CN111657146A - Induction culture medium and induction culture method for culture breeding of green Chinese onion flowers - Google Patents
Induction culture medium and induction culture method for culture breeding of green Chinese onion flowers Download PDFInfo
- Publication number
- CN111657146A CN111657146A CN202010614533.4A CN202010614533A CN111657146A CN 111657146 A CN111657146 A CN 111657146A CN 202010614533 A CN202010614533 A CN 202010614533A CN 111657146 A CN111657146 A CN 111657146A
- Authority
- CN
- China
- Prior art keywords
- anther
- culture
- induction
- onion
- green chinese
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention provides an induction culture medium for culture breeding of green Chinese onion flowers, which comprises KNO (potassium dihydrogen phosphate)3、CaCl2·2H2O、（NH4）2SO4、NaH2PO4·H2O、MgSO4·7H2O、MnSO4·4H2O、ZnSO4·7H2O、H3BO3、KI、Na2MoO4·2H2O、CuSO4·5H2O、CoCl2·6H2O, Fe-EDDHA, corn stigma extract, hydrolyzed casein, inositol, sodium pyruvate, fumaric acid, aspartic acid, glycine, sodium humate, riboflavin, vitamin B1, vitamin B6, D-biotin, choline, salicylic acid, sodium butyrate, paclobutrazol, KT, 6-BA, p-hydroxybenzoic acid, sucrose, maltose and plant gel. The invention also provides a green Chinese onion anther induction culture method, which comprises the step of inoculating the green Chinese onion anther after uniform magnetic field treatment to the induction culture medium for culture, wherein the culture medium can be prepared byThe callus induction efficiency of the onion anther can be obviously improved, and the establishment of a onion anther culture breeding system is promoted.
Description
Technical Field
The invention belongs to the high and new technical field of vegetable cultivation, and particularly relates to an induction culture medium for onion anther induction culture breeding and a onion anther induction culture method.
Background
Scallion (academic name:Allium.fistulosum L.var.gigantum Makino) The green onion is the next variety of the Allium (Allium) in the Allium of the Liliaceae, the leaves are cylindrical and hollow, the leaf sheath and stem parts comprise pseudostems, and the plants grow in two or three years and are vegetables widely cultivated in China. The literature describes that the origin of onion is siberia, northwest and northeast china and central asia. The Chinese green Chinese onion has a long history, and a large number of local varieties are formed due to strong adaptability and wide cultivation region. The scallion is a seasoning which is often eaten by people all the year round, is a vegetable with rich nutrition, contains phytoncin, and can improve appetiteHas effects in promoting digestion, killing bacteria, and relieving inflammation. The research proves that the scallion has the functions of reducing blood fat, blood sugar and blood pressure and nourishing brain, and the sulfide contained in the scallion can reduce the deposition of cholesterol in blood vessels of a human body and prevent thrombus, so the edible value of the scallion is more and more valued by people in the world. The traditional Chinese medicine considers that the scallion is warm in nature and pungent in taste, and has the effects of dispelling cold, invigorating stomach, eliminating phlegm, sterilizing, benefiting lung, activating yang, inducing sweat, relieving exterior syndrome, promoting lactation, stopping bleeding, relieving pain and treating injury.
The final purpose of breeding is to breed a new variety with high quality, disease resistance and high yield. Quality is an indispensable important goal of any crop breeding program. For vegetable crops, quality breeding is more and more important, and improving vegetable quality has become an important research topic for vegetable researchers. The green Chinese onions are typical cross-pollinated crops, and the time for breeding a good inbred line is at least 6-10 years. From the 70 s of the 20 th century, the modern biotechnology mainly based on cell engineering and genetic engineering is applied to the improvement of crop varieties, and a new way for improving the varieties of vegetable crops is developed. The organic combination of biotechnology and conventional breeding technology has the advantages of multiple creative variations, strong breeding purpose, short breeding time, stable progeny selection and the like, and provides a new technical means for breeding high-yield, high-resistance, multi-resistance and high-quality varieties.
Anther culture breeding, namely anther in vitro culture breeding, one of the modern breeding means is a breeding method for obtaining homozygous diploid by cultivating pollen into haploid plants and then carrying out natural or artificial doubling on chromosomes. The homozygous diploid generated by chromosome doubling is very stable in heredity and does not have character separation, so that the flower breeding seeds can separate offspring stably at an extremely early stage and shorten the breeding period. Meanwhile, the homozygous diploid plant after the anther culture and doubling can eliminate the interference caused by covering recessive factors by dominant factors in heterozygotes, and improve the accuracy and reliability of selection. Because the pollen plant is formed by directly doubling haploids, recessive character is homozygously expressed, and the pollen plant is derived from F1Or F2The current generation strains all show rich diversity, such as plant height, growth period, fertility, resistance and the like. These traitsThe cross of the plants forms a flower culture line with various morphological characteristics, so that the flower culture can fully utilize the germplasm resources of the plants and obtain diversity of traits. Thus greatly shortening the breeding period and enriching the germplasm resources. The obvious advantages of anther culture breeding and anther culture breeding arouse great interest of breeding workers in various countries in the world. China began to breed flowers in the 70 th of the 20 th century, and has achieved huge achievements so far. The flower breeding seeds are combined with conventional crossbreeding, distant crossbreeding, mutation breeding and transgenic technology to develop a set of breeding technology system.
The anther isolated culture is to inoculate anther which develops to a certain stage on an artificial culture medium to change the development way of pollen grains in the anther to form pollen embryo or pollen callus, and then directly develop the embryo into a plant or differentiate the callus into the plant. There are many factors that affect the anther culture efficiency, including the genotype (variety or type) of the test plant, pretreatment, the development period of the pollen microspore, the culture medium, the culture method, the culture conditions, and the like. Scholars at home and abroad research factors influencing the cultivation of vegetable anthers, remarkable progress is made, the anther induction rate of part of vegetable materials is improved by optimizing a culture medium and improving a cultivation method, and a seed cultivation system of part of vegetable seed flowers is established. However, the research on the culture of the green Chinese onion anther is slow, and the research on the breeding of the green Chinese onion by using the technology is rarely reported, mainly because the induction rate of the anther callus is low and unstable, and the application of the culture technology of the green Chinese onion anther in breeding is directly limited.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to provide an induction culture medium for onion anther culture breeding and an induction culture method, which can obviously improve the callus induction efficiency of the onion anther and improve the culture efficiency of the onion anther.
In order to achieve the purpose, the invention adopts the following technical scheme:
an induction culture medium for culture breeding of green Chinese onion flowers is characterized in that the formula of the culture medium is as follows (calculated by 1L):
KNO31500-1700mg，CaCl2·2H2O 310-340mg，（NH4）2SO4127-153mg，NaH2PO4·H2O100-130mg，MgSO4·7H2O 160-190mg，MnSO4·4H2O 4.0-7.0mg，ZnSO4·7H2O 1.5-2.5mg，H3BO35.0-7.0mg，KI 1.0-2.0mg，Na2MoO4·2H2O 0.2-0.3mg，CuSO4·5H2O 0.02-0.03mg，CoCl2·6H20.02-0.03mg of O, 26.4-30.2mg of Fe-EDDHA, 30-50ml of corn stigma extract, 150mg of hydrolyzed casein 120, 90-120mg of inositol, 15.5-21.3mg of sodium pyruvate, 20-25mg of fumaric acid, 2.0-4.0mg of aspartic acid, 1.0-3.0mg of glycine, 8-12mg of sodium humate, 0.2-0.4mg of riboflavin, 10.3-0.5 mg of vitamin B, 60.5-0.7mg of vitamin B, 0.8-1.0mg of D-biotin, 2.6-3.0mg of choline, 0.08-0.12mg of salicylic acid, 8-12mmol of sodium butyrate, 0.4-0.6mg of paclobutrazol, 0.4-0.6mg of KT, 0.4-0.6mg of 6-BA, 0.9-1.1mg of beta-cyclodextrin, 0.8-1.2g of p-hydroxybenzoic acid, 25-35g of sucrose, 25-35g of maltose and 6-8g of plant gel.
The pH of the medium was 5.8.
The corn stigma extracting solution is prepared by the following method: soaking corn stigma in NaOH solution for 15min, washing to neutral, adding clear water at a ratio of 1:10, heating to boil, keeping the temperature for 30min, standing for 24h, treating with ultrasonic wave of 300W power and 40kHz frequency for 10min, filtering with double-layer gauze, and collecting filtrate.
The method comprises the following steps:
1) material taking and pretreatment: collecting buds of green Chinese onions in the morning on a sunny day, carrying the buds to a laboratory by using an ice box, carrying out microscopic examination to select the buds of which the pollen development period is a mononuclear border period, wetting the buds by using water, putting the buds into a culture dish filled with test paper, and carrying out low-temperature pretreatment at 4 ℃ for 2-3 days;
2) and (3) sterilization: cleaning buds, putting the cleaned buds into a triangular flask, adding 0.1% mercuric chloride, adding 2 drops of Tween, putting the triangular flask on a shaking table, shaking for 10-12 min, wiping the triangular flask with 75% ethanol by volume fraction, disinfecting, putting the triangular flask into a super-clean workbench, pouring out disinfectant, and washing with sterile water for 3-4 times until no foam exists in the flask;
3) inoculation and culture: removing anther from sterilized bud with tweezers, cutting filament, inoculating to induction culture medium, treating in uniform magnetic field for 30min, and dark culturing in 25 deg.C constant temperature incubator until callus is formed.
The buds of the green Chinese onions collected in the step 1) are unopened or just opened.
The microscopic examination method in the step 1) comprises the following steps: the anther is stripped from the flower bud and placed on a glass slide, a small drop of kappaucin staining solution is added, the anther is mashed by forceps, the microspore is squeezed out, the residue is removed, and then the glass slide is covered and placed under a microscope to observe the development period of the microspore.
The magnetic field intensity in the step 3) is 400 mT.
Compared with the prior art, the invention has the positive effects that:
1) the induction culture medium provided by the invention is developed aiming at the growth characteristics of the pollen of the green Chinese onion, and is firstly researched and adjusted according to the content proportions of major elements, trace elements, organic matters and the organic matters in the culture medium; secondly, additional components such as corn stigma extracting solution, hydrolyzed casein, sodium pyruvate, fumaric acid, sodium humate, salicylic acid, sodium butyrate, p-hydroxybenzoic acid and the like are discovered and tested, and the callus induction rate can be improved under the appropriate concentration; thirdly, the hormone combination of paclobutrazol, KT and 6-BA is determined to have better induction effect than the hormone combination of 2,4-D and 6-BA studied by the predecessor.
2) The invention treats the onion anther in a uniform magnetic field of 400mT and then carries out induction culture, which can obviously improve the callus induction rate.
3) The method can effectively improve the callus induction rate of the onion anther culture, and can provide a theoretical basis for the establishment of the onion anther culture system and the research of anther culture breeding.
Detailed Description
In order to more fully explain the practice of the present invention, the following preparation method working examples are provided. These examples are merely illustrative and do not limit the scope of the invention.
Example 1
The test materials are Yishui scallion and nutmeg harlequin glorybower, and the seeds are purchased from the market and planted in the field.
The onion anther induction culture is carried out according to the following method:
1) material taking and pretreatment: collecting unopened or just opened buds of the green Chinese onions in the morning on a sunny day, carrying the buds to a laboratory by using an ice box, screening out the buds with the pollen development period being the mononuclear border period by using a microscopic examination, wetting the buds by using water, putting the buds into a culture dish filled with test paper, and pretreating the buds at a low temperature of 4 ℃ for 2-3 days. The specific method of microscopic examination is as follows: the anther is stripped from the flower bud and placed on a glass slide, a small drop of kappaucin staining solution is added, the anther is mashed by forceps, the microspore is squeezed out, the residue is removed, and then the glass slide is covered and placed under a microscope to observe the development period of the microspore.
2) And (3) sterilization: putting the flower buds into a triangular flask, adding 0.1% mercuric chloride, adding 2 drops of Tween, placing the triangular flask on a shaking table, shaking for 10-12 min, wiping the triangular flask with 75% ethanol by volume fraction, disinfecting, placing the triangular flask into a super-clean workbench, pouring out disinfectant, and washing with sterile water for 3-4 times until no foam exists in the flask.
3) Inoculation and culture: and (3) stripping anthers from the sterilized buds by using tweezers, cutting filaments, inoculating the anthers onto an induction culture medium, placing the induction culture medium into a constant-temperature incubator at 25 ℃ for dark culture for 30-40 days, and observing and counting the callus induction rate, wherein the callus induction rate = the number of generated callus/the number of inoculated anthers multiplied by 100%.
The formulation of the induction medium was as follows (1L): KNO31600mg，CaCl2·2H2O 325mg，（NH4）2SO4140mg，NaH2PO4·H2O 115mg，MgSO4·7H2O 175mg，MnSO4·4H2O 5.5mg，ZnSO4·7H2O 2.0mg，H3BO36.0mg，KI 1.5mg，Na2MoO4·2H2O 0.25mg，CuSO4·5H2O 0.025mg，CoCl2·6H2O0.025mg，Fe-EDDHA 28.3mg, 40ml of corn stigma extract, 135mg of hydrolyzed casein, 105mg of inositol, 18.4mg of sodium pyruvate, 22.5mg of fumaric acid, 3.0mg of aspartic acid, 2.0mg of glycine, 10mg of sodium humate, 0.3mg of riboflavin, 10.4 mg of vitamin B, 60.6 mg of vitamin B, 0.9mg of D-biotin, 2.8mg of choline, 0.1mg of salicylic acid, 0.5mg of paclobutrazol, 0.5mg of KT, 1.0mg of 6-BA, 30g of sucrose, 30g of maltose, 7g of plant gel and 5.8 of pH value.
The corn stigma extracting solution is prepared by the following method: soaking corn stigma in NaOH solution for 15min, washing to neutral, adding clear water at a ratio of 1:10, heating to boil, keeping the temperature for 30min, standing for 24h, treating with ultrasonic wave of 300W power and 40kHz frequency for 10min, filtering with double-layer gauze, and collecting filtrate.
Factor analysis for influencing callus induction effect of green Chinese onion anther
1. Influence of magnetic field treatment on callus induction rate of green Chinese onion anther
Inoculating green Chinese onion anther onto an induction culture medium, respectively placing the green Chinese onion anther in uniform magnetic fields with the strength of 200mT, 400mT, 600mT and 800mT for treating for 30min, taking untreated anther as a control, placing the green Chinese onion anther in a constant-temperature incubator at 25 ℃ for dark culture, and counting the callus induction rate, wherein the results are shown in the following table 1.
As can be seen from Table 1, the induction rate of the callus produced by the magnetic field treatment is higher than that of the untreated control, and when the magnetic field strength is 400mT, the induction rates of the callus of the two test materials are highest, namely, Yishui shallot 27.15% and Qiumu phoenix tree 31.30%; along with the increase of the magnetic field intensity, the callus induction rate is reduced, so that the uniform magnetic field with the magnetic field intensity of 400mT can obtain better effect when being used for treating the green Chinese onion anther. Proper magnetic field treatment can improve the callus induction rate of the green Chinese onion, and the analysis reason is probably that the magnetic field treatment can influence various enzyme activities in plants, so that the physiological state of the pollen is improved, and the initiation of the androgenesis is facilitated.
2. Influence of sodium butyrate on callus induction rate of green Chinese onion anther
Adding sodium butyrate into an induction culture medium to enable the concentration of the sodium butyrate to be 1mmol/L, 5mmol/L, 10mmol/L and 20mmol/L, taking the induction culture medium without the sodium butyrate as a control, inoculating the allium fistulosum anther onto the induction culture medium, placing the allium fistulosum anther into a constant-temperature incubator at 25 ℃ for dark culture, and counting the induction rate of the callus, wherein the result is shown in the following table 2.
As is clear from Table 2, the induction rate of sodium butyrate to anther-derived calli of the test material was basically increased, and then increased to the maximum value and then decreased. Compared with a control, the influence of the concentration of the sodium butyrate of 1mmol/L on the callus induction rate is small; when the concentration of the sodium butyrate is 10mmol/L, the callus inductivity of the two test materials reaches the highest, namely 28.84% of Yishui green Chinese onion and 33.61% of Qiu Dasytong tree; the callus induction rate of the two test materials was lower at a sodium butyrate concentration of 20mmol/L than at 10mmol/L, and therefore 10mmol/L was an appropriate concentration of sodium butyrate.
3. Influence of p-hydroxybenzoic acid on callus induction rate of green Chinese onion anther
P-hydroxybenzoic acid was added to the induction medium to make the concentration 0.5g/L, 1.5g/L, 2.0g/L, 3.0g/L, the induction medium without p-hydroxybenzoic acid was used as a control, the Allium fistulosum anther was inoculated to the above induction medium, and placed in a constant temperature incubator at 25 ℃ for dark culture, and the callus induction rate was counted, and the results are shown in Table 3 below.
As can be seen from Table 3, the rate of induction of the anther-derived callus by p-hydroxybenzoic acid was basically increased to the maximum level and then decreased. Compared with a control, when the concentration of the p-hydroxybenzoic acid is 1.0g/L, the callus induction rates of the two test materials reach the highest, namely 26.98% of Yishui scallion and 29.47% of Zhangqida phoenix tree; when the concentration of p-hydroxybenzoic acid was 3.0g/L, the callus induction rate of both test materials was significantly reduced, wherein the callus induction rate of Allium fistulosum Yishui was already lower than that of the control, indicating that this concentration has a negative effect on callus induction, so 1.0g/L is an appropriate concentration of p-hydroxybenzoic acid.
Example 2 Effect of different Induction Medium on callus induction rate of Allium fistulosum anther
The induction culture medium of the invention: KNO31600mg/L，CaCl2·2H2O 325mg/L，（NH4）2SO4140mg/L，NaH2PO4·H2O 115mg/L，MgSO4·7H2O 175mg/L，MnSO4·4H2O 5.5mg/L，ZnSO4·7H2O 2.0mg/L，H3BO36.0mg/L，KI 1.5mg/L，Na2MoO4·2H2O 0.25mg/L，CuSO4·5H2O 0.025mg/L，CoCl2·6H2O0.025 mg/L, Fe-EDDHA 28.3mg/L, corn stigma extract 40ml/L, hydrolyzed casein 135mg/L, inositol 105mg/L, sodium pyruvate 18.4mg/L, fumaric acid 22.5mg/L, aspartic acid 3.0mg/L, glycine 2.0mg/L, sodium humate 10mg/L, riboflavin 0.3mg/L, vitamin B10.4mg/L, vitamin B60.6mg/L, D-biotin 0.9mg/L, choline 2.8mg/L, salicylic acid 0.1mg/L, sodium butyrate 10mmol/L, paclobutrazol 0.5mg/L, KT0.5 mg/L, 6-BA 1.0mg/L, 1.0g/L of p-hydroxybenzoic acid, 30g/L of sucrose, 30g/L of maltose, 7g/L of plant gel and 5.8 of pH value.
Control induction medium: b5 minimal medium +2, 4-D2.5 mg/L +6-BA 0.5mg/L + glutathione 800mg/L + sucrose 60g/L + agar 6g/L, pH 5.8 (Wangwangjie, Zhangguoqian, et al, first report on cultivation research of anther of green Chinese onion).
The Allium fistulosum anthers were stripped according to the method of example 1, inoculated onto the induction medium of the present invention and the control, respectively, treated in a uniform magnetic field with a magnetic field strength of 400mT for 30min, placed in a constant temperature incubator at 25 ℃ for dark culture, and the callus induction rate was counted, and the results are shown in Table 4 below.
As can be seen from Table 4, the callus induction rates of the same test materials were significantly different in different induction media. On the induction culture medium, the callus induction rate of the Yishui scallion is 34.29 percent, which is 3.23 times of that of a control culture medium, and the callus induction rate of the Zhang harlequin glorybower leaf is 37.53 percent, which is 2.46 times of that of the control culture medium, so that the induction culture medium can effectively improve the callus induction rate of scallion anther culture, and further improve the culture efficiency of the scallion anther. The induction culture medium provided by the invention is developed aiming at the growth characteristics of the pollen of the green Chinese onion, and is firstly researched and adjusted according to the content proportions of major elements, trace elements, organic matters and the organic matters in the culture medium; secondly, additional components such as corn stigma extracting solution, hydrolyzed casein, sodium pyruvate, fumaric acid, sodium humate, salicylic acid, sodium butyrate, p-hydroxybenzoic acid and the like are discovered and tested, and the callus induction rate can be improved under the appropriate concentration; thirdly, the hormone combination of paclobutrazol, KT and 6-BA is adopted, so that the induction effect is better.
The above embodiments are only for illustrating the technical idea of the present invention, and the protection scope of the present invention cannot be limited thereby, and any modification made on the basis of the technical scheme according to the technical idea proposed by the present invention falls within the protection scope of the present invention; the technology not related to the invention can be realized by the prior art.
Claims (7)
1. An induction culture medium for culture breeding of green Chinese onion flowers is characterized in that the formula of the culture medium is as follows (calculated by 1L):
KNO31500-1700mg，CaCl2·2H2O 310-340mg，（NH4）2SO4127-153mg，NaH2PO4·H2O100-130mg，MgSO4·7H2O 160-190mg，MnSO4·4H2O 4.0-7.0mg，ZnSO4·7H2O 1.5-2.5mg，H3BO35.0-7.0mg，KI 1.0-2.0mg，Na2MoO4·2H2O 0.2-0.3mg，CuSO4·5H2O 0.02-0.03mg，CoCl2·6H20.02-0.03mg of O, 26.4-30.2mg of Fe-EDDHA, 30-50ml of corn stigma extract, 150mg of hydrolyzed casein 120, 90-120mg of inositol, 15.5-21.3mg of sodium pyruvate, 20-25mg of fumaric acid, 2.0-4.0mg of aspartic acid, 1.0-3.0mg of glycine, 8-12mg of sodium humate, 0.2-0.4mg of riboflavin, 10.3-0.5 mg of vitamin B, 60.5-0.7mg of vitamin B, 0.8-1.0mg of D-biotin, 2.6-3.0mg of choline, 0.08-0.12mg of salicylic acid, 8-12mmol of sodium butyrate, 0.4-0.6mg of paclobutrazol, 0.4-0.6mg of KT, 0.4-0.6mg of 6-BA, 0.9-1.1mg of beta-cyclodextrin, 0.8-1.2g of p-hydroxybenzoic acid, 25-35g of sucrose, 25-35g of maltose and 6-8g of plant gel.
2. The induction medium for cultivating green Chinese onion flowers as claimed in claim 1, wherein the pH value of the medium is 5.8.
3. The induction medium for the culture of the onion flowers as claimed in claim 1, wherein the corn silk extract is prepared by the following method: soaking corn stigma in NaOH solution for 15min, washing to neutral, adding clear water at a ratio of 1:10, heating to boil, keeping the temperature for 30min, standing for 24h, treating with ultrasonic wave of 300W power and 40kHz frequency for 10min, filtering with double-layer gauze, and collecting filtrate.
4. The onion anther induction culture method is characterized by comprising the following steps:
1) material taking and pretreatment: collecting buds of green Chinese onions in the morning on a sunny day, carrying the buds to a laboratory by using an ice box, carrying out microscopic examination to select the buds of which the pollen development period is a mononuclear border period, wetting the buds by using water, putting the buds into a culture dish filled with test paper, and carrying out low-temperature pretreatment at 4 ℃ for 2-3 days;
2) and (3) sterilization: cleaning buds, putting the cleaned buds into a triangular flask, adding 0.1% mercuric chloride, adding 2 drops of Tween, putting the triangular flask on a shaking table, shaking for 10-12 min, wiping the triangular flask with 75% ethanol by volume fraction, disinfecting, putting the triangular flask into a super-clean workbench, pouring out disinfectant, and washing with sterile water for 3-4 times until no foam exists in the flask;
3) inoculation and culture: removing anther from sterilized bud with tweezers, cutting filament, inoculating to induction culture medium, treating in uniform magnetic field for 30min, and dark culturing in 25 deg.C constant temperature incubator until callus is formed.
5. The method for induction culture of green Chinese onion anther as claimed in claim 4, wherein the green Chinese onion buds collected in step 1) are unopened or just opened.
6. The onion anther induction culture method as claimed in claim 4, wherein the microscopic examination method in the step 1) is as follows: the anther is stripped from the flower bud and placed on a glass slide, a small drop of kappaucin staining solution is added, the anther is mashed by forceps, the microspore is squeezed out, the residue is removed, and then the glass slide is covered and placed under a microscope to observe the development period of the microspore.
7. The method for anther induction culture of green Chinese onion as claimed in claim 4, wherein said magnetic field strength in step 3) is 400 mT.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010614533.4A CN111657146A (en) | 2020-06-30 | 2020-06-30 | Induction culture medium and induction culture method for culture breeding of green Chinese onion flowers |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010614533.4A CN111657146A (en) | 2020-06-30 | 2020-06-30 | Induction culture medium and induction culture method for culture breeding of green Chinese onion flowers |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111657146A true CN111657146A (en) | 2020-09-15 |
Family
ID=72390516
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010614533.4A Pending CN111657146A (en) | 2020-06-30 | 2020-06-30 | Induction culture medium and induction culture method for culture breeding of green Chinese onion flowers |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111657146A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111657147A (en) * | 2020-06-30 | 2020-09-15 | 富阿丽 | Differentiation culture medium and differentiation culture method for culture breeding of green Chinese onion flowers |
| CN112042543A (en) * | 2020-10-22 | 2020-12-08 | 郭爱英 | Method for obtaining haploid plant through in vitro culture of pear unfertilized ovule |
| CN112568124A (en) * | 2020-12-16 | 2021-03-30 | 山西农业大学 | Substrate for tissue culture of mohair architectonica grass and preparation method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104488717A (en) * | 2014-12-22 | 2015-04-08 | 北京市农林科学院 | Method for obtaining multiple-ploidy regeneration plant by culturing flower bud of green Chinese onion |
| CN105794647A (en) * | 2016-04-10 | 2016-07-27 | 沈阳农业大学 | Method for culturing Brassica rapa microspores with sodium butyrate-NaB as histone deacetylase inhibitor |
-
2020
- 2020-06-30 CN CN202010614533.4A patent/CN111657146A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104488717A (en) * | 2014-12-22 | 2015-04-08 | 北京市农林科学院 | Method for obtaining multiple-ploidy regeneration plant by culturing flower bud of green Chinese onion |
| CN105794647A (en) * | 2016-04-10 | 2016-07-27 | 沈阳农业大学 | Method for culturing Brassica rapa microspores with sodium butyrate-NaB as histone deacetylase inhibitor |
Non-Patent Citations (7)
| Title |
|---|
| SAIJU,H.K.等: "Anther culture of Allium fistulosum L", 《PHYTOMORPHOLOGY》 * |
| 刘敏: "《花卉组织培养与工厂化生产》", 30 June 2002, 地质出版社 * |
| 张小玲等: "磁场预处理对籼稻花药培养影响研究初报", 《核农学报》 * |
| 张小玲等: "磁场预处理对花椰菜花药培养的影响", 《上海农业学报》 * |
| 梁海曼等: "丁酸钠预培养对水稻花药绿苗形成的影响", 《杭州大学学报》 * |
| 王文洁等: "大葱花药培养研究初报", 《北方园艺》 * |
| 陈世昌等: "《植物组织培养（第3版）》", 30 June 2016, 重庆大学出版社 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111657147A (en) * | 2020-06-30 | 2020-09-15 | 富阿丽 | Differentiation culture medium and differentiation culture method for culture breeding of green Chinese onion flowers |
| CN112042543A (en) * | 2020-10-22 | 2020-12-08 | 郭爱英 | Method for obtaining haploid plant through in vitro culture of pear unfertilized ovule |
| CN112568124A (en) * | 2020-12-16 | 2021-03-30 | 山西农业大学 | Substrate for tissue culture of mohair architectonica grass and preparation method and application thereof |
| CN112568124B (en) * | 2020-12-16 | 2022-09-13 | 山西农业大学 | Substrate for tissue culture of mohair architectonica grass and preparation method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111657146A (en) | Induction culture medium and induction culture method for culture breeding of green Chinese onion flowers | |
| Moradi et al. | Micropropagation of strawberry by multiple shoots regeneration tissue cultures | |
| CN110547200B (en) | Marigold pollen differentiation medium and differentiation culture method | |
| CN105941152A (en) | Asparagus all-male breeding method | |
| CN115281081A (en) | Breeding method of miniature test tube detoxified seed ginger | |
| CN102613074A (en) | Breeding method of tetraploid radish | |
| CN101548646B (en) | Method for rapidly propagating aralia elata through somatic embryo and secondary somatic embryogenesis | |
| CN109819892B (en) | Tissue culture method of good single plant of tsaoko | |
| CN109258469B (en) | Method for inducing regeneration plants of Chinese capsicum leaves with stalks | |
| CN107410024A (en) | Avocado callus induction method and method for promoting bud differentiation of avocado callus | |
| CN115606503B (en) | Tissue culture method of aster | |
| CN112119913A (en) | Virus-free propagation method for sweet potatoes | |
| CN107439211B (en) | Method for shortening eggplant germination time | |
| CN103109745A (en) | Method for removing tobacco mosaic virus and rapidly cultivating non-toxic seedling in test tube | |
| CN105010123B (en) | The method and culture medium of strawberry distant hybrid are obtained by rescue isolated culture | |
| CN110199884A (en) | A kind of breeding method of setting seeds under quinoa tissue-cultured seedling aseptic condition | |
| CN115968786A (en) | Culture medium and culture method for tea tree tissue culture | |
| KR102624679B1 (en) | Method for mass production of Cnidium officinale clones through slice culture | |
| Prabhuling et al. | In Vitro Regeneration in Pomegranate (Punica granatum L.) cv. Bhagwa using Double Nodal Segments | |
| CN108064694B (en) | Tissue culture rapid propagation method of yellow water branches | |
| Mahdi et al. | In vitro regeneration protocol for anupam and chini champa: two banana (Musa sapientum) cultivars of Bangladesh | |
| CN107155897B (en) | A kind of muskmelon anther induced medium and Anther culture breeding method | |
| CN108719046B (en) | Method for induced cultivation of hybrid liquidambar formosana tetraploid | |
| KR20210050893A (en) | Lily tissue culture Method | |
| KR100302206B1 (en) | In-flight mass production and forge transplanting method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20200915 |
|
| WD01 | Invention patent application deemed withdrawn after publication |