CN103416309B - A kind of broccoli cells in-vitro quick proliferation method - Google Patents
A kind of broccoli cells in-vitro quick proliferation method Download PDFInfo
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Abstract
The present invention relates to a kind of broccoli cells in-vitro quick proliferation method, the method comprises the following steps: (1) prepare MS minimal medium; (2) broccoli cells in-vitro obtains and inoculation method: will obtain aseptic seed after broccoli seed irrigation and disinfection then; Aseptic seed is inoculated on MS minimal medium, obtains aseptic seedling after cultivating 14d; Cut plumule and be inoculated into face up on MS inducing culture, under dark, temperature are 24 ~ 26 DEG C of conditions, dedifferentiation is cultivated 30d and is namely obtained broccoli cells in-vitro; (3) broccoli cells in-vitro enrichment procedure: broccoli cells in-vitro be inoculated on MS proliferated culture medium, cultivates 15 ~ 20d under dark, temperature are 24 ~ 26 DEG C of conditions.The inventive method is easy, quick, can the fast breeding of directed regulation and control cells in-vitro and growth, improves the multiplication rate of cell, thus breaks away from broccoli self-sow rule, shorten the breeding cycle.
Description
Technical field
The present invention relates to plant biotechnology field, particularly relate to a kind of broccoli cells in-vitro quick proliferation method.
Background technology
Broccoli (
brassicaoleraceal.var
italicaplenck) belong to the Cruciferae tomb that rues and belong to brassica specie, be one, biennial herb plant.Broccoli is with the vitality of tanacity, irreplaceable nutritive value, medical value, be described as " anti-cancer, anticancer rising star " fashionable world out of edibility.The agricultural research institute of Japan represents that isothiocyanate can stop the growth of black cancer cell.Wherein, sulforaphen is the class isothiocyanate that the anticancer vigor that finds in vegetables is up to now the strongest, and its antitumaous effect is proved fully in the breast cancer of rat, skin.In recent years, the research of extracting sulforaphen from broccoli seed, Different Organs is wider, but from these materials, extract sulforaphen can only depend on plant itself, be seriously subject to the multifactorial restrictions such as cost is high, content is low, season, plant growing cycle, land resources.The basic method addressed this problem is to create a kind of broccoli cells in-vitro quick proliferation method.
But, want the object reaching fast breeding, the internal and external factors such as broccoli kind, medium, condition of culture and growth cycle must be considered, wherein the selection of induction and proliferated culture medium and hormone concentration and media thereof plays decisive role, to such an extent as to cells in-vitro induction is carried out to broccoli organ material and fast breeding condition is optimized, find the culture environment of best inducation and proliferation, the optimization completing the rapid, high volume enrichment procedure of broccoli cell is very important.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of broccoli cells in-vitro quick proliferation method effectively efficiently.
For solving the problem, a kind of broccoli cells in-vitro quick proliferation method of the present invention, comprises the following steps:
(1) prepare MS minimal medium:
First in the beaker of 1000mL, the agar of 4.5g ~ 5.5g being added temperature is in the distilled water of 85 ~ 95 DEG C, being stirred well to described agar to dissolving completely, obtaining aqueous agar solution; Then in described aqueous agar solution, add macroelement mother liquor 20mL, micro-mother liquor 20mL, mother liquid of iron salt 20mL, Calcisolution 20mL, magnesium salts mother liquor 20mL, organic mother liquor 20mL, inositol mother liquor 20mL successively, the sucrose of 30g is added after abundant stirring, be stirred to after sucrose dissolves completely and be settled to 1000mL scale place with distilled water, after stirring evenly, to add concentration be 0.1mol/LNaOH or concentration is 0.1mol/LHCl adjust ph to 5.8 ~ 6.0; Finally in 100mL triangular flask, every 40 ~ 60mL is packed as one bottle, and after 121 DEG C of sterilizing 20min, horizontal rest cooling 4h under room temperature, obtains MS minimal medium;
(2) broccoli cells in-vitro obtains and inoculation method:
To broccoli seed with after clear water flushing 20 ~ 40min, be first the alcohol disinfecting 0.25 ~ 0.75min of 75% by mass concentration then, then be 1%NaClO sterilization 2 ~ 6min by mass concentration, obtain aseptic seed; Described aseptic seed sterile water is rinsed and is washed 4 ~ 5 times and be inoculated on described MS minimal medium, intensity of illumination be 4000 ~ 6000LX, temperature obtains aseptic seedling after cultivating 14d under being 24 ~ 26 DEG C of conditions, wherein inoculates 5 ~ 8 seeds in MS minimal medium described in every 40 ~ 60mL; Cut plumule that length is 5mm and be inoculated into face up on MS inducing culture, wherein inoculating 4 ~ 6 plumules in MS inducing culture described in every 40 ~ 60mL, under dark, temperature are 24 ~ 26 DEG C of conditions, dedifferentiation is cultivated 30d and is namely obtained broccoli cells in-vitro;
(3) broccoli cells in-vitro enrichment procedure:
Described broccoli cells in-vitro is inoculated on MS proliferated culture medium, under dark, temperature are 24 ~ 26 DEG C of conditions, cultivate 15 ~ 20d, wherein inoculate 4 ~ 6 pieces in MS proliferated culture medium described in every 40 ~ 60mL and the described broccoli cells in-vitro of every block 0.05 ~ 0.1g.
Described step (2) in MS inducing culture refer to first in the beaker of 1000mL, the agar of 4.5g ~ 5.5g being added temperature is in the distilled water of 85 ~ 95 DEG C, is stirred well to described agar to dissolving completely, obtains aqueous agar solution; Then in described aqueous agar solution, add macroelement mother liquor 20mL, micro-mother liquor 20mL, mother liquid of iron salt 20mL, Calcisolution 20mL, magnesium salts mother liquor 20mL, organic mother liquor 20mL, inositol mother liquor 20mL successively, the sucrose of 30g is added after abundant stirring, be stirred to sucrose to dissolve completely, obtain MS minimal medium mixed liquor; Secondly the 6-benzyl aminoadenine (6-BA) of 2.0mg and 2 of 1.0mg are added at described MS minimal medium mixed liquor, 4-dichlorphenoxyacetic acid (2,4-D), 1000mL scale place is settled to afterwards with distilled water, after stirring evenly, to add concentration be 0.1mol/LNaOH or concentration is 0.1mol/LHCl adjust ph to 5.8 ~ 6.0; Finally in 100mL triangular flask, every 40 ~ 60mL is packed as one bottle, and after 121 DEG C of sterilizing 20min, under room temperature, horizontal rest cools 4h and get final product.
Described step (3) in MS proliferated culture medium refer to first in the beaker of 1000mL, it is in the distilled water of 85 ~ 95 DEG C that the agar of 4.5g ~ 5.5g and 200mg caseinhydrolysate are added temperature respectively, being stirred well to described agar and described caseinhydrolysate to dissolving completely, obtaining the agar proteins aqueous solution; Then in the described agar proteins aqueous solution, macroelement mother liquor 20mL, micro-mother liquor 20mL, mother liquid of iron salt 20mL, Calcisolution 20mL, magnesium salts mother liquor 20mL, organic mother liquor 20mL, inositol mother liquor 20mL is added successively, 30g sucrose is added after abundant stirring, be stirred to sucrose to dissolve completely, obtain MS minimal medium mixed liquor; Secondly the 6-benzyl aminoadenine (6-BA) of 1.7mg and the methyl α-naphthyl acetate (NAA) of 0.9mg is added at described MS minimal medium mixed liquor, 1000mL scale place is settled to afterwards with distilled water, after stirring evenly, to add concentration be 0.1mol/LNaOH or concentration is 0.1mol/LHCl adjust ph to 5.8 ~ 6.0; Finally in 100mL triangular flask, every 40 ~ 60mL is packed as one bottle, and after 121 DEG C of sterilizing 20min, under room temperature, horizontal rest cools 4h and get final product.
Described macroelement mother liquor refers to containing ammonium nitrate 82500mg/L, potassium nitrate 95000mg/L, the mixed liquor of potassium dihydrogen phosphate 8500mg/L.
Described micro-mother liquor refers to containing MnSO
44H
2o1115mg/L, ZnSO
47H
2o430mg/L, boric acid 248mg/L, potassium iodide 41.5mg/L, Na
2moO
42H
2o12.5mg/L, CuSO
45H
2o1.25mg/L, CoCl
26H
2the mixed liquor of O1.25mg/L.
Described mother liquid of iron salt refers to containing FeSO
47H
2o1390mg/L, Na
2 . eDTA
. 2H
2the mixed liquor of O1865mg/L.
Described magnesium salts mother liquor refers to containing MgSO
47H
2the solution of O18500mg/L.
Described Calcisolution refers to containing CaCl
22H
2the solution of O22000mg/L.
Described organic mother liquor refers to containing nicotinic acid 25mg/L, puridoxine hydrochloride (Cobastab
6) 25mg/L, thiamine hydrochloride (Cobastab
1) 25mg/L, the mixed liquor of glycine 100mg/L.
Described inositol mother liquor refers to the solution containing inositol 5000mg/L.
The present invention compared with prior art has the following advantages:
1, because the present invention carries out cell chulture by plant tissue culture technique, the isolated growth condition of artificial adjustment broccoli cell, therefore, can the fast breeding of directed regulation and control cells in-vitro and growth, improve the multiplication rate of cell, thus break away from broccoli self-sow rule, shorten the breeding cycle.
2, because the present invention can the growth of In vitro Regulation cell, thus can carry out the metabolic regulation of secondary metabolites sulforaphen synthesis to it, improve the ability of cell synthetic radish thionin, reduce the production cost of sulforaphen.
3, the inventive method is easy, quick.
Embodiment
embodiment 1a kind of broccoli cells in-vitro quick proliferation method, comprises the following steps:
(1) prepare MS minimal medium:
First in the beaker of 1000mL, the agar of 4.5g ~ 5.5g being added temperature is in the distilled water of 85 ~ 95 DEG C, being stirred well to agar to dissolving completely, obtaining aqueous agar solution; Then in aqueous agar solution, add macroelement mother liquor 20mL, micro-mother liquor 20mL, mother liquid of iron salt 20mL, Calcisolution 20mL, magnesium salts mother liquor 20mL, organic mother liquor 20mL, inositol mother liquor 20mL successively, the sucrose of 30g is added after abundant stirring, be stirred to after sucrose dissolves completely and be settled to 1000mL scale place with distilled water, after stirring evenly, to add concentration be 0.1mol/LNaOH or concentration is 0.1mol/LHCl adjust ph to 5.8 ~ 6.0; Finally in 100mL triangular flask, every 40 ~ 60mL is packed as one bottle, and after 121 DEG C of sterilizing 20min, horizontal rest cooling 4h under room temperature, obtains MS minimal medium.
(2) broccoli cells in-vitro obtains and inoculation method:
To broccoli seed with after clear water flushing 20min, be first the alcohol disinfecting 0.25min of 75% by mass concentration then, then be 1%NaClO sterilization 2min by mass concentration, obtain aseptic seed; Aseptic seed sterile water is rinsed and is washed 4 ~ 5 times and be inoculated on described MS minimal medium, intensity of illumination be 4000LX, temperature obtains aseptic seedling after cultivating 14d under being 24 DEG C of conditions, wherein inoculation 5 seeds in every 40mLMS minimal medium; Cut plumule that length is 5mm and be inoculated into face up on MS inducing culture, wherein inoculation 4 plumules in every 40mLMS inducing culture, under dark, temperature are 24 DEG C of conditions, dedifferentiation is cultivated 30d and is namely obtained broccoli cells in-vitro.
(3) broccoli cells in-vitro enrichment procedure:
Broccoli cells in-vitro is inoculated on MS proliferated culture medium, cultivates 15d in dark, temperature under being 24 DEG C of conditions, wherein inoculate 4 pieces in every 40mLMS proliferated culture medium and the broccoli cells in-vitro of every block 0.05g.
embodiment 2a kind of broccoli cells in-vitro quick proliferation method, comprises the following steps:
(1) prepare MS minimal medium same
embodiment 1.
(2) broccoli cells in-vitro obtains and inoculation method:
To broccoli seed with after clear water flushing 40min, be first the alcohol disinfecting 0.75min of 75% by mass concentration then, then be 1%NaClO sterilization 6min by mass concentration, obtain aseptic seed; Aseptic seed sterile water is rinsed and is washed 4 ~ 5 times and be inoculated on described MS minimal medium, intensity of illumination be 6000LX, temperature obtains aseptic seedling after cultivating 14d under being 26 DEG C of conditions, wherein inoculation 8 seeds in every 60mLMS minimal medium; Cut plumule that length is 5mm and be inoculated into face up on MS inducing culture, wherein inoculation 6 plumules in every 60mLMS inducing culture, under dark, temperature are 26 DEG C of conditions, dedifferentiation is cultivated 30d and is namely obtained broccoli cells in-vitro.
(3) broccoli cells in-vitro enrichment procedure:
Broccoli cells in-vitro is inoculated on MS proliferated culture medium, cultivates 20d in dark, temperature under being 26 DEG C of conditions, wherein inoculate 6 pieces in every 60mLMS proliferated culture medium and the broccoli cells in-vitro of every block 0.1g.
embodiment 3a kind of broccoli cells in-vitro quick proliferation method, comprises the following steps:
(1) prepare MS minimal medium same
embodiment 1.
(2) broccoli cells in-vitro obtains and inoculation method:
To broccoli seed with after clear water flushing 30min, be first the alcohol disinfecting 0.50min of 75% by mass concentration then, then be 1%NaClO sterilization 4min by mass concentration, obtain aseptic seed; Aseptic seed sterile water is rinsed and is washed 4 ~ 5 times and be inoculated on described MS minimal medium, intensity of illumination be 5000LX, temperature obtains aseptic seedling after cultivating 14d under being 25 DEG C of conditions, wherein inoculation 6 seeds in every 50mLMS minimal medium; Cut plumule that length is 5mm and be inoculated into face up on MS inducing culture, wherein inoculation 5 plumules in every 50mLMS inducing culture, under dark, temperature are 25 DEG C of conditions, dedifferentiation is cultivated 30d and is namely obtained broccoli cells in-vitro.
(3) broccoli cells in-vitro enrichment procedure:
Broccoli cells in-vitro is inoculated on MS proliferated culture medium, cultivates 18d in dark, temperature under being 25 DEG C of conditions, wherein inoculate 5 pieces in every 50mLMS proliferated culture medium and the broccoli cells in-vitro of every block 0.08g.
Above-mentioned
embodiment 1 ~ 3in, MS inducing culture refers to first in the beaker of 1000mL, and the agar of 4.5g ~ 5.5g being added temperature is in the distilled water of 85 ~ 95 DEG C, being stirred well to described agar to dissolving completely, obtaining aqueous agar solution; Then in described aqueous agar solution, add macroelement mother liquor 20mL, micro-mother liquor 20mL, mother liquid of iron salt 20mL, Calcisolution 20mL, magnesium salts mother liquor 20mL, organic mother liquor 20mL, inositol mother liquor 20mL successively, the sucrose of 30g is added after abundant stirring, be stirred to sucrose to dissolve completely, obtain MS minimal medium mixed liquor; Secondly the 6-benzyl aminoadenine (6-BA) of 2.0mg and 2 of 1.0mg are added at described MS minimal medium mixed liquor, 4-dichlorphenoxyacetic acid (2,4-D), 1000mL scale place is settled to afterwards with distilled water, after stirring evenly, to add concentration be 0.1mol/LNaOH or concentration is 0.1mol/LHCl adjust ph to 5.8 ~ 6.0; Finally in 100mL triangular flask, every 40 ~ 60mL is packed as one bottle, and after 121 DEG C of sterilizing 20min, under room temperature, horizontal rest cools 4h and get final product.
MS proliferated culture medium refers to first in the beaker of 1000mL, it is in the distilled water of 85 ~ 95 DEG C that the agar of 4.5g ~ 5.5g and 200mg caseinhydrolysate are added temperature respectively, being stirred well to described agar and described caseinhydrolysate to dissolving completely, obtaining the agar proteins aqueous solution; Then in the described agar proteins aqueous solution, macroelement mother liquor 20mL, micro-mother liquor 20mL, mother liquid of iron salt 20mL, Calcisolution 20mL, magnesium salts mother liquor 20mL, organic mother liquor 20mL, inositol mother liquor 20mL is added successively, 30g sucrose is added after abundant stirring, be stirred to sucrose to dissolve completely, obtain MS minimal medium mixed liquor; Secondly the 6-benzyl aminoadenine (6-BA) of 1.7mg and the methyl α-naphthyl acetate (NAA) of 0.9mg is added at described MS minimal medium mixed liquor, 1000mL scale place is settled to afterwards with distilled water, after stirring evenly, to add concentration be 0.1mol/LNaOH or concentration is 0.1mol/LHCl adjust ph to 5.8 ~ 6.0; Finally in 100mL triangular flask, every 40 ~ 60mL is packed as one bottle, and after 121 DEG C of sterilizing 20min, under room temperature, horizontal rest cools 4h and get final product.
Wherein: macroelement mother liquor refers to containing ammonium nitrate 82500mg/L, potassium nitrate 95000mg/L, the mixed liquor of potassium dihydrogen phosphate 8500mg/L.
Trace element mother liquor refers to containing MnSO
44H
2o1115mg/L, ZnSO
47H
2o430mg/L, boric acid 248mg/L, potassium iodide 41.5mg/L, Na
2moO
42H
2o12.5mg/L, CuSO
45H
2o1.25mg/L, CoCl
26H
2the mixed liquor of O1.25mg/L.
Mother liquid of iron salt refers to containing FeSO
47H
2o1390mg/L, Na
2 . eDTA
. 2H
2the mixed liquor of O1865mg/L.
Magnesium salts mother liquor refers to containing MgSO
47H
2the solution of O18500mg/L.
Calcisolution refers to containing CaCl
22H
2the solution of O22000mg/L.
Organic mother liquor refers to containing nicotinic acid 25mg/L, puridoxine hydrochloride (Cobastab
6) 25mg/L, thiamine hydrochloride (Cobastab
1) 25mg/L, the mixed liquor of glycine 100mg/L.
Inositol mother liquor refers to the solution containing inositol 5000mg/L.
Claims (1)
1. a broccoli cells in-vitro quick proliferation method, comprises the following steps:
(1) prepare MS minimal medium:
First in the beaker of 1000mL, the agar of 4.5g ~ 5.5g being added temperature is in the distilled water of 85 ~ 95 DEG C, being stirred well to described agar to dissolving completely, obtaining aqueous agar solution; Then in described aqueous agar solution, add macroelement mother liquor 20mL, micro-mother liquor 20mL, mother liquid of iron salt 20mL, Calcisolution 20mL, magnesium salts mother liquor 20mL, organic mother liquor 20mL, inositol mother liquor 20mL successively, the sucrose of 30g is added after abundant stirring, be stirred to after sucrose dissolves completely and be settled to 1000mL scale place with distilled water, after stirring evenly, to add concentration be 0.1mol/LNaOH or concentration is 0.1mol/LHCl adjust ph to 5.8 ~ 6.0; Finally in 100mL triangular flask, every 40 ~ 60mL is packed as one bottle, and after 121 DEG C of sterilizing 20min, horizontal rest cooling 4h under room temperature, obtains MS minimal medium;
Described macroelement mother liquor refers to containing ammonium nitrate 82500mg/L, potassium nitrate 95000mg/L, the mixed liquor of potassium dihydrogen phosphate 8500mg/L; Described micro-mother liquor refers to containing MnSO
44H
2o1115mg/L, ZnSO
47H
2o430mg/L, boric acid 248mg/L, potassium iodide 41.5mg/L, Na
2moO
42H
2o12.5mg/L, CuSO
45H
2o1.25mg/L, CoCl
26H
2the mixed liquor of O1.25mg/L; Described mother liquid of iron salt refers to containing FeSO
47H
2o1390mg/L, Na
2 . eDTA
. 2H
2the mixed liquor of O1865mg/L; Described magnesium salts mother liquor refers to containing MgSO
47H
2the solution of O18500mg/L; Described Calcisolution refers to containing CaCl
22H
2the solution of O22000mg/L; Described organic mother liquor refers to containing nicotinic acid 25mg/L, puridoxine hydrochloride 25mg/L, thiamine hydrochloride 25mg/L, the mixed liquor of glycine 100mg/L; Described inositol mother liquor refers to the solution containing inositol 5000mg/L;
(2) broccoli cells in-vitro obtains and inoculation method:
To broccoli seed with after clear water flushing 20 ~ 40min, be first the alcohol disinfecting 0.25 ~ 0.75min of 75% by mass concentration then, then be 1%NaClO sterilization 2 ~ 6min by mass concentration, obtain aseptic seed; Described aseptic seed sterile water is rinsed and is washed 4 ~ 5 times and be inoculated on described MS minimal medium, intensity of illumination be 4000 ~ 6000LX, temperature obtains aseptic seedling after cultivating 14d under being 24 ~ 26 DEG C of conditions, wherein inoculates 5 ~ 8 seeds in MS minimal medium described in every 40 ~ 60mL; Cut plumule that length is 5mm and be inoculated into face up on MS inducing culture, wherein inoculating 4 ~ 6 plumules in MS inducing culture described in every 40 ~ 60mL, under dark, temperature are 24 ~ 26 DEG C of conditions, dedifferentiation is cultivated 30d and is namely obtained broccoli cells in-vitro;
Described MS inducing culture refers to first in the beaker of 1000mL, and the agar of 4.5g ~ 5.5g being added temperature is in the distilled water of 85 ~ 95 DEG C, being stirred well to described agar to dissolving completely, obtaining aqueous agar solution; Then in described aqueous agar solution, add macroelement mother liquor 20mL, micro-mother liquor 20mL, mother liquid of iron salt 20mL, Calcisolution 20mL, magnesium salts mother liquor 20mL, organic mother liquor 20mL, inositol mother liquor 20mL successively, the sucrose of 30g is added after abundant stirring, be stirred to sucrose to dissolve completely, obtain MS minimal medium mixed liquor; Secondly the 6-benzyl aminoadenine of 2.0mg and 2 of 1.0mg are added at described MS minimal medium mixed liquor, 4-dichlorphenoxyacetic acid, be settled to 1000mL scale place with distilled water afterwards, after stirring evenly, to add concentration be 0.1mol/LNaOH or concentration is 0.1mol/LHCl adjust ph to 5.8 ~ 6.0; Finally in 100mL triangular flask, every 40 ~ 60mL is packed as one bottle, and after 121 DEG C of sterilizing 20min, under room temperature, horizontal rest cools 4h and get final product;
(3) broccoli cells in-vitro enrichment procedure:
Described broccoli cells in-vitro is inoculated on MS proliferated culture medium, under dark, temperature are 24 ~ 26 DEG C of conditions, cultivate 15 ~ 20d and get final product, wherein inoculating 4 ~ 6 pieces in MS proliferated culture medium described in every 40 ~ 60mL and the described broccoli cells in-vitro of every block 0.05 ~ 0.1g;
Described MS proliferated culture medium refers to first in the beaker of 1000mL, it is in the distilled water of 85 ~ 95 DEG C that the agar of 4.5g ~ 5.5g and 200mg caseinhydrolysate are added temperature respectively, being stirred well to described agar and described caseinhydrolysate to dissolving completely, obtaining the agar proteins aqueous solution; Then in the described agar proteins aqueous solution, macroelement mother liquor 20mL, micro-mother liquor 20mL, mother liquid of iron salt 20mL, Calcisolution 20mL, magnesium salts mother liquor 20mL, organic mother liquor 20mL, inositol mother liquor 20mL is added successively, 30g sucrose is added after abundant stirring, be stirred to sucrose to dissolve completely, obtain MS minimal medium mixed liquor; Secondly the 6-benzyl aminoadenine of 1.7mg and the methyl α-naphthyl acetate of 0.9mg is added at described MS minimal medium mixed liquor, 1000mL scale place is settled to afterwards with distilled water, after stirring evenly, to add concentration be 0.1mol/LNaOH or concentration is 0.1mol/LHCl adjust ph to 5.8 ~ 6.0; Finally in 100mL triangular flask, every 40 ~ 60mL is packed as one bottle, and after 121 DEG C of sterilizing 20min, under room temperature, horizontal rest cools 4h and get final product.
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| CN111034616B (en) * | 2019-12-25 | 2022-05-06 | 甘肃农业大学 | Broccoli hairy root high sulforaphane glycoside and sulforaphane synthesis release culture method |
| CN111758571A (en) * | 2020-07-23 | 2020-10-13 | 温州科技职业学院 | Special culture medium for promoting quick growth of broccoli and preparation method thereof |
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| CN1552198A (en) * | 2003-06-05 | 2004-12-08 | 天津科润农业科技股份有限公司 | Method for culturing cauliflower regenerative tree by free microspore culture technology |
| CN101617630A (en) * | 2009-08-13 | 2010-01-06 | 浙江省农业科学院 | Culture method of high diplont rate sporule regeneration plant of broccoli |
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| US7256328B2 (en) * | 2005-08-31 | 2007-08-14 | Sakata Seed Corporation | Inbred broccoli line GKO-1 |
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