CN101283669B - Breeding method for obtaining triploid grape by embryo rescue and ploidy early identification - Google Patents

Abstract

The invention discloses a breeding technology for obtaining triploid grapes by embryo rescue and ploidy early identification, which aims to adopt the embryo rescue technology to cultivate the triploid grapes, overcome the defect of early abortion of hybrid embryos of diploid and tetraploid conventional breeding, obtain hybrid seedlings, and perform the ploidy early identification on obtained hybrid offspring by combining flow cytometry and chromosome counting by a wall-removing hypotonic method, thereby improving the breeding efficiency of the triploid grapes, accelerating the breeding process of triploid grape varieties and opening a wide prospect for cultivating large-grain seedless grape varieties.

Description

A Breeding Method for Obtaining Triploid Grapes Using Embryo Rescue and Early Identification of Ploidy

technical field

The invention relates to a plant breeding method, in particular to a high-efficiency breeding method for obtaining triploid grape hybrid seedlings by using embryo rescue culture, and carrying out systematic and comprehensive early ploidy identification on the obtained hybrid progeny, and belongs to the field of biotechnology.

Background technique

Polyploidy breeding is an important way to cultivate new varieties of fruit trees. Its advantages lie in the characteristics of vigorous growth, thick branches, thick leaves, large fruits, high yield and strong adaptability. Budding, artificial chemical mutagenesis, artificial sexual hybridization, endosperm culture and anther culture, etc. Among them, the hybridization between diploid varieties and tetraploids is the most widely used, and results have been obtained in watermelons, bananas, and oranges, and triploid seedless fruits have been obtained. Grape is the second largest fruit in the world, and its seedless varieties have always been an important direction of current international consumption, and the acquisition of new varieties of large seedless grapes has long attracted the attention of breeders. In addition to the many advantages of polyploid plants, triploid grapes have seedless or few seeds. Therefore, triploid grape breeding has opened up a new way to cultivate large seedless grape varieties. According to research reports, Japan has bred triploid seedless grape varieties Wei Ling, Della Wang, Mi Wu Seed, Kai Meiling, and Xia Hei, and my country has bred triploid seedless grape varieties, and began to promote them in production.

Triploid grape varieties are produced through hybridization between diploid and tetraploid varieties, natural hybridization, budding of triploid varieties, endosperm culture and anther culture, but in fact, the most effective way is still to use Diploids and tetraploids cross directly. However, the biggest obstacle to diploid and tetraploid hybridization is poor affinity, low fruit setting rate, early abortion of hybrid embryos or disintegration of endosperm, low viability of obtained triploid hybrid seeds, and difficulty in obtaining hybrid offspring. Embryo rescue technology can prevent the early abortion of triploid hybrid immature embryos and form triploid plants.

Since the last century, through ovule culture, Japanese scholar Hiroyuki Yamashita has obtained triploid plants by crossing tetraploids and diploids of seeded grapes ("Journal of Horticultural Society" 1993, 62 (2): 249-255 ), then Li Shicheng ("Journal of Shanghai Agricultural Science" 1998, 14(4): 13-17), Pan Chunyun ("Journal of Shandong Agricultural University" 1998, 29(3): 299-302), Xu Haiying ("Journal of Fruit Tree" 2001 , 18(6): 317-320), Guo Yinshan ("Journal of Shenyang Agricultural University" 2005, 36(5): 606-608) and others also used embryo rescue technology to overcome the conventional breeding of grape diploid and tetraploid. Due to the obstacles of early abortion of hybrid embryos and low breeding efficiency, triploid hybrid seedlings have been successfully bred, but there is little systematic comprehensive ploidy detection of the resulting hybrid seedlings, and a complete and efficient breeding system is lacking.

Usually polyploid identification methods include plant trait identification, cytological identification, molecular biological identification and other methods, while grape polyploidy is mostly identified by the number of chromosomes under a microscope, and it is also determined by flow cytometry. The chromosome counting method is intuitive and effective , but the material is limited to the shoot tip and root tip, and the operation is complicated. Flow cytometry is used to identify the DNA content of the plant to be tested. It can detect tens of thousands of cells in a short period of time, and can ensure the population characteristics of biological cells. The measurement is fast and the result is reliable, but it can only provide semi-quantitative measurement. result.

Contents of the invention

The purpose of the present invention is aimed at the current situation of triploid grape breeding and the advantages and disadvantages of various polyploid identification methods, using embryo rescue culture to obtain triploid grape hybrid seedlings, combined with flow cytometry, wall removal and hypotonic method chromosome counting , carry out systematic and comprehensive early identification of ploidy on the obtained hybrid progeny, and improve the breeding efficiency of triploid grapes.

Implementation steps of the present invention are as follows:

a. Grape Breeding Field Hybridization

Take the pollen, pick the normally developed inflorescence one day before the flowering of the male parent of the tetraploid variety or at the beginning of flowering, remove the top third part, pick off the corolla and anthers, sieve them with gauze, and dry them under the fluorescent lamp Dried until the anthers split, sieved again, poured into a mortar, ground to powder, put into a clean vial, sealed, placed in a desiccator filled with anhydrous CaCl ₂ , stored at a low temperature of 4°C; detasselled for pollination , select a number of inflorescences that develop identically on the female parent tree of European seedless grape varieties, artificially emasculate the inflorescences 3-4 days before flowering, and immediately spray the inflorescences with clean water, bag them and mark them, when the stigmas begin to secrete drop-shaped mucus At the same time, use absorbent cotton to pick up pollen for artificial pollination, and pollinate continuously for 3 times, once a day;

b. Embryo rescue culture and seedling formation

After 40-55 days of cross-castration and pollination, the ovules in the hybrid ear are stripped for embryo rescue culture, the ear is washed and placed on an ultra-clean workbench, soaked in 75% ethanol for 1 min, rinsed with sterile water for 3-5 times, and then used Sterilize with 0.1% mercuric chloride for 6 minutes, rinse with sterile water for 3-5 times, cut the fruit grains, take out the ovules and inoculate them into a 100ml Erlenmeyer flask with 40ml medium inside, inoculate 10-20 grains per bottle, and use B5 as the basic culture medium for ovules Base, add 0.1-1.0mg/LBA, 1.5-2.5mg/LIAA, 0.25-0.75mg/LGA ₃ , 6% sucrose, 0.6% agar, 0.1% activated carbon; Embryos are inoculated in embryo germination medium, with WP as the basic medium, supplemented with 0.1-0.5mg/LIBA, 2% sucrose, 0.6% agar, 0.1% activated carbon; continue to culture for 10-15 days, transfer to seedling medium for For subculture, use 1/2MS as the basic medium, add 0.1-0.3mg/LIBA, 2% sucrose, 0.6% agar, 0.1% activated carbon, and carry out subsequent ploidy identification after the leaves grow in the seedling medium. The culture conditions are temperature 25°C±2°C, light 16h per day, and light intensity 2000lux;

c. Semi-quantitative analysis of grape hybrid offspring by flow cytometry

The preliminary identification of the ploidy of the hybrid offspring obtained by embryo rescue culture uses flow cytometry to measure its DNA content, take 1-2 pieces of young leaves or a small amount of callus tissue into a petri dish, add 0.5-1.0ml of DNA Extract liquid HR-A, cut it with a blade and let it stand for 2-5 minutes, then filter the sample through a 30 μm Partec Celltrics ^TM microporous membrane into the test tube, then add 2ml of staining liquid HR-B, and then load the sample In the Partec flow cytometer, the DNA content distribution map is automatically generated by the flow cytometer. First, the fluorescence value FL on the horizontal axis of the analysis peak produced by the parent breed is set, the parameters are fixed, and then the hybridization is performed sequentially. The fluorescence value of the seedlings was measured;

d. Identify the assay results obtained by counting the chromosomes by the wall-removing hypotonic method

After taking the root tip or shoot tip of the hybrid test tube seedling on the ultra-clean workbench, immediately put it into a saturated p-dichlorobenzene solution and treat it at room temperature for 2h-3h; remove the treatment solution, rinse it with distilled water several times, and inject the newly prepared methanol : glacial acetic acid=3:1 fixative solution, treated at normal temperature for 3h-5h; remove the fixative solution, rinse with distilled water, add 3.5% cellulase and pectinase 1:1 mixed enzyme solution, mixed enzyme solution and The ratio of materials is 30:1, enzymatic hydrolysis at 37°C for 20-30min; rinse with distilled water and stain in carbo fuchsin staining solution for 30min-3h; take a small piece of material and put it on a glass slide soaked in 70% ethanol On the slide, drop a drop of carbo fuchsin staining solution, mash it with tweezers and cover the cover glass, then press the corner and beat the cover glass evenly with a wooden stick until the material is dispersed into a mist, and finally wipe it with absorbent paper Suck off the excess staining solution, dry it on an alcohol lamp, and take pictures with an OLMPUS-BX51 microscopic digital photography system;

e. Embryo-rescued seedling hardening and transplanting

In spring, select strong seedlings with 4-5 roots, root length 3-5cm, 4-5 leaves, strong stems, no callus at the base, and exercise under strong light for 1 week; rinse the seedlings with sterile water and transplant them to sterilized plants. The perlite of good bacteria: peat = 1:1 mixed matrix, harden the seedlings in the culture room, cover the seedlings with plastic cups, water 1000 times of carbendazim and 1/8MS nutrient solution every 5-10 days, and timely Remove moldy leaves and stems, control the temperature in the greenhouse at 18-25°C, relative humidity 60-70%, and light intensity 500-1000lux; the seedlings to be transplanted will grow new roots and leaves, and the ground temperature will rise in spring When it reaches 18-23 ℃, move to the field. Immediately after transplanting, set up a shading net and pour enough root water, gradually uncover the shading net, and timely water and prevent diseases and insect pests.

Adopt the method of the present invention, carry out in vitro culture to hybrid combination Ruby Seedless × Fujiminori, Aimo Seedless × Hei Aolin, the result embryo germination rate reaches 23.1%, 22.2% respectively, and the seedling rate reaches 87.5%, 87.5%, respectively. 75%, therefore, through the present invention, more hybrid offspring are obtained, the breeding efficiency is improved, and the breeding process of triploid grape varieties is accelerated.

Detailed ways

The operating method of the present invention will be further described below in conjunction with specific embodiments

a. The steps of grape breeding field hybridization are as follows:

a.1 In the early stage of flowering, pick the inflorescences of Fujiminori and Black Olin, remove the top third part, pick off the corolla and anthers, sieve them with gauze, and dry them under the fluorescent lamp until the anthers split , pour it into a mortar after sieving again, grind it into powder, put it into a clean vial, seal it, put it in a desiccator filled with anhydrous CaCl ₂ , and store it at a low temperature of 4°C;

a.2 3 days before flowering, select 10 spikes of inflorescences that develop identically on the trees of the female parent varieties Ruby Seedless and Aimo Seedless, and carry out artificial emasculation, then immediately spray the inflorescences with clear water, bag them and put them on a tag. When the stigma begins to secrete drop-shaped mucus, use absorbent cotton to pick up pollen for artificial pollination, and pollinate continuously for 3 times, once a day;

b. The steps of embryo rescue culture and seedling formation are as follows:

b.1 On 48d and 47d after pollination, the fruit ears of the hybrid combination Ruby Seedless×Fujiminori and Aimo Seedless×Hei Aolin were taken for in vitro culture, and the fruit ears were washed and placed on the ultra-clean workbench with 75% ethanol Soak for 1 min, rinse with sterile water for 3 times, then sterilize with 0.1% mercury liter for 6 min, rinse with sterile water for 3 times, cut the fruit grains, take out the ovules and inoculate them in a 100ml Erlenmeyer flask with 40ml of medium inside, and inoculate 10 grains in each bottle. The ovule medium adopts B5 as the basic medium, supplemented with 0.5mg/LBA, 2.0mg/LIAA, 0.5mg/LGA ₃ , 6% sucrose, 0.6% agar, and 0.1% activated carbon; Take naked embryos and inoculate them in embryo germination medium, with WP as the basic medium, add 0.1mg/LIBA, 2% sucrose, 0.6% agar, 0.1% activated carbon; continue to culture for two weeks, transfer to seedling medium for subculture Culture, using 1/2MS as the basic medium, add 0.1mg/LIBA, 2% sucrose, 0.6% agar, 0.1% activated carbon, and carry out subsequent ploidy identification after the leaves grow in the seedling medium. The culture conditions are: The temperature is 25℃±2℃, the light is 16h per day, and the light intensity is 2000lux;

c. Semi-quantitative analysis of grape hybrid offspring by flow cytometry:

c.1 The DNA content of the hybrid offspring obtained by embryo rescue culture was measured by flow cytometry, and its ploidy was initially identified. Take 1 seedling obtained from the hybrid combination Ruby Seedless × Fujiminori and Aimo Seedless × Hei Aolin -Put 2 pieces of young leaves or a small amount of callus into a petri dish, add 1.0ml of DNA extraction solution HR-A, chop it up with a blade and let it stand for 3 minutes, then pass the sample through a 30μm Partec Celltrics ^TM microporous membrane Filter it into a test tube, add 2ml of staining solution HR-B, and then load the sample on the Partec flow cytometer. The DNA content distribution map is automatically generated by the flow cytometer. The fluorescence value FL of the analysis peak on the horizontal axis is set, the parameters are fixed, and then the fluorescence values of the hybrid seedlings are measured in sequence;

d. Implement the chromosome counting method of removing the wall and hypotonicity method to the gained hybrid seedling:

d.1 Preparation of reagents used, 3.5% mixed enzyme solution: weigh 0.7g of cellulase and pectinase, add 20ml of distilled water, and store in a refrigerator at 4°C; pretreatment solution: weigh 5g of p-dichlorobenzene saturated crystals and put Put it into a brown reagent bottle, add 100ml of distilled water heated to 45°C, shake for 5 minutes, let it cool down and store it at room temperature; fixative: volume ratio of glacial acetic acid to methanol = 1:3; carbo magenta staining solution: first Prepare three stock solutions, stock solution A: dissolve 3g of basic fuchsin in 100ml of 70% alcohol, stock solution B: take 10ml of stock solution A and add it to 90ml of 5% carbolic acid aqueous solution, stock solution C: take 55ml of stock solution B, add 6ml of glacial acetic acid and 6ml of formosan Formalin, stock solution A and stock solution C are stored for a long time, and stock solution B is limited to use within two weeks. Then take 20ml of stock solution C, add 90ml of 45% glacial acetic acid, and add 1.8g of sorbitol to make carbo fuchsin at a concentration of 20%. Dyeing solution, use after two weeks;

d.2 After taking the root tip or shoot tip of the hybrid test tube seedling on the ultra-clean workbench, immediately put it into a saturated p-dichlorobenzene solution and treat it at room temperature for 2 hours;

d.3 Remove the treatment solution, rinse with distilled water several times, inject the newly prepared fixative solution of methanol: glacial acetic acid = 3:1, and treat for 4 hours at room temperature;

d.4 Remove the fixative, rinse with distilled water, add 3.5% mixed enzyme solution of cellulase and pectinase, the ratio of mixed enzyme solution and material is 30:1, and enzymolyze at a constant temperature of 37°C for 30 minutes;

d.5 Rinse with distilled water and stain in carbo fuchsin staining solution for 1 hour;

d.6 Take a small piece of material and place it on a glass slide soaked in 70% ethanol, drop a drop of carbo fuchsin staining solution, mash it with tweezers and cover it with a cover glass, then press a corner with a wooden small Beat the cover glass evenly with a rod until the material is dispersed into a mist, and finally absorb the excess staining solution with absorbent paper, dry it on an alcohol lamp, and take pictures with an OLMPUS-BX51 digital microphotography system;

e. Carry out hardening and transplanting of embryo rescued seedlings

e. In the middle and late March of the following year, select strong seedlings with 4-5 roots, root length 3-5cm, 4-5 leaves, thick stems, no callus at the base, and after 1 week of strong light exercise, the seedlings are used After rinsing with sterile water, transplant them into the sterilized perlite: peat = 1:1 mixed matrix, harden the seedlings in the cultivation room, and cover the seedlings with plastic cups for moisturizing.

e.2 Water 1000 times of carbendazim and 1/8MS nutrient solution every week, and remove moldy leaves and stems in time. The temperature of the greenhouse is controlled at 22°C, the relative humidity is 60%, and the light intensity is 1000lux.

e.3 The seedlings to be transplanted will grow new roots and new leaves, and they will be moved to the field when the ground temperature rises to 2°C in spring. Shade nets, and timely watering and pest control.

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.