrate that the component proportion of the culture medium prescription that the present invention provides is optimum combination, this The green plant regeneration effect of japonica rice anther callus is had by the culture medium of bright offer than the japonica rice flower pesticide division culture medium of forefathers Significantly improve.Our result of study is for further genralrlization and applies japonica rice haploid breeding and theoretical research without being suspected to have relatively Big realistic meaning and practical value.
Those skilled in the art can be according to present disclosure and the art technology grasped in the present invention Holding and make replacement or modification, but these are replaced or modification is all not regarded as a departure from present inventive concept, these are replaced or modification All in claimed interest field.

Claims (1)

1. a japonica rice flower pesticide division culture medium, it is characterised in that the formula of this culture medium is as follows:
KNO32820～3080mg/L, C8H7O2K 1800～2000mg/L, (NH4)2SO4400～460mg/L, KH2PO4 320 ～380mg/L, CaCl2100～120mg/L, MgSO480～90mg/L, Na2-EDTA 30～35mg/L, FeSO4∙7H2O 22 ～26mg/L, MnSO4∙H2O 2.8～3.2mg/L, ZnSO4∙H2O 1.2～1.4mg/L, H3BO31.3～1.5mg/L, KI 0.6～0.7mg/L, CuSO4∙5H2O 0.04～0.06mg/L, Na2MoO4∙2H2O 0.14～0.18mg/L, CoCl2∙6H2O 0.14～0.18mg/L, glycerol 0.8～1.0mg/L, glycine 1.8～2.2mg/L, folic acid 0.35～0.45mg/L, first Base nitroso ureas 1.1～1.3mg/L, alanine 1.1～1.3mg/L, lysine 1.3～1.5mg/L, vitamin B1 4.5 ～5.5mg/L, vitamin B6 0.9～1.1mg/L, acidum nicotinicum 0.9～1.1mg/L, sucrose 23～27g/L, plant coagulates Glue 5～6g/L；
NAA 0.045～0.055mg/L, 6-BA 1.8～2.2mg/L, Prohexadione calcium 0.6～0.8mg/L, sorbitol 23～ 27g/L, L-PROLINE 100～140mg/L, hydrolyzed protein 0.9～1.1g/L, colchicine 0.18～0.22mg/L, biological Element 0.07～0.09mg/L, pH 5.8-6.0.
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