CN109924127B - Polyploid breeding method based on negative pressure technology - Google Patents
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Abstract
The invention discloses a polyploid breeding method based on a negative pressure technology, which comprises the following steps: collecting seeds from the end of 10 months to the beginning of 11 months, germinating by sand storage, and selecting germinated seedlings; dissolving colchicine, streptomycin, sodium nitrate, monopotassium phosphate, ethylene diamine tetraacetic acid and lithium chloride in water, then adding silica sol, stirring and mixing to prepare a mutagen; placing the selected germinated seedlings in a culture dish containing a mutagen, and placing the culture dish in a negative pressure tank of a negative pressure system for treatment; and (4) culturing the treated seedlings in a solid culture medium, and transplanting after new leaves grow. The plant obtained by the method provided by the invention has thick leaves, deep leaf color, high survival rate, simple operation and low cost.
Description
Technical Field
The invention relates to the technical field of agriculture, in particular to a polyploid breeding method based on a negative pressure technology.
Background art:
the breeding is a technology for cultivating a new variety of excellent animals and plants by creating genetic variation and improving genetic characteristics. It is based on genetics as theory and applies many disciplinary knowledge of ecology, physiology, biochemistry, pathology and biology statistics. Has very important significance for developing animal husbandry and planting industry. The goal of plant breeding is to obtain high-yield, stable-yield, high-quality and high-efficiency crops. However, the specific breeding objective should be adjusted according to the development of production by comprehensively considering the current situation of local varieties, breeding basis, natural environment, cultivation system, cultivation level, economic conditions, etc. And the breeding target is specified by using a plurality of varieties with larger planting areas or representatives in the local area as standards and defining the direction needing to be maintained or improved, improved or overcome.
The main breeding methods for crops at present mainly comprise: mutation breeding: the mutation breeding refers to a breeding method for obtaining a new biological variety by using an artificial mutation method, and the main mutation methods are radiation mutation, laser, chemical substance mutation, space-induced mutation and the like. The method can effectively improve variation frequency, quicken breeding process, greatly improve certain crop shapes, and has wide variation range, but the favorable variation is less, and the direction and the property of mutagenesis can not be controlled. Second, cross breeding is a breeding method in which individual organisms of the same species (or different species) having different genetic compositions are crossed to obtain a desired phenotype. The shape of the crop prepared by the method is controllable, but the time is long, and excellent characters need to be found in time in the breeding process. And thirdly, haploid breeding: the method is a breeding method which obtains a haploid plant by utilizing an anther isolated culture technology and induces the chromosome of the haploid plant to be doubled so as to obtain a required pure line plant. The method mainly utilizes chromosome variation, has short breeding time, and is mainly realized by a tissue culture method. However, the method is technically complicated and requires a rain crossbreeding combination. And fourthly, polyploid breeding, wherein the method is mainly obtained by utilizing chromosome variation, a new variety can be cultivated, and the cultivated plant has large organ, high yield and rich nutrition. However, the plant cultivated by the method has low fruiting rate. Fifthly, cell engineering breeding: the method is to obtain hybrid cells by cell fusion and to cultivate hybrid plants by tissue culture method by utilizing the totipotency of the cells. The method can overcome the incompatibility of distant hybridization and purposefully cultivate excellent varieties, but the technology is also relatively complex and has great difficulty. Sixthly, breeding by genetic engineering.
Patent (CN 201710317959.1) discloses a cucumber breeding method based on induced polyploidy and an identification method of polyploid plant, comprising: breeding the germinated cucumber seeds into seedlings, and inducing growth points of the seedlings by using colchicine solution; and then carrying out plant morphology identification, stomata identification, flow cytometry identification and cytology identification on the diploid plants and the variant plants. The method can effectively breed the novel cucumber polyploid line suitable for production and application, thereby improving the economic benefit. Patent (cn201010241253. x) discloses a kelp breeding method based on induction of polyploid gametophytes, comprising the following steps: firstly, processing haploid kelp gametophyte cells by using a mitosis inhibitor to double chromosomes in the cells, so that the haploid kelp gametophyte cells are induced into polyploid kelp gametophyte cells; then, a kelp gametocyte hybridization technology is utilized to cultivate polyploid kelp fronds. The invention has the advantages that the gene content of the kelp can be improved through polyploid breeding, and the added quantitative character gene can enhance the physiological activity of the kelp, thereby improving the economic character of the kelp; the chromosome doubling method of the invention can be applied to the cultivation of homologous polyploids and also can be applied to the cultivation of heterologous polyploids. The patent (CN 201410848092.9) discloses a centella asiatica polyploid and an induced breeding method thereof, comprising the following steps: transplanting the wild snowgrass to a culture medium; preparing an inducer solution; the inducer solution is sulfamethoxazole with the concentration of 30-50 mg/L, and 1-3 wt% of dimethyl sulfoxide is added; wrapping absorbent cotton at growing points of clumpy leaves of centella asiatica, and infiltrating the absorbent cotton with an inducer solution; treating for 2-4 days under natural illumination conditions; and (4) carrying out asexual propagation and propagation preservation on the sample which is detected as the tetraploid by the chromosome microscope. The invention carries out living body induction on the centella asiatica, not only has high induction conversion rate, but also greatly improves the content of medicinal effective substances in the polyploid centella asiatica. The patent (CN 93110536.6) discloses a breeding method of polyploid Chinese cabbage, which comprises inducing diploid Chinese cabbage with colchicine solution, selecting mutant strains, continuously separating 4 generations to obtain mutation strains of the fourth generation, namely mutation strain 1 and mutation strain 2, and performing combined seed collection on the two mutation strains to breed a tetraploid variety, namely 'Hot Excellent No. 2'. According to field tests and analysis and determination results, the Hongyou No. 2 Chinese cabbage has the advantages of fast growth, high nutrient content, good commodity quality, strong disease resistance and high temperature resistance of the polyploid Chinese cabbage, and is an ideal variety for summer planting. Although the prior art can obtain high-quality varieties, the cultivated seedlings have poor stress resistance, and the induced transformation rate needs to be further improved.
The invention content is as follows:
the technical problems to be solved by the invention are that the variety cultivated in the breeding process in the prior art has poor stress resistance, low induced transformation rate, high mortality rate and poor quality. In order to solve the technical problems, the invention provides a polyploid breeding method based on a negative pressure technology.
In order to better solve the technical problems, the invention adopts the following technical scheme:
a polyploid breeding method based on negative pressure technology comprises the following steps:
(1) collecting crape myrtle seeds from the end of 10 months to the beginning of 11 months, germinating by sand storage, and selecting germinated seedlings;
(2) dissolving colchicine, streptomycin, sodium nitrate, monopotassium phosphate, ethylene diamine tetraacetic acid and lithium chloride in water, then adding silica sol, stirring and mixing to prepare a mutagen;
(3) placing the selected germinated seedlings in a culture dish containing a mutagen, and placing the culture dish in a negative pressure tank of a negative pressure system for treatment;
(4) and (4) culturing the treated seedlings in a solid culture medium, and transplanting after new leaves grow.
Preferably, in step (2), the mutagen comprises the following components in parts by weight: 0.1-0.4 part of colchicine, 0.2-0.6 part of streptomycin, 1-5 parts of sodium nitrate, 1-3 parts of monopotassium phosphate, 0.1-0.3 part of ethylene diamine tetraacetic acid, 0.02-0.03 part of lithium chloride, 0.15-0.33 part of silica sol and 40-60 parts of water.
Preferably, in the step (2), the colloidal particle size of the silica sol is 10-20 nm.
Preferably, in the step (3), the pressure in the negative pressure treatment is-40 to-50 KPa, and the treatment time is 8 to 15 min.
Preferably, in step (3), the negative pressure system includes a pressure pump, a negative pressure tank, a negative pressure gauge, an air inlet valve, an air outlet valve, an air filter, and a culture dish.
Preferably, in step (4), the composition of the solid medium is specifically: MS minimal medium, 3-7mg/L amino acid, 6.5g/L agar, 0.35mg/L vitamin B1 and 80-100mg/L banana puree.
As a preferred mode of the above-mentioned technical means, in the step (4), the specific conditions for the culture in the solid medium are: the culture temperature is 22 ℃, the photoperiod is 15h/9h white/black period, and the light intensity is 1800-.
Preferably, in the step (4), the transplanted seedling is subjected to post-management.
As the optimization of the technical scheme, the later-period management comprises additional fertilization, watering, weeding and pest control.
The invention has the following beneficial effects:
in order to improve the survival rate of seedlings, firstly, the germinated seedlings of seeds are induced by a mutagen, and a negative pressure condition is adopted during treatment, so that the negative pressure slightly damages the germinated seedlings of the seeds, the rapid penetration of the cells of the germinated seedlings of the seeds to the mutagen is increased, the growth and the development of the germinated seedlings are positively influenced, and the survival rate after seedling transplantation is improved to a certain extent. The method is key to the size of the negative pressure value of the technology, if the negative pressure value is too small, the influence on the growth and development of the seedlings is small, but if the negative pressure value is too large, irreversible damage can be caused to the germinated seedlings, so that the survival rate of the transplanted seedlings is seriously influenced, the negative pressure value is limited to be-40 to-50 KPa, the processing time is 8 to 15min, and the survival rate of the transplanted seedlings is greatly improved.
The invention also adds a certain amount of nano silica sol into the mutagen, which is a colloidal solution formed by uniformly dispersing amorphous SiO2 particles in water, and silicon element can effectively enter cells of the transplanted seedlings under the negative pressure condition, thereby improving the stress resistance of the transplanted seedlings and increasing the survival rate of the transplanted seedlings. The method is simple to operate, low in cost, economical and environment-friendly.
The specific implementation mode is as follows:
in order to better understand the present invention, the following examples further illustrate the invention, the examples are only used for explaining the invention, not to constitute any limitation of the invention.
The breeding method of the present invention will be further described with lagerstroemia indica as an example.
Example 1
A polyploid breeding method based on negative pressure technology comprises the following steps:
(1) collecting the lagerstroemia indica seeds from the bottom of 10 months to the beginning of 11 months, germinating by sand storage, and selecting the germinated lagerstroemia indica seedlings;
(2) dissolving 0.1 part of colchicine, 0.2 part of streptomycin, 1 part of sodium nitrate, 1 part of monopotassium phosphate, 0.1 part of ethylenediamine tetraacetic acid and 0.02 part of lithium chloride in 40 parts of water by weight, adding 0.15 part of silica sol, and stirring and mixing to prepare a mutagen;
(3) placing the selected lagerstroemia indica germinating seedlings into a culture dish containing a mutagen, placing the culture dish into a negative pressure tank of a negative pressure system, and treating for 8min under the pressure of-40 to-50 KPa;
(4) the treated crape myrtle seedlings are placed in a MS basic culture medium, a culture medium containing 3mg/L amino acid, 6.5g/L agar, 0.35mg/L vitamin B1 and 80mg/L banana puree for culture, after new leaves grow out, the seedlings are transplanted, after the seedlings are transplanted, later-stage management such as topdressing, watering, weeding, pest control and the like is carried out, after the seedlings are transplanted for one month, 500 plants are adopted as research objects, the survival rate is determined to be 97.4%, and the tetraploid inductivity is 76.5%.
Example 2
A polyploid breeding method based on negative pressure technology comprises the following steps:
(1) collecting the lagerstroemia indica seeds from the bottom of 10 months to the beginning of 11 months, germinating by sand storage, and selecting the germinated lagerstroemia indica seedlings;
(2) dissolving 0.4 part of colchicine, 0.6 part of streptomycin, 5 parts of sodium nitrate, 3 parts of monopotassium phosphate, 0.3 part of ethylenediamine tetraacetic acid and 0.03 part of lithium chloride in 60 parts of water by weight, adding 0.33 part of silica sol, and stirring and mixing to prepare a mutagen;
(3) placing the selected lagerstroemia indica germinating seedlings into a culture dish containing a mutagen, placing the culture dish into a negative pressure tank of a negative pressure system, and treating for 15min under the pressure of-40 to-50 KPa;
(4) the treated crape myrtle seedlings are placed in a culture medium of MS basic culture medium, 7mg/L amino acid, 6.5g/L agar, 0.35mg/L vitamin B1 and 100mg/L banana puree for culture, after new leaves grow out, the seedlings are transplanted, after the seedlings are transplanted, the post-management of topdressing, watering, weeding, pest control and the like is carried out, after the seedlings are transplanted for one month, 500 plants are adopted as research objects, the survival rate is determined to be 96.8%, and the tetraploid inductivity is 77.2%.
Example 3
A polyploid breeding method based on negative pressure technology comprises the following steps:
(1) collecting the lagerstroemia indica seeds from the bottom of 10 months to the beginning of 11 months, germinating by sand storage, and selecting the germinated lagerstroemia indica seedlings;
(2) dissolving 0.12 part of colchicine, 0.3 part of streptomycin, 2 parts of sodium nitrate, 1.5 parts of monopotassium phosphate, 0.15 part of ethylenediamine tetraacetic acid and 0.022 part of lithium chloride in 45 parts of water by weight, adding 0.17 part of silica sol, and stirring and mixing to prepare a mutagen;
(3) placing the selected lagerstroemia indica germinating seedlings into a culture dish containing a mutagen, placing the culture dish into a negative pressure tank of a negative pressure system, and treating for 10min under the pressure of-40 to-50 KPa;
(4) the treated crape myrtle seedlings are placed in a MS basic culture medium, a culture medium containing 4mg/L amino acid, 6.5g/L agar, 0.35mg/L vitamin B1 and 85mg/L banana puree for culture, after new leaves grow out, the seedlings are transplanted, after the seedlings are transplanted, later-stage management such as topdressing, watering, weeding, pest control and the like is carried out, after the seedlings are transplanted for one month, 500 plants are adopted as research objects, the survival rate is determined to be 98.2%, and the tetraploid inductivity is 76.8%.
Example 4
A polyploid breeding method based on negative pressure technology comprises the following steps:
(1) collecting the lagerstroemia indica seeds from the bottom of 10 months to the beginning of 11 months, germinating by sand storage, and selecting the germinated lagerstroemia indica seedlings;
(2) dissolving 0.3 part of colchicine, 0.4 part of streptomycin, 2.5 parts of sodium nitrate, 2 parts of monopotassium phosphate, 0.2 part of ethylenediamine tetraacetic acid and 0.024 part of lithium chloride in 50 parts of water by weight, adding 0.2 part of silica sol, stirring and mixing to prepare a mutagen;
(3) placing the selected lagerstroemia indica germinating seedlings into a culture dish containing a mutagen, placing the culture dish into a negative pressure tank of a negative pressure system, and treating for 11min under the pressure of-40 to-50 KPa;
(4) the treated crape myrtle seedlings are placed in a MS basic culture medium, a culture medium containing 5mg/L amino acid, 6.5g/L agar, 0.35mg/L vitamin B1 and 90mg/L banana puree for culture, after new leaves grow out, the seedlings are transplanted, after the seedlings are transplanted, later-stage management such as topdressing, watering, weeding, pest control and the like is carried out, after the seedlings are transplanted for one month, 500 plants are adopted as research objects, the survival rate is determined to be 98.6%, and the tetraploid inductivity is 77.5%.
Example 5
A polyploid breeding method based on negative pressure technology comprises the following steps:
(1) collecting the lagerstroemia indica seeds from the bottom of 10 months to the beginning of 11 months, germinating by sand storage, and selecting the germinated lagerstroemia indica seedlings;
(2) dissolving 0.3 part of colchicine, 0.5 part of streptomycin, 4 parts of sodium nitrate, 2.5 parts of monopotassium phosphate, 0.25 part of ethylenediamine tetraacetic acid and 0.026 part of lithium chloride in 55 parts of water by weight, adding 0.25 part of silica sol, and stirring and mixing to prepare a mutagen;
(3) placing the selected lagerstroemia indica germinating seedlings into a culture dish containing a mutagen, placing the culture dish into a negative pressure tank of a negative pressure system, and treating for 12min under the pressure of-40 to-50 KPa;
(4) the treated crape myrtle seedlings are placed in a MS basic culture medium, 6mg/L amino acid, 6.5g/L agar, 0.35mg/L vitamin B1 and 90mg/L banana puree culture medium for culture, after new leaves grow out, the seedlings are transplanted, after the seedlings are transplanted, later period management such as additional fertilization, watering, weeding, pest control and the like is carried out, after the seedlings are transplanted for one month, 500 plants are adopted as research objects, the survival rate is determined to be 97.6%, and the tetraploid inductivity is 77.0%.
Example 6
A polyploid breeding method based on negative pressure technology comprises the following steps:
(1) collecting the lagerstroemia indica seeds from the bottom of 10 months to the beginning of 11 months, germinating by sand storage, and selecting the germinated lagerstroemia indica seedlings;
(2) dissolving 0.35 part of colchicine, 0.55 part of streptomycin, 4.5 parts of sodium nitrate, 3 parts of monopotassium phosphate, 0.3 part of ethylenediamine tetraacetic acid and 0.028 part of lithium chloride in 55 parts of water by weight, adding 0.3 part of silica sol, and stirring and mixing to prepare a mutagen;
(3) placing the selected lagerstroemia indica germinating seedlings into a culture dish containing a mutagen, placing the culture dish into a negative pressure tank of a negative pressure system, and treating for 13min under the pressure of-40 to-50 KPa;
(4) the treated crape myrtle seedlings are placed in a MS basic culture medium, 6mg/L amino acid, 6.5g/L agar, 0.35mg/L vitamin B1 and 95mg/L banana mud for culture, after new leaves grow out, the seedlings are transplanted, after the transplanted seedlings are subjected to post-management such as topdressing, watering, weeding, pest control and the like, after the seedlings are transplanted for one month, 500 plants are adopted as research objects, the survival rate is determined to be 98.2%, and the tetraploid inductivity is 76.9%.
Comparative example 1
A polyploid breeding method based on negative pressure technology comprises the following steps:
(1) collecting the lagerstroemia indica seeds from the bottom of 10 months to the beginning of 11 months, germinating by sand storage, and selecting the germinated lagerstroemia indica seedlings;
(2) dissolving 0.35 part of colchicine, 0.55 part of streptomycin, 4.5 parts of sodium nitrate, 3 parts of monopotassium phosphate, 0.3 part of ethylenediamine tetraacetic acid and 0.028 part of lithium chloride in 55 parts of water by weight, adding 0.3 part of silica sol, and stirring and mixing to prepare a mutagen;
(3) placing the selected lagerstroemia indica germinating seedlings in a culture dish containing mutagen, and treating for 13min at normal temperature and normal pressure;
(4) the treated crape myrtle seedlings are placed in a culture medium of MS basic culture medium, 6mg/L amino acid, 6.5g/L agar, 0.35mg/L vitamin B1 and 95mg/L banana mud for culture, after new leaves grow out, the seedlings are transplanted, after the seedlings are transplanted, the post-management of topdressing, watering, weeding, pest control and the like is carried out, after the seedlings are transplanted for one month, 500 plants are adopted as research objects, the survival rate is determined to be 83.2%, and the tetraploid inductivity is 59.2%.
Comparative example 2
A polyploid breeding method based on negative pressure technology comprises the following steps:
(1) collecting the lagerstroemia indica seeds from the bottom of 10 months to the beginning of 11 months, germinating by sand storage, and selecting the germinated lagerstroemia indica seedlings;
(2) dissolving 0.35 part of colchicine, 0.55 part of streptomycin, 4.5 parts of sodium nitrate, 3 parts of monopotassium phosphate, 0.3 part of ethylene diamine tetraacetic acid and 0.028 part of lithium chloride in 55 parts of water by weight to prepare a mutagen;
(3) placing the selected lagerstroemia indica germinating seedlings into a culture dish containing a mutagen, placing the culture dish into a negative pressure tank of a negative pressure system, and treating for 13min under the pressure of-40 to-50 KPa;
(4) the treated crape myrtle seedlings are placed in a MS basic culture medium, 6mg/L amino acid, 6.5g/L agar, 0.35mg/L vitamin B1 and 95mg/L banana puree culture medium for culture, after new leaves grow out, the seedlings are transplanted, after the seedlings are transplanted, the post-management of topdressing, watering, weeding, pest control and the like is carried out, after the seedlings are transplanted for one month, 500 plants are adopted as research objects, the survival rate is determined to be 88.3%, and the tetraploid inductivity is 61.2%.
It can be seen from the above examples and comparative examples that the induction treatment of the germinated seedlings under negative pressure can effectively improve the survival rate of the transplanted seedlings and the polyploid induction rate, mainly because the negative pressure under certain conditions has certain damage to the explant tissues, thereby increasing the absorption of the mutagen and improving the induction rate, but the negative pressure value selection is large, which can cause great damage to the explant tissues and can influence the survival rate of the seedlings. The addition of the nano silica sol also improves the survival rate of the seedlings to a certain extent.
Although specific embodiments of the invention have been described, many other forms and modifications of the invention will be apparent to those skilled in the art. It is to be understood that the appended claims and this invention generally cover all such obvious forms and modifications which are within the true spirit and scope of the present invention.
Claims (7)
1. A polyploid breeding method based on a negative pressure technology is characterized by comprising the following steps:
(1) collecting crape myrtle seeds from the end of 10 months to the beginning of 11 months, germinating by sand storage, and selecting germinated seedlings;
(2) dissolving 0.1-0.4 part of colchicine, 0.2-0.6 part of streptomycin, 1-5 parts of sodium nitrate, 1-3 parts of monopotassium phosphate, 0.1-0.3 part of ethylenediamine tetraacetic acid and 0.02-0.03 part of lithium chloride in 40-60 parts of water by weight, and then adding 0.15-0.33 part of silica sol, stirring and mixing to prepare a mutagen;
(3) placing the selected germinated seedlings in a culture dish containing a mutagen, and placing the culture dish in a negative pressure tank of a negative pressure system for processing for 8-15min under-40 to-50 Kpa;
(4) and (4) culturing the treated seedlings in a solid culture medium, and transplanting after new leaves grow.
2. The negative pressure technology-based polyploid breeding method according to claim 1, wherein in the step (2), the colloidal particle size of the silica sol is 10-20 nm.
3. The polyploid breeding method based on the negative pressure technology as claimed in claim 1, wherein in the step (3), the negative pressure system comprises a pressure pump, a negative pressure tank, a negative pressure gauge, an air inlet valve, an air outlet valve, an air filter and a culture dish.
4. The method for breeding polyploids based on the negative pressure technology, according to claim 1, wherein in the step (4), the composition of the solid medium is specifically as follows: MS minimal medium, 3-7mg/L amino acid, 6.5g/L agar, 0.35mg/L vitamin B1 and 80-100mg/L banana puree.
5. The method for breeding polyploids based on the negative pressure technology, as claimed in claim 1, wherein in step (4), the specific conditions for culturing in the solid medium are: the culture temperature is 22 ℃, the photoperiod is 15h/9h white/black period, and the light intensity is 1800-.
6. The method for breeding polyploids based on the negative pressure technology as claimed in claim 1, wherein in step (4), the post-management of the transplanted seedlings is required.
7. The method of claim 6, wherein the post management comprises additional manuring, watering, weeding, and pest control.
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| CN103749292A (en) * | 2014-01-23 | 2014-04-30 | 程钰翔 | Method for culturing Acanthopanax senticosus tetraploid |
| CN108432536A (en) * | 2018-03-22 | 2018-08-24 | 合肥申沃园艺有限公司 | A kind of crape myrtle implantation methods resistant to lodging |
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| CN103749292A (en) * | 2014-01-23 | 2014-04-30 | 程钰翔 | Method for culturing Acanthopanax senticosus tetraploid |
| CN108432536A (en) * | 2018-03-22 | 2018-08-24 | 合肥申沃园艺有限公司 | A kind of crape myrtle implantation methods resistant to lodging |
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