CN101283669B - Breeding technique of obtaining triploid grape and ploidy early identification using embryo - Google Patents
Breeding technique of obtaining triploid grape and ploidy early identification using embryo Download PDFInfo
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Abstract
The invention discloses a culture method for triploid grape culture by embryo rescue and early identification of ploidy. The invention aims to cultivate triploid grape by embryo rescue and overcome the problem of seed abortion of hybrid embryo between diploid and tetraploid grape cultivars in conventional breeding. After a hybrid seed is obtained, chromosome count is performed by the combination of flow cytometry and wall degradation hypotonic method to identify the ploidy of the hybrid progeny. The invention increases the breeding efficiency of triploid grape, accelerates the breed selection of triploid grape variety and provides a new approach to the culture of seedless grape variety.
Description
Technical field
The present invention relates to plant breeding method, particularly relate to and utilize embryo to save cultivation acquisition triploid grape hybridization seedling, the gained hybrid generation is carried out system and the high-efficient breeding method of the early stage evaluation of comprehensive ploidy, belong to biological technical field.
Background technology
Polyploid breeding is the important channel of cultivating fruit tree of new species; Its superiority is that the growth that is shown is vigorous, branch is thick, leaf is thick, fruit is big, output is high and characteristics such as adaptability is strong, and polyploid generation approach has nature bud mutation, artificial mutagenesis, artificial sexual hybridization, endosperm cultivation and anther culture etc.Utilize wherein that to hybridize application between liploid variety and the tetraploid the widest, in watermelon, banana, citrus, bear fruit, obtained the triploid seedless fruit.Grape is as global second largest fruit, and its seedless variety is the important directions of current international consumption always, and obtains the attention that big grain currant new varieties enjoy the breeding scholar for a long time.Triploid grape is except that the many superiority with polyploid plant, and its fruit is seedless or lack nuclear, so the new way of cultivating big grain currant kind has been opened up in the triploid grape breeding.According to research reports, Japan bred the triploid currant kind tail tinkling of pieces of jade, wear draw that king, honey are seedless, the striking Mei Ling of first, summer be black, it is red seedless morning that China has bred triploid, and on producing, begin to promote.
The triploid grape kind is cultivated through bud mutation, the endosperm of dliploid and tetraploid intervarietal cross, natural crossing, triploid kind and number of ways generation such as anther culture; But in fact, valid approach remains and utilizes dliploid and tetraploid direct cross.But the biggest obstacle of dliploid and tetraploid hybridization is that affinity is poor, and percentage of fertile fruit is low, and early stage abortion of hybrid embryo or endosperm disintegrate, and it is low to obtain the triploid hydrid seed vigor, is difficult to obtain filial generation.Utilize embryo rescue techniques, can stop the early stage abortion of triploid hydrid rataria, form triploid.
Since eighties of last century, to cultivate through ovule, abundant utilization has the tetraploid and the diploid hybrid of nuclear grape under the Japanese scholar mountain; Obtained triploid (" horticulture can journal 1993,62 (2): 249-255), sincere (" the Shanghai Agricultural journal " 1998 of Lee's generation thereafter; 14 (4): 13-17), Pan Chunyun (" Shandong Agricultural University's journal " 1998; 29 (3): 299-302), Xu Haiying (" fruit tree journal " 2001,18 (6): 317-320), Guo Yinshan (" Agricultural University Of Shenyang's journal " 2005,36 (5): 606-608) wait and also to adopt embryo rescue techniques; The early stage abortion of hybridization embryo, obstacle that breeding efficiency is low have been overcome in grape dliploid and the tetraploid conventional breeding; Success bred triploid hybrid seedling, but gained hybridization seedling is carried out comprehensive ploidy detecting of rare system lacks complete breeding system efficiently.
Usually the method for polyploid evaluation has methods such as plant trait evaluation, cytological Identification, molecular biology identification; And the grape polyploid adopts the microscopically chromosome number to identify more; Also utilize flow cytometer to measure; Chromosome counting method is intuitively effective, but material only limits to the stem apex and the tip of a root, and complicated operation.Flow cytometer is that plant dna content to be measured is identified, can detect thousands of cells at short notice, can guarantee to obtain the population characteristic of biological cell, measure fast, and reliable results, but only semiquantitative measurement result can be provided.
Summary of the invention
The object of the invention is exactly the pluses and minuses to present situation of triploid grape breeding now and various polyploid authentication methods; Utilize embryo to save and cultivate acquisition triploid grape hybrid seedling; In conjunction with flow cytometry, remove the hypotonic method chromosome counting of wall; To the gained hybrid generation carry out system and comprehensively ploidy identify in early days, improve the breeding efficiency of triploid grape.
Implementation step of the present invention is following:
A. grape breeding field hybridization
Take pollen, bloomed preceding 1 day or come into bloom, adopt down normotrophic inflorescence in tetraploid kind male parent; Remove the part at top 1/3rd, take behind corolla and the flower pesticide, dry under fluorescent light to flower pesticide and split with after the gauze sieve; Pour in the mortar behind the sieve once more, be ground to powdery, pack into and handle clean bottle; Seal, be placed on anhydrous CaCl is housed
2Drier in, 4 ℃ of low temperature are preserved; Castrate pollination, select the inflorescence of growth unanimity on the European currant kind stock tree some, bloom and carried out artificial emasculation in preceding 3-4 days; Spray inflorescence with clear water immediately afterwards; And the bagging and the mark of listing, when beginning on the column cap to secrete drops mucus, dip in absorbent cotton and to get pollen and carry out artificial pollination; Pollination is 3 times continuously, every day 1 time;
B. embryo is saved and is cultivated and the one-tenth seedling
Hybridize and castrate pollination after 40-55 days, the ovule that strips in the hybridization fruit ear carries out embryo redemption cultivation, and fruit ear is cleaned on the rearmounted superclean bench; 75% alcohol immersion 1min; Aseptic water washing 3-5 time is again with 0.1% mercuric chloride sterilization 6min, aseptic water washing 3-5 time; Cutting fruit grain taking-up ovule is inoculated in the 100ml triangular flask of interior dress 40ml medium; Every bottle graft kind 10-20 grain, it is minimal medium that ovules culture medium adopts B5, additional 0.1-1.0mg/LBA, 1.5-2.5mg/LIAA, 0.25-0.75mg/LGA
3, 6% sucrose, 0.6% agar, 0.1% active carbon; After cultivating for 8 weeks, under aseptic condition, stripping naked embryo and be inoculated in the embryo germination medium, is minimal medium with WP, additional 0.1-0.5mg/LIBA, 2% sucrose, 0.6% agar, 0.1% active carbon; Continue to cultivate 10-15 days; Be transferred to into the seedling medium and carry out successive transfer culture, employing 1/2MS is a minimal medium, additional 0.1-0.3mg/LIBA, 2% sucrose, 0.6% agar, 0.1% active carbon; Carrying out follow-up ploidy after in becoming the seedling medium, growing blade identifies; Condition of culture is 25 ℃ ± 2 ℃ of temperature, illumination 16h every day, luminous intensity 2000lux;
C. flow cytometer carries out semi-quantitative analysis to the grape hybrid generation
Saving the Preliminary Identification of cultivating the filial generation ploidy that obtains through embryo adopts flow cytometry to measure its dna content; Get the tender blade of 1-2 sheet children or a small amount of callus and put into culture dish; The DNA extraction liquid HR-A that adds 0.5-1.0ml; After leaving standstill processing 2-5min after the blade chopping, with the Partec Celltrics of sample through 30 μ m
TMMicro-pore-film filtration is in testing tube; Add 2ml dyeing liquor HR-B again, be splined on the Partec flow cytometer with being about to sample, the content distribution figure of DNA is generated by flow cytometer automatically; Wherein, The fluorescent value FL of analysis peak on transverse axis that earlier this kind of father and mother is produced sets, and preset parameter is measured the fluorescent value of hybridization seedling more successively;
D. through going the hypotonic method chromosome counting of wall that the mensuration result of gained is identified
Behind the tip of a root or stem apex of getting the hybridization test-tube plantlet on the superclean bench, drop into immediately and handle 2h-3h in the saturated paracide solution under the normal temperature; Remove treatment fluid, inject the methyl alcohol of new preparation with the distilled water flushing number all over the back: the fixer of glacial acetic acid=3: 1, normal temperature is handled 3h-5h down; Remove fixer, clean with distilled water flushing, the cellulase and 1: 1 the mixed enzyme solution of pectase of adding 3.5%, the ratio of mixed enzyme solution and material is 30: 1, enzymolysis 20-30min under 37 ℃ of constant temperature; 30min-3h dyes in the carbolfuchsin dyeing liquor behind the distilled water flushing; Getting a small pieces of material is placed on the slide of 70% alcohol immersion; Drip a last carbolfuchsin dyeing liquor; Smash the back covered to pieces with tweezers, press one jiao then and evenly beat cover glass, nebulize to dispersion of materials with wooden spillikin; Inhale with blotting paper at last and remove unnecessary dyeing liquor, on the alcolhol burner take a picture with the micro-digital photography of OLMPUS-BX51 type system in dry back;
E. embryo is saved the acclimatization and transplants of seedling
Select 4-5 bar root in strong sprout spring, the long 3-5cm of root, 4-5 sheet leaf, the stem stalk is sturdy, and base portion does not have callus, and high light tempered for 1 week; Seedling is transplanted to the perlite of the bacterium of having gone out after with aseptic water washing: in the matrix of the peat composed of rotten mosses=1: 1 mixing; Culturing room's refining seedling covers plastic cup on seedling, carbendazim and 1/8MS nutrient solution that pouring in every 5-10 days is 1000 times; And in time remove mouldy blade and cane; Greenhouse temperature is controlled at 18-25 ℃, relative moisture 60-70%, intensity of illumination 500-1000lux; Wait that the seedling of transplanting out grows Xin Gen and young leaves, move to the land for growing field crops when ground temperature is increased to 18-23 ℃, take shading screen after the transplanting immediately and irritate sufficient normal root water, open shading screen gradually, and in time water and the diseases prevention worm in spring.
Adopt method of the present invention; To the hybrid combination ruby seedless * rattan harvests, likes that not seedless * black Olympic carries out cultured in vitro, the embryo germination rate reaches 23.1%, 22.2% respectively as a result, planting percent then reaches 87.5%, 75% respectively; Therefore; Obtain more filial generation through the present invention, improved breeding efficiency, quickened the seed selection process of triploid grape kind.
Embodiment
Further specify method of operating of the present invention below in conjunction with specific embodiment
A. the step of grape breeding field hybridization is following:
A.1 coming into bloom, adopting down rattan and harvest, deceive the normotrophic inflorescence of Olympic, removing the part at top 1/3rd; Take behind corolla and the flower pesticide with after the gauze sieve, dry under fluorescent light to flower pesticide and split, pour in the mortar behind the sieve once more; Grind powdery; Pack into and handle clean bottle, seal, be placed on anhydrous CaCl is housed
2Drier in, 4 ℃ of low temperature are preserved;
A.2 bloomed preceding 3 days, and selected inflorescence 10 fringes that maternal kind ruby is seedless, like to grow on the not seedless tree body unanimity, carry out artificial emasculation; Spray inflorescence with clear water immediately afterwards; And the bagging and the mark of listing, when beginning on the column cap to secrete drops mucus, dip in absorbent cotton and to get pollen and carry out artificial pollination; Pollination is 3 times continuously, every day 1 time;
B. the step of embryo redemption cultivation and one-tenth seedling is as follows:
B.1 respectively at pollination back 48d, 47d take the hybrid combination ruby seedless * rattan harvests, likes that the fruit ear of not seedless * black Olympic carries out cultured in vitro; Fruit ear is cleaned on the rearmounted superclean bench 75% alcohol immersion 1min, aseptic water washing 3 times; Again with 0.1% mercuric chloride sterilization 6min; Aseptic water washing 3 times cuts fruit grain taking-up ovule and is inoculated in the 100ml triangular flask of interior dress 40ml medium 10 of every bottle graft kinds; It is minimal medium that ovules culture medium adopts B5, additional 0.5mg/LBA, 2.0mg/LIAA, 0.5mg/LGA
3, 6% sucrose, 0.6% agar, 0.1% active carbon; After cultivating for 8 weeks, under aseptic condition, stripping naked embryo and be inoculated in the embryo germination medium, is minimal medium with WP, additional 0.1mg/LIBA, 2% sucrose, 0.6% agar, 0.1% active carbon; Continue to cultivate for two weeks; Be transferred to into the seedling medium and carry out successive transfer culture, employing 1/2MS is a minimal medium, additional 0.1mg/LIBA, 2% sucrose, 0.6% agar, 0.1% active carbon; Carrying out follow-up ploidy after in becoming the seedling medium, growing blade identifies; Condition of culture is: 25 ℃ ± 2 ℃ of temperature, illumination 16h every day, luminous intensity 2000lux;
C. utilize flow cytometer that the grape hybrid generation is carried out semi-quantitative analysis:
C.1 save through embryo and cultivate the filial generation that obtains; Adopt flow cytometry to measure its dna content; Its ploidy of Preliminary Identification, get the hybrid combination ruby seedless * rattan harvests, likes that tender blade or a small amount of callus of 1-2 sheet children of not seedless * black Olympic gained seedling put into culture dish, add 1.0ml DNA extraction liquid HR-A; After leaving standstill processing 3min after the blade chopping, with the Partec Celltrics of sample through 30 μ m
TMMicro-pore-film filtration is in testing tube; Add 2ml dyeing liquor HR-B again, be splined on the Partec flow cytometer with being about to sample, the content distribution figure of DNA is generated by flow cytometer automatically; Wherein, The fluorescent value FL of analysis peak on transverse axis that earlier this kind of father and mother is produced sets, and preset parameter is measured the fluorescent value of hybridization seedling more successively;
D. gained hybridization seedling is implemented to go the hypotonic method chromosome counting of wall:
D.1 prepare agents useful for same, 3.5% mixed enzyme solution: take by weighing cellulase, each 0.7g of pectase, add 20ml distilled water, preserve in 4 ℃ of refrigerators; Pretreatment fluid: take by weighing the full crystallization of 5g paracide and put into brown reagent bottle, add 100ml and heated to 45 ℃ distilled water, vibration 5min leaves standstill cooling back room temperature and deposits; Fixer: the volume ratio of glacial acetic acid and methyl alcohol=1: 3; The carbolfuchsin dyeing liquor: be made into three kinds of stostes earlier, stoste A:3g basic fuchsin is dissolved in 100ml 70% alcohol, stoste B: get stoste A10ml and join in the 90ml5% aqua carbolisata solution; Stoste C: get stoste B55ml, add 6ml glacial acetic acid and 6ml formalin, wherein stoste A and stoste C long preservation; Stoste B limit was used in two weeks, got stoste C20ml again, added 45% glacial acetic acid 90ml; Add sorbierite 1.8g again, be made into the carbolfuchsin dyeing liquor of 20% concentration, place the back use of two weeks;
D.2 behind the tip of a root or stem apex of getting the hybridization test-tube plantlet on the superclean bench, drop into immediately and handle 2h in the saturated paracide solution under the normal temperature;
D.3 remove treatment fluid, inject the methyl alcohol of new preparation with the distilled water flushing number all over the back: the fixer of glacial acetic acid=3: 1, normal temperature is handled 4h down;
D.4 remove fixer, clean with distilled water flushing, the cellulase and the pectase mixed enzyme solution of adding 3.5%, the ratio of mixed enzyme solution and material is 30: 1, enzymolysis 30min under 37 ℃ of constant temperature;
D.5 1h dyes in the carbolfuchsin dyeing liquor behind the distilled water flushing;
D.6 getting a small pieces of material is placed on the slide of 70% alcohol immersion; Drip a last carbolfuchsin dyeing liquor; Smash the back covered to pieces with tweezers, press one jiao then and evenly beat cover glass, nebulize to dispersion of materials with wooden spillikin; Inhale with blotting paper at last and remove unnecessary dyeing liquor, on the alcolhol burner take a picture with the micro-digital photography of OLMPUS-BX51 type system in dry back;
E. carry out embryo and save the acclimatization and transplants of seedling
E.1 mid or late March is selected 4-5 bar root in strong sprout, the long 3-5cm of root, 4-5 sheet leaf next year; The stem stalk is sturdy, and base portion does not have callus, after high light tempered for 1 week; Seedling is transplanted to the perlite of the bacterium of having gone out after with aseptic water washing: in the matrix of the peat composed of rotten mosses=1: 1 mixing; Culturing room's refining seedling, the cover plastic cup is in order to preserve moisture on seedling
E.2 water 1000 times carbendazim and 1/8MS nutrient solution weekly, and in time remove mouldy blade and cane, greenhouse temperature is controlled at 22 ℃, relative moisture 60%, and intensity of illumination 1000lux,
E.3 wait that the seedling of transplanting out grows Xin Gen and young leaves, move to the land for growing field crops when ground temperature is increased to 2 ℃, take shading screen after the transplanting immediately and irritate sufficient normal root water, open shading screen gradually, and in time water and the diseases prevention worm in spring.
Claims (2)
1. utilize embryo to save and obtain triploid grape and the early stage breeding method of identifying of ploidy, it is characterized in that carrying out according to the following steps:
1.a grape breeding field hybridization
Take pollen, bloomed preceding 1 day or come into bloom, adopt down normotrophic inflorescence in tetraploid kind male parent; Remove the part at top 1/3rd, take behind corolla and the flower pesticide, dry under fluorescent light to flower pesticide and split with after the gauze sieve; Pour in the mortar behind the sieve once more, be ground to powdery, pack into and handle clean bottle; Seal, be placed on anhydrous CaCl is housed
2Drier in, 4 ℃ of low temperature are preserved; Castrate pollination, select the inflorescence of growth unanimity on the European currant kind stock tree some, bloom and carried out artificial emasculation in preceding 3-4 days; Spray inflorescence with clear water immediately afterwards; And the bagging and the mark of listing, when beginning on the column cap to secrete drops mucus, dip in absorbent cotton and to get pollen and carry out artificial pollination; Pollination is 3 times continuously, every day 1 time;
1.b saving, cultivates and the one-tenth seedling embryo
Hybridize and castrate pollination after 40-55 days, the ovule that strips in the hybridization fruit ear carries out embryo redemption cultivation, and fruit ear is cleaned on the rearmounted superclean bench; 75% alcohol immersion 1min; Aseptic water washing 3-5 time is again with 0.1% mercuric chloride sterilization 6min, aseptic water washing 3-5 time; Cutting fruit grain taking-up ovule is inoculated in the 100ml triangular flask of interior dress 40ml medium; Every bottle graft kind 10-20 grain, it is minimal medium that ovules culture medium adopts B5, additional 0.1-1.0mg/LBA, 1.5-2.5mg/LIAA, 0.25-0.75mg/LGA
3, 6% sucrose, 0.6% agar, 0.1% active carbon; After cultivating for 8 weeks, under aseptic condition, stripping naked embryo and be inoculated in the embryo germination medium, is minimal medium with WP, additional 0.1-0.5mg/LIBA, 2% sucrose, 0.6% agar, 0.1% active carbon; Continue to cultivate 10-15 days; Be transferred to into the seedling medium and carry out successive transfer culture, employing 1/2MS is a minimal medium, additional 0.1-0.3mg/LIBA, 2% sucrose, 0.6% agar, 0.1% active carbon; Carrying out follow-up ploidy after in becoming the seedling medium, growing blade identifies; Condition of culture is 25 ℃ ± 2 ℃ of temperature, illumination 16h every day, luminous intensity 2000lux;
1.c flow cytometer carries out semi-quantitative analysis to the grape hybrid generation
Saving the Preliminary Identification of cultivating the filial generation ploidy that obtains through embryo adopts flow cytometry to measure its dna content; Get the tender blade of 1-2 sheet children or a small amount of callus and put into culture dish; The DNA extraction liquid HR-A that adds 0.5-1.0ml; After leaving standstill processing 2-5min after the blade chopping, with the Partec Celltrics of sample through 30 μ m
TMMicro-pore-film filtration is in testing tube; Add 2ml dyeing liquor HR-B again, be splined on the Partec flow cytometer with being about to sample, the content distribution figure of DNA is generated by flow cytometer automatically; Wherein, The fluorescent value FL of analysis peak on transverse axis that earlier this kind of father and mother is produced sets, and preset parameter is measured the fluorescent value of hybridization seedling more successively;
1.d the mensuration result of gained is identified through removing the hypotonic method chromosome counting of wall
Behind the tip of a root or stem apex of getting the hybridization test-tube plantlet on the superclean bench, drop into immediately and handle 2h-3h in the saturated paracide solution under the normal temperature; Remove treatment fluid, inject the volume ratio methyl alcohol of preparation with the distilled water flushing number all over the back: the fixer of glacial acetic acid=3: 1, normal temperature is handled 3h-5h down; Remove fixer, clean with distilled water flushing, the cellulase and 1: 1 the mixed enzyme solution of pectase volume ratio of adding mass volume ratio 3.5%, the ratio of mixed enzyme solution and material is 30: 1, enzymolysis 20-30min under 37 ℃ of constant temperature; 30min-3h dyes in the carbolfuchsin dyeing liquor behind the distilled water flushing; Getting a small pieces of material is placed on the slide of 70% alcohol immersion; Drip a last carbolfuchsin dyeing liquor; Smash the back covered to pieces with tweezers, press one jiao then and evenly beat cover glass, nebulize to dispersion of materials with wooden spillikin; Inhale with blotting paper at last and remove unnecessary dyeing liquor, on the alcolhol burner take a picture with the micro-digital photography of OLMPUS-BX51 type system in dry back;
1.e embryo is saved the acclimatization and transplants of seedling
Select 4-5 bar root in strong sprout spring, the long 3-5cm of root, 4-5 sheet leaf, the stem stalk is sturdy, and base portion does not have callus, and high light tempered for 1 week; Seedling is transplanted to the perlite of the bacterium of having gone out after with aseptic water washing: in the matrix of the peat composed of rotten mosses=1: 1 mixing; Culturing room's refining seedling covers plastic cup on seedling, carbendazim and 1/8MS nutrient solution that pouring in every 5-10 days is 1000 times; And in time remove mouldy blade and cane; Greenhouse temperature is controlled at 18-25 ℃, relative moisture 60-70%, intensity of illumination 500-1000lux; Wait that the seedling of transplanting out grows Xin Gen and young leaves, move to the land for growing field crops when ground temperature is increased to 18-23 ℃, take shading screen after the transplanting immediately and irritate sufficient normal root water, open shading screen gradually, and in time water and the diseases prevention worm in spring.
2. the embryo that utilizes according to claim 1 is saved acquisition triploid grape and the early stage breeding method of identifying of ploidy, it is characterized in that:
2.a grape breeding field hybridization is to adopt that rattan is harvested, the black normotrophic inflorescence of Olympic coming into bloom; Bloomed preceding 3 days, and selected inflorescence 10 fringes that maternal kind ruby is seedless, like to grow on the not seedless tree body unanimity, carry out artificial emasculation;
2.b embryo save to be cultivated and become seedling be respectively at pollination back 48d, 47d take the hybrid combination ruby seedless * rattan harvests, likes that the fruit ear of not seedless * black Olympic carries out cultured in vitro; The about 1min of 75% alcohol immersion; Aseptic water washing 3 times is again with 0.1% mercuric chloride sterilization 6min, aseptic water washing 3 times; Cutting fruit grain taking-up ovule is inoculated in the 100ml triangular flask of interior dress 40ml medium; 10 of every bottle graft kinds, it is minimal medium that ovules culture medium adopts B5, additional 0.5mg/LBA, 2.0mg/LIAA, 0.5mg/LGA
3, 6% sucrose, 0.6% agar, 0.1% active carbon; After cultivating for 8 weeks, under aseptic condition, stripping naked embryo and be inoculated in the embryo germination medium, is minimal medium with WP, additional 0.1mg/LIBA, 2% sucrose, 0.6% agar, 0.1% active carbon; Continue to cultivate for two weeks; Be transferred to into the seedling medium and carry out successive transfer culture, employing 1/2MS is a minimal medium, additional 0.1mg/LIBA, 2% sucrose, 0.6% agar, 0.1% active carbon; Carrying out follow-up ploidy after in becoming the seedling medium, growing blade identifies; Condition of culture is: 25 ℃ ± 2 ℃ of temperature, illumination 16h every day, luminous intensity 2000lux;
2.c utilize flow cytometer that the grape hybrid generation is carried out semi-quantitative analysis: get the hybrid combination ruby seedless * rattan harvests, likes that tender blade or a small amount of callus of 1-2 sheet children of not seedless * black Olympic gained seedling put into culture dish; Add 1.0ml DNA extraction liquid HR-A; After leaving standstill processing 3min after the blade chopping, with the Partec Celltrics of sample through 30 μ m
TMMicro-pore-film filtration adds 2ml dyeing liquor HR-B again in testing tube;
2.d gained hybridization seedling is implemented to go the hypotonic method chromosome counting of wall:
2.d.1 the preparation agents useful for same, 3.5% mixed enzyme solution: take by weighing cellulase, each 0.7g of pectase, add 20ml distilled water, preserve in 4 ℃ of refrigerators; Pretreatment fluid: take by weighing the full crystallization of 5g paracide and put into brown reagent bottle, add 100ml and heated to 45 ℃ distilled water, vibration 5min leaves standstill cooling back room temperature and deposits; Fixer: the volume ratio of glacial acetic acid and methyl alcohol=1: 3; The carbolfuchsin dyeing liquor: be made into three kinds of stostes earlier, stoste A:3g basic fuchsin is dissolved in 100ml 70% alcohol, stoste B: get stoste A 10ml and join in the 90ml 5% aqua carbolisata solution; Stoste C: get stoste B55ml, add 6ml glacial acetic acid and 6ml formalin, wherein stoste A and stoste C long preservation; Stoste B limit was used in two weeks, got stoste C 20ml again, added 45% glacial acetic acid 90ml; Add sorbierite 1.8g again, be made into the carbolfuchsin dyeing liquor of 20% concentration, place the back use of two weeks;
2.d.2 behind the tip of a root or stem apex of getting the hybridization test-tube plantlet on the superclean bench, drop into immediately and handle 2h in the saturated paracide solution under the normal temperature; Remove treatment fluid, inject fixer with the distilled water flushing number all over the back, normal temperature is handled 4h down; Remove fixer, clean with distilled water flushing, enzymolysis 30min under 37 ℃ of constant temperature; 1h dyes in the carbolfuchsin dyeing liquor behind the distilled water flushing;
2.e carry out the acclimatization and transplants that embryo is saved seedling: next year mid or late March selection 4-5 bar root in strong sprout, the long 3-5cm of root, 4-5 sheet leaf; The stem stalk is sturdy, and base portion does not have callus, after high light tempered for 1 week; Seedling is transplanted to the perlite of the bacterium of having gone out after with aseptic water washing: in the matrix of the peat composed of rotten mosses=1: 1 mixing, culturing room's refining seedling, the cover plastic cup is in order to preserve moisture on seedling; Water 1000 times carbendazim and 1/8MS nutrient solution weekly; Greenhouse temperature is controlled at 22 ℃, relative moisture 60%, intensity of illumination 1000lux.
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| CN103308361B (en) * | 2013-06-08 | 2015-09-30 | 吉林省拓华生物科技有限公司 | A kind of chromosome flaking method |
| GB201504569D0 (en) * | 2015-03-18 | 2015-05-06 | Oxford Cancer Biomarkers Ltd | Tissue sample analysis technique |
| CN104782474B (en) * | 2015-03-27 | 2017-05-31 | 浙江省农业科学院 | It is a kind of to improve the method that currant bybrid embryo saves seedling |
| CN104871974B (en) * | 2015-05-25 | 2017-06-16 | 中国农业科学院郑州果树研究所 | It is a kind of to induce currant rataria that the method and special culture media of somatic embryo occur |
| RU2631329C2 (en) * | 2015-10-15 | 2017-09-21 | Федеральное государственное бюджетное образовательное учреждение высшего профессионального образования "Горский государственный аграрный университет" | Method of rooting vines |
| CN105850710A (en) * | 2016-03-30 | 2016-08-17 | 山东省葡萄研究院 | Rapid seed breeding method for seedless grapes |
| CN107969237A (en) * | 2017-11-24 | 2018-05-01 | 犍为县九妹家庭农场 | A kind of high-quality grapes selection based on cross-pollination |
| CN109906868A (en) * | 2019-04-03 | 2019-06-21 | 新疆农业科学院园艺作物研究所 | A kind of method of new grape variety quickly breeding |
| CN116530414A (en) * | 2023-05-22 | 2023-08-04 | 西北农林科技大学 | Method for creating polyploid seedless germplasm by utilizing clustered bud cluster stem tips |
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