CN107980620B - Composition for chromosome doubling of corn haploid seedlings - Google Patents
Composition for chromosome doubling of corn haploid seedlings Download PDFInfo
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Abstract
The invention relates to a composition for doubling chromosomes of corn haploid seedlings, which is used for doubling plant chromosomes by using a compound formula prepared from a plurality of cell mitosis inhibitors and tubulin inhibitors. The key point and key point of the invention are three compounds with different functions of doubling agent, auxiliary agent and antidote, which have the functions of improving doubling effect and reducing drug damage. Can solve the problems that the damage to the processed plant material is large in the process of doubling the plant chromosome by using colchicine, the processed material is often abnormal in growth, deformed and even dead, and the doubling success rate is low. More importantly, the colchicine can solve the problems that colchicine has strong toxicity and potential carcinogenic effect on mammals including human beings and has great harm to the health of operators and the environmental safety.
Description
Technical Field
The invention relates to a doubling agent formula and a doubling method for plant chromosomes, belongs to the fields of plant biology, plant biotechnology and plant breeding, and particularly relates to a composition for doubling the chromosomes of corn haploid seedlings.
Background
Ploidy changes in plants are an important research topic in plant biology research, plant biotechnology, and plant breeding. Compared with diploid, the autopolyploid has the characteristics of certain organ enlargement or metabolite content improvement, and has excellent breeding utilization value for crops aiming at harvesting nutritive organs and asexual propagation crops; artificially creating polyploids can also recombine the wild species and the genetic materials of cultivated species to breed novel crops; haploid doubling can obtain pure lines of genotype homozygous plants and shorten the breeding period; obtaining odd polyploids (such as triploid) can cultivate seedless fruits such as seedless watermelons, seedless grapes, seedless oranges and the like, can also cultivate plant species without seeds, and can be mainly used for urban landscaping such as poplar and willow trees and the like and the cultivation of fast-growing forest varieties to obtain varieties without flying floc; the allopolyploids obtained by crossing different closely related species can be used for breeding new species, new crops and can be used for plant evolution research. Chromosome doubling is therefore of great theoretical research and commercial value. These studies all rely on a common technique-namely chromosome doubling. The chromosome doubling technology adopted in the past is to use colchicine as a doubling agent, but the doubling effect of the colchicine on plants is difficult to meet the needs of the research, the main problems are that the colchicine has great damage to the treated plant materials, abnormal growth, malformation and even death of the treated materials can be caused frequently, and in addition, the doubling success rate of the colchicine is low. In addition, colchicine is extremely toxic, has greater toxicity and potential carcinogenic effect on mammals including human beings, and has great harm to the health and environmental safety of operators. Therefore, the invention provides a high-efficiency and low-toxicity plant chromosome doubling method capable of replacing colchicine, which has important significance.
Disclosure of Invention
The invention aims to provide a plant chromosome doubling method which is lower in toxicity, higher in efficiency and wide in application range than colchicine.
The plant chromosome doubling method provided by the invention is used for doubling the plant chromosome by using a compound formula prepared from a plurality of cell mitosis inhibitors and tubulin inhibitors.
The effective components of the doubling agent are the following compounds: butralin (chemical name: N-sec-butyl-4-tert-butyl-2, 6-dinitroaniline; English name: Butralin; molecular formula: C)14H21N3O4(ii) a Molecular weight: 295.3342), glufosinate (chemical name: O-methyl-O- (2-nitro-4-methylphenyl) -N-isopropylthiophosphonamide; english name: amiperofos methyl, AMP; the molecular formula is as follows: c11H17N2O4PS; molecular weight: 304.3), asulam (chemical name: 3, 5-dinitro-N ', N' -dipropylsulfonamide; 3, 5-dinitro-N4, N4-propylsulfonamide; english name: oryzalin; the molecular formula is as follows: c12H18N4O6S; molecular weight: 346.3595), nocodazole (chemical name: methyl- (5-thenoyl-2-benzimidazolyl) carbamic acid; english name: nocodazole; the molecular formula is as follows: c14H11N3O3S; molecular weight: 301.3204), trifluralin (chemical name: 2, 6-dinitro-N, N-di-N-propyl-4-trifluoromethylaniline; english name: trifluralin; the molecular formula is as follows: c13H16F3N3O4(ii) a Molecular weight: 335.28) Aminotrifluralin (chemical name: 5-dipropylamino- α, α, α -4, 6-dinitro-o-toluidine; the name of English: procymidon; the molecular formula is as follows: c13H17N4O4(ii) a Molecular weight: 350.3), dithiopyr (chemical name: s, S' -dimethyl-2-difluoromethyl-4-isobutyl-6-trifluoromethylpyridine-3, 5-dithioformate; english name: dithiopyr; the molecular formula is as follows: c15H16F5NO2S2(ii) a Molecular weight: 401.4), dichlormid (chemical name: n, N-dimethyl-2, 2-diphenylacetamide; english name: DIPHENAMID, respectively; the molecular formula is as follows: c16H17NO; molecular weight: 239.31), pretilachlor (chemical name: 2-chloro-N- (2, 6-diethylphenyl) -N- (2-propoxyethyl) acetamide; english name: pretilachlor; the molecular formula is as follows: c17H26ClNO2(ii) a Molecular weight: 311.85), propyzamide (chemical name: 3, 5-dichloro-N- (1, 1-dimethylpropynyl) benzamide; english name: (ii) a propylzamide; the molecular formula is as follows: c12H11Cl2NO; molecular weight: 256.13), mefenacet (chemical name: 2- (1, 3-benzothiazol-2-yloxy) -N-methylacetanilide; english name: mefenacet; the molecular formula is as follows: c16H14N2O2S; molecular weight: 298.3596), metolachlor (chemical name: 2-chloro-2 ', 6' -diethyl-N- (2-propoxyethyl) acetanilide; english name: metalachlor; the molecular formula is as follows: c15H22ClNO2(ii) a Molecular weight: 283.79), chlorpropham (chemical name: isopropyl N- (3-chlorophenyl) carbamate; english name: (ii) Chloropropham; the molecular formula is as follows: c10H12ClNO2(ii) a Molecular weight: 213.66), pendimethalin (chemical name: n- (1-ethylpropyl) -2, 6-dinitro-3, 4-dimethylaniline; english name: penlimethhalin; the molecular formula is as follows: c13H19N3O4(ii) a Molecular weight: 281.31), anilox (chemical name: isopropyl N-phenyl carbamate; english name: propham; the molecular formula is as follows: c10H13NO2(ii) a Molecular weight: 179.22); the above compound is a doubling agent used in the present invention;
the doubling agent compound needs Ethylene Glycol (EG) and/or Dimethyl sulfoxide (English name: Dimethyl sulfoxide) and/or toluene and/or xylene as an auxiliary agent, and has the functions of promoting agent dissolution and enhancing agent permeation in plant tissues and cells, and promoting the agent to reach stem tips, bud tips, root tips and other growing tissues and parts with growing points, inflorescences and the like capable of generating reproductive organs and progeny so as to achieve the doubling effect. Commercial pesticide adjuvants which are commercially available, including organosilicon adjuvants, adhesives and emulsifiers, are also available, such as TWEEN20, TWEEN60 and TWEEN80, which are the adjuvants used in the present invention;
the doubling treatment also comprises the use of potassium phosphate and/or dipotassium hydrogen phosphate and/or potassium dihydrogen phosphate, which can relieve the toxicity of the medicament, reduce the adverse effects of slow growth, stagnation, abnormality, malformation and death of the treated material caused by medicament damage, provide phosphorus and potassium nutrient elements necessary for the growth of the treated plant material, and relieve the symptoms of phosphorus deficiency and potassium deficiency after the doubling treatment, and the compound is used as an antidote in the method. The antidote can also be added into nutrient solution containing other inorganic and organic nutrient components, including MS culture solution, Hoagland nutrient solution and various water culture nutrient solution with various changed components, and the use mode provides more full and comprehensive nutrient components for the doubled material, but the core component is 0.1-2% of potassium phosphate and/or dipotassium hydrogen phosphate and/or potassium dihydrogen phosphate in percentage by mass;
the double-layer processing method is characterized in that when the processing parts in the double-layer processing are whole plants, roots and materials needing transplanting, ABT rooting powder is used, the concentration of the ABT rooting powder is 10-25 ppm (parts per million), and the ABT rooting powder has the functions of promoting the differentiation and growth of roots and improving the survival rate of processed plants. The compounds (medicine and reagent) are plant growth regulator. The growth regulator can be other auxin compounds containing plant growth regulator promoters besides ABT rooting powder, and comprises compounds containing indoleacetic acid, indolebutyric acid, naphthylacetic acid and compound sodium nitrophenolate (such as potassium indolebutyrate and/or sodium naphthylacetate);
the key point of the invention is the use of the compound medicament, and the matched use of the four different compounds (doubling agent, auxiliary agent, antidote and growth regulator) is the core and key of the invention, and can play the roles of improving the doubling effect and reducing the drug damage. The doubling agent is used in a form of being dissolved in water, and the final concentration range of each compound unit of butralin, glufosinate-methyl, benazolin, nocodazole, trifluralin, prodiamine, dithiopyr, dibenzamide, pretilachlor, pentyne, mefenacet, metolachlor, pendimethalin and anilide is 0.01 mu M-10 mM; the final concentration ranges of the ethylene glycol, the dimethyl sulfoxide, the toluene and the xylene are 0.02-5% (V/V, volume percentage content); the final concentration of potassium phosphate, dipotassium hydrogen phosphate and potassium dihydrogen phosphate is 0.1-2% (W/V, mass percentage content); the concentration of ABT rooting powder and analogues thereof is 10-25 ppm (parts per million); the concentration used can be any concentration within this continuous range and includes both values;
the doubling agent can also be used in the form of a seed coating agent, and the weight volume percentage content of each compound single product in the coating agent is 0.0001-1%;
the pharmaceutical compounds are commercially available, unless otherwise specified, and include those available from laboratory consumable suppliers, chemical plants, chemical reagent (pharmaceutical) companies, pesticide manufacturers and their agents, pesticide companies, various agricultural stores, or agricultural markets. Different preparation modes comprise analytical pure grade, chemical pure grade and various commercial preparations with different concentrations and different contents;
the compound doubling agent is not required to be prepared in the same formula for all the medicines, and because different medicines have different damage effects on different kinds of plants and different effects on monocotyledons and dicotyledons, the doubling effect and the survival rate need to be comprehensively balanced and considered in application, and different medicines are selected for different plants to be prepared into a combined formula. In particular, the doubling effect and the damage degree of the doubling agent used in the invention on monocotyledons and dicotyledons and perennial and annual plants are obviously different, so that different compound combinations and concentration combinations are adopted according to different plant categories;
the combination of compounds can be any and all possible combinations of 2 and more than 2 of any of the aforementioned doubling agents, adjuvants, antidotes and growth regulators;
the plants usable in the present invention range from all plants which can undergo mitosis and meiosis, and the treatments for doubling include the whole plants, parts of plants, organs, tissues and cells, etc. of these plants; modifications to the present method can also be used for chromosome doubling in animals and fungi that have mitotic and meiotic capabilities. These plant ranges include, but are not limited to: watermelon, cucumber, melon, capsicum, eggplant, tomato, potato, arabidopsis thaliana, sweet potato, citrus, grapefruit, sugarcane, rubber tree, banana, mango, pseudo-ginseng, corn, rice, wheat, cotton, soybean, rape, cabbage, grape, apple, pear, papaya, peanut, sorghum, tobacco, alfalfa, cherry, poplar, willow, cassava, oil palm, barley, oat, sunflower, millet, beet, lotus, lily, peach, plum, apricot, chrysanthemum, tea tree, pepper, pumpkin, wax gourd, towel gourd, balsam pear, kidney bean, cowpea, broad bean, chickpea, lupin, clover, ginseng, licorice, walnut, kiwi, lychee, longan, red date, pseudo-ginseng, persimmon, cauliflower, cabbage, spinach, strawberry, blueberry, mulberry, rose, orchid, rose, moth orchid, tang cattail, pink, barley, peony, sorghum, banana, mango, sorghum, rice, wheat, rice, wheat, Chinese yew, ginkgo, lettuce, leek, scallion, onion, plum blossom and dandelion; the application range of the invention also includes doubling of chromosomes of various ploidy plants and obtaining of doubled materials, including aneuploid, haploid, diploid, triploid, tetraploid, pentaploid, hexaploid, heptaploid, octaploid, nonaploid, decaploid, dodecaploid and hexadecploid.
The doubling treatment method comprises the selection and combination of factors such as treatment positions, treatment time, treatment materials, treatment modes and the like:
treatment of the sites: the stem apical meristem, the flower meristem and the immature embryo in the development process of the plant are taken as main objects, and the systemic property and the conductivity effect can be utilized to enable the medicament to reach the growing point through the treatment of non-growing tissues such as root stems and leaves;
treatment time: from 1min to 50 days, the optimum time varies depending on the kind of plant, the material to be treated and the treatment method;
the treatment method comprises the following steps: doubling chromosomes and producing reproducible offspring by various modes of action of direct and indirect contact;
a. treatment of ungerminated seeds: the double treatment can be carried out by adopting the modes of seed soaking, seed coating, spraying, smearing, culture medium adding, absorbent paper, wrapping various materials with adsorbability and receptivity to liquid and the like;
b. treatment of germinated (germinated) seeds or/and seedlings: the method can adopt the modes of medicine application such as dipping, smearing, spraying, dripping and the like, and can adopt the method of exposing the growth points according to the requirements during treatment, including stripping/cutting organs and tissues such as peripheral leaves, leaf sheaths, cotyledons, young leaves and the like so as to improve the doubling effect; the medicament can be directly injected to the accessory of the growing point by adopting a micro-injection mode;
c. treatment of roots: the method can adopt modes of medicament dipping, water culture, sand culture, soil addition, artificial matrix addition, culture medium addition, spraying and the like, and the treatment process can be continuous treatment, intermittent treatment or short-term intermittent treatment or/and one-time treatment;
d. young growing points, bud tips, stem tips and inflorescence tissue treatment: the method of administration such as dropping, coating (smearing) or contacting absorbent cotton containing solution, filter paper and various materials with adsorption capacity with the material to be treated can be adopted, and thickening agent, tackifier and the like are added into the medicament in the using process to enhance the attachment capacity of the medicament; the medicament can be directly injected to the growing point or/and the vicinity of the reproductive organs by a micro-injection mode;
e. treatment of immature seeds and immature embryos: the doubling purpose can be achieved by adopting the modes of dipping, smearing, spraying, dropping, injecting, coating, adding a culture medium, introducing a pollen tube, introducing a filament, introducing a floral organ and the like.
The invention has the following positive effects: compared with the doubling method using colchicine, the method has the advantages of low concentration and dosage of the used medicament compound, low toxicity, low residue, easy degradation and small environmental pollution, particularly low toxicity to human and other animals, low field death rate of the treated plants, low incidence rate of abnormal plant growth and higher doubling rate, and is an effective chromosome doubling method.
Detailed Description
The methods provided below are examples illustrating the effectiveness of the methods of the invention and are not limiting or/and limiting of the types, concentrations, combinations, and ranges of use of the compound agents used in the methods.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents, and the like used in the following examples are commercially available, unless otherwise specified, and include those available from suppliers of laboratory consumables, chemical plants, chemical reagent (pharmaceutical) companies, agricultural chemicals manufacturers and their agents, agricultural chemicals companies, various agricultural stores, or agricultural markets.
The molar concentrations of the reagent solutions in the examples described below were calculated from the content of the active ingredient of the compound that was actually purchased and prepared.
Example A monocot Secale chromosome doubling
First, reagent preparation
1. Preparation of doubling agent mother liquor
The doubling agent needs to be prepared into mother liquor respectively and independently, when in use, the doubling agent is diluted according to the formula requirement, each mother liquor is uniformly prepared according to the following method, and the preparation method of the reagent/medicament is a conventional technology adopted in a common laboratory:
weighing 50ml of dimethyl sulfoxide into a volumetric flask of 1000ml, calculating and weighing a corresponding amount of solid medicines according to the final volume of 1000ml and the final concentration of 10mM and the effective content of each medicine, adding the solid medicines into the dimethyl sulfoxide, and slowly stirring until the solid medicines are completely dissolved; adding 200ml of glycol, and fully stirring until the mixture is dissolved uniformly; adding distilled water to a constant volume of 1000ml, fully stirring and uniformly mixing, filling into a brown bottle, and putting into a refrigerator at 4 ℃ for later use;
2. preparation of composite doubling agent
Adding 500ml of sterilized distilled water into a volumetric flask of 1000ml, respectively sucking the reagent mother liquor into the distilled water by a liquid-moving machine, wherein the sucking amount is 3ml of methylaminophosphine, 3ml of bisphenyloxamide, 3ml of butralin, 1ml of trifluralin, 1ml of pretilachlor and 1ml of asulam, and then fixing the volume to 1000ml by using the distilled water. The final concentration of the components in the solution is 30 mu M of methylaminophosphine, 30 mu M of dibenzamide, 30 mu M of butralin, 10 mu M of trifluralin, 10 mu M of pretilachlor and 10 mu M of oryzalin.
Preparation and pretreatment of plant material to be doubled-seed treatment and germination
Taking rye seeds with germination capacity, soaking the rye seeds with clear water at room temperature (about 18-25 ℃) overnight (14-18 hours) to ensure that the seeds are swelled; then, the rye seeds are placed into a germination box paved with germination paper or filter paper, are subjected to moisture-preserving culture in a 20-25 ℃ artificial climate chamber, an automatic temperature control incubator or at room temperature, and are regularly washed with clean water for 3 times every morning, noon and evening until the root length reaches 0.5-1.0 cm.
Triple, double processing
Putting the germinated rye seeds into a 200ml beaker, adding a doubling agent, and continuously culturing for 6-8 hours at constant temperature or room temperature (18-25 ℃) when the doubling agent is required to completely submerge the buds and the radicles. Then cleaning with clear water and continuously culturing for 8-24 hours.
Detection of four, doubling effect-cytology detection
And (5) taking root tips, observing the number of cell chromosomes by adopting a temporary tabletting method under an optical microscope, and checking the doubling effect. The non-doubled, normal diploid root tip cells of rye have only 14 chromosomes, while the tetraploid root tip cells that are successfully doubled have 28 chromosomes.
Five, double effect
Taking 100 bud root tips subjected to doubling treatment for carrying out doubling effect test, wherein the doubling rate reaches 100%; the doubling effect of colchicine reaches 100%. The method has no obvious difference from colchicine in the doubling effect of the rye root tip.
Example chromosome doubling of pea plants of the dicotyledonous plants
First, reagent preparation
1. Preparation of doubling agent mother liquor
Same as example one
2. Preparation of composite doubling agent
Adding 500ml of sterilized distilled water into a volumetric flask of 1000ml, respectively sucking the reagent mother liquor into the distilled water by a liquid-moving machine, wherein the sucking amount is 5ml of butralin, 3ml of glufosinate-methyl, 3ml of prodiamine, 1ml of pentyne, 1ml of mefenacet and 1ml of dithiopyr, and then fixing the volume to 1000ml by using the distilled water. The final concentration of the components in the solution is 50 mu M of butralin, 30 mu M of glufosinate-methyl, 30 mu M of prodiamine, 10 mu M of penetryn, 10 mu M of mefenacet and 10 mu M of dithiopyr.
Preparation and pretreatment of plant material to be doubled-seed treatment and germination
Soaking pea seeds with germination capacity in clear water at room temperature (about 18-25 ℃) overnight (14-18 hours) to ensure that the seeds are swelled; then, pea seeds are placed into a germination box paved with germination paper or filter paper, are subjected to moisture preservation culture in a 20-DEG C artificial climate chamber and an automatic temperature control incubator, and are regularly washed with clean water for 3 times every morning, noon and evening until the radicle breaks through the seed coat and the root length reaches 0.5-1.0 cm, and the process generally needs 2 days.
Triple, double processing
Putting the germinated pea seeds into a 200ml beaker, adding the compound doubling agent prepared in the example, and continuously culturing for 6-8 hours under the condition of constant temperature or room temperature (18-25 ℃) if the agent is required to completely submerge the germinated peas and the radicles. Then cleaning the seeds with clear water, and putting the seeds back into a germination box to continue culturing for 48 to 72 hours.
Four, doubling effect assay-morphological and cytological assays
After the pea seedlings which are subjected to double treatment are taken out, obvious morphological change can be found, and the remarkable characteristics are that the development of buds and roots is shortened and coarsened, particularly, the root tips are enlarged and swollen, and the shapes are short and thick; and (5) taking root tips, observing the number of cell chromosomes by adopting a temporary tabletting method under an optical microscope, and checking the doubling effect. The non-doubled, normal pea diploid root tip cells have only 7 pairs (14) of chromosomes, whereas the root tip cells that are successfully doubled have 28 or more chromosomes.
Five, double effect
Taking 100 bud root tips subjected to doubling treatment for carrying out doubling effect test, wherein the doubling rate reaches 100%;
the first and second examples are only to test the effect of the compound doubling agent on monocotyledons and dicotyledons, so detoxification treatment and late seedling culture and transplantation are not carried out; if the seedling is required to be cultured and transplanted later, detoxification treatment and rooting treatment are required.
But the method has the advantages that:
1. the treatment time is short, colchicine needs to be treated for about 26 hours, the method only needs about 6 hours, and the double effect can be further improved and the treatment time can be shortened by increasing the concentration of the medicament and the treatment temperature;
2. the doubling agent compound used in the method has no strong toxicity, and colchicine is strong toxicity, so the harm to the health of operators and the environment is far less than that of the original treatment method using colchicine as the doubling agent.
EXAMPLE chromosome doubling of triple maize haploid embryos
First, reagent preparation
1. Preparation of reagent mother liquor
The same as in the first embodiment;
2. preparation of composite doubling agent
Adding 60ml of sterilized distilled water into a 100ml volumetric flask, respectively sucking the reagent mother liquor into the distilled water by a liquid-moving machine, wherein the sucking amount is 0.5ml of methylaminophosphine, 0.5ml of nocodazole, 0.3ml of phenylaniline, 0.3ml of chlorpropham, 0.1ml of pentynil, 0.1ml of pendimethalin and 0.1ml of metolachlor, and then fixing the volume to 100ml by the distilled water. The final concentration of the components in the solution is 50 mu M of methylaminophosphine, 50 mu M of nocodazole, 30 mu M of phenylaniline, 30 mu M of chlorpropham, 10 mu M of penoxsulam, 10 mu M of pendimethalin and 10 mu M of metolachlor.
3. Preparing an antidote: adding 0.2g of monopotassium phosphate into 100ml of distilled water to prepare a 0.2% aqueous solution, and then adding 0.5g of urea;
preparing an ABT rooting powder solution: the ABT rooting powder No. 6 is used in the example, is produced by Beijing Zhonglin Jialin science and technology Limited company, and is prepared into an aqueous solution with the concentration of 20ppm (parts per million) according to the requirements of the specification provided on a packing box;
preparation and pretreatment of plant material to be doubled
1. Induction of haploids
The embryo used in the experiment is obtained by taking normal diploid corn as a female parent and haploid inducing material with female parent haploid inducing capacity as a male parent through an artificial hybridization method. Before the female parent silks grow out, the female parent silks need to be bagged to prevent pollen pollution; after the female parent female ear filaments grow out, the female parent female ear is pollinated with male parent pollen to obtain heterozygous diploid grains and haploid grains. The method for controlling pollination and preventing foreign pollen pollution is the conventional maize pollination technology which is generally adopted in maize heredity and breeding. The pollination method can produce 1.5-23% haploid grains, the proportion of which is different according to different induction lines, and the rest is mainly diploid heterozygous grains. The female parent can be a hybrid, a local variety, a comprehensive variety, an inbred line, an artificially synthesized population and the like, and can also be other self-propagating zea materials whether in a genetic homozygous state or not; the male parent inducing material may be haploid inducing line such as Stock 6, Nongda high inducing No. 1, RWS, KEMS, KMS, ZMS, CAUHI1, CAUHI5, etc. or hybrid of different inducing lines, including single cross, double cross, triple cross, comprehensive cross, etc. and their progeny material of different separation generations. The haploid induction line used in the experiment is Nongda GaoChing No. 1 (the public can obtain from Chinese agriculture university, and the non-patent documents recorded with the material are Liu Zhi Zeng, Song Tong Ming 2000, breeding and identification of the corn high-frequency parthenogenesis haploid induction line, the crop science report 26 (5): 570-574);
2. obtaining of immature embryos
And 5-30 days after pollination, taking down the immature fruit cluster of the female parent, removing bracts and filaments, sterilizing the surface of the fruit cluster with hypochlorous acid or hydrogen peroxide for 20min, and manually taking out the young embryo with a sterilized scalpel. The taken-out young embryos are placed into sterilized pure water for short-term storage, and after a certain number of the obtained young embryos are obtained, the obtained young embryos are subjected to doubling treatment;
triple, double processing
After the water content of the taken-out immature embryos is controlled to be dry, putting the immature embryos into a culture dish, adding the doubling agent until the immature embryos are submerged, and treating for 20-30 min; then pouring out the liquid medicine, repeatedly washing with sterile water for at least 3 times, and washing to remove the residual liquid medicine;
four, double post-treatment
Detoxification treatment: soaking the young embryo in 0.2% (W/V) potassium dihydrogen phosphate solution for 20 min;
culturing the young embryo: uniformly and tidily placing the plumule subjected to doubling treatment on a culture dish containing a total nutrient medium, taking the fact that the plumule faces downwards and the scutellum faces upwards, carrying out dark culture at 26 ℃ for 6-10 days (generally 7 days), then carrying out illumination culture for 2-3 weeks, and continuing to culture until healthy buds and roots grow out of the plantlet, so as to obtain the seedling with survival ability. In the process, the young embryo or the young seedling is divided into diploid and haploid according to the existence of the color marker, the purple is diploid, and the purple-free is probably haploid.
Fifth, transplanting seedlings
When transplanting seedlings, the seedlings can be divided into haploids and diploids again through marking characters such as colors, and the haploids and the diploids are transplanted respectively. Soaking the roots in ABT rooting powder solution for 2 hours before transplanting; then transplanting the seedlings into a culture pot filled with nutrient soil for acclimatization, and transplanting the seedlings into a greenhouse or open field after one week. And (4) carrying out normal cultivation management on the transplanted plants, bagging before flowering, and selfing after flowering until mature seeds are obtained.
Sixth, cytological examination
When the length of the radicle reaches 1-2 cm, one root tip is taken to detect the chromosome number of the cell, and the doubling effect is tested. The diploid plant (cell) with successful doubling has 20 chromosomes, the haploid plant (cell) without successful doubling has 10 chromosomes, and the tetraploid plant (cell) has 40 chromosomes.
Seventhly, field verification
Statistics of plant pollen scattering rate in corn flowering period and fructification plant rate in mature period
The young seedlings are differentiated by color marks whether the young embryos before treatment are haploid or diploid, the haploid is completely transplanted, and only 50 plants are transplanted in the diploid. The above seedlings were transplanted into the greenhouse. And (3) after seedling delaying, carrying out normal management measures of greenhouse cultivation, counting the haploid pollen strain rate in the flowering period, carrying out selfing pollination, distinguishing the haploid and the diploid according to the color separation of grains on the fruit cluster after the plant is mature, and counting the maturing rate of the haploid plant. The obtained seed progeny adopts a planting mode of ear rows, and the doubling effect of the haploid is judged according to the uniformity of morphological characters in the rows and the existence of purple marks of the seeds and the separation condition. The test result shows that: the haploid multiplying power (powder scattering rate) of the method can reach 44.4-64.7%, and the average is 51.9% (different materials); the doubling rate is 36.4-62.5%, and the average rate is 48.1% (due to the difference of materials, the data is the result of small-scale experiments). 2 plants are found to be tetraploid plants in the offspring of the diploid plants, which shows that the method has better chromosome doubling effect.
TABLE-results of the doubling of immature embryo culture
| Ear numbering | Number of immature embryos | Number of haploid embryos | Number of grown seedlings | Number of transplanted seedlings | Number of loose powder plants | Number of fructification plants |
| 1 | 121 | 19 | 17 | 17 | 11 | 4 |
| 2 | 136 | 21 | 18 | 18 | 8 | 4 |
| 3 | 128 | 18 | 18 | 17 | 8 | 5 |
| Total up to | 385 | 58 | 53 | 52 | 27 | 13 |
| Ratio of | 15.06％ | 91.3％ | 98.1％ | 51.9％ | 48.1％ |
In the practical application process, the method can be modified and optimized to be more efficient. When the immature embryos are obtained, haploids and diploids can be distinguished according to the color of the immature embryos, the immature embryos with purple marks are diploids, the immature embryos without purple marks can be haploids, and only the immature embryos without purple marks are left; during the culture of the young embryo, the young embryo or the plant showing the purple marker is continuously eliminated, and the efficiency of identifying and doubling the haploid can be improved.
EXAMPLE quadruple maize haploid seedling chromosome doubling
First, reagent preparation
1. Preparation of reagent mother liquor
The same as in example one.
2. Preparation of composite doubling agent
Adding 10L of tap water, well water, river water, canal water or other clear water into a 15L agricultural sprayer, respectively measuring the reagent mother liquor by using a measuring cylinder, adding the reagent mother liquor into the sprayer, adding 75ml of methylaminophosphine, 75ml of nocodazole, 45ml of benazolin, 45ml of chlorpropham, 30ml of trifluralin, 15ml of pencyamine, 15ml of metolachlor and 15ml of pendimethalin, adding 50ml of dimethyl sulfoxide, adding water to 15L scale marks, and uniformly stirring and mixing. The final concentration of the components in the solution is 50 mu M of methylaminophosphine, 50 mu M of nocodazole, 30 mu M of oryzaline, 30 mu M of chlorpropham, 20 mu M of trifluralin, 10 mu M of pentyne-sodium, 10 mu M of metolachlor and 10 mu M of pendimethalin.
3. Preparing an antidote: same as example three;
preparing an ABT rooting powder solution: same as example three;
preparation and pretreatment of plant material to be doubled
Method for obtaining haploid
1. Induction of haploids
Same as the three phases of the example
2. Selection of haploid seeds
After the seeds are mature, the ears are harvested. The haploid and the diploid are distinguished on the fruit cluster according to the color marks on the grains, the seeds with the purple marks are diploid, the seeds without the purple marks can be haploid, and only the haploid seeds are reserved for the next test;
3. planting the obtained haploid seeds in a field, wherein the field management is the same as that of common corn;
triple, double processing and double post processing
1. When the haploidy seeds under sowing emerge and the seedlings grow to 1 leaf and 1 core period, uniformly spraying the haploidy seeds from the upper parts of the seedlings downwards by using a sprayer before sunset every evening in sunny days, and requiring that liquid medicine is fully distributed on the plants, particularly in the corn cores and flows downwards along the seedlings;
2. spraying antidote and rooting powder with sprayer in the morning of the second day;
3. spraying doubling agent every night, spraying antidote and rooting powder the next morning, and repeating the operation until the seedling grows to 6 leaves;
fourth, field verification of doubling effect
Statistics of pollen scattering rate of corn plants in flowering period and fructification rate of corn plants in mature period
After the doubling treatment, the plant is normally subjected to field management, the haploid pollen-loosing plant rate is counted in the flowering period and self-pollinated, and the maturing rate of the haploid plant is counted after the plant is mature. The test result shows that: the haploid multiplying power (powder scattering rate) of the method can reach 60.2 percent, and the average rate is 58.8 percent (different due to different materials); the maturing rate after doubling can reach 45.4%, and the average maturing rate is 43.4% (different due to different materials). Compared with the doubling method adopting colchicine, the method has the advantages that the used medicament compound has small pollution to the environment, low toxicity, low residue and easy degradation, the treated field death rate is low, the abnormal growth rate of plants is low, the operation is simple, the doubling rate is higher, and the method is an effective method for doubling chromosomes.
Results of seedling doubling test
| Repetition of | Treatment of | Number of plants surviving | Number of loose powder plants | Number of fructification plants | Survival rate (%) | Powder scattering ratio (%) | Percentage of fruit set (%) |
| 1 | 260 | 201 | 153 | 115 | 77.3 | 58.8 | 44.2 |
| 2 | 260 | 207 | 149 | 118 | 79.6 | 57.3 | 45.4 |
| 3 | 249 | 199 | 150 | 101 | 79.9 | 60.2 | 40.6 |
| Total up to | 769 | 608 | 452 | 334 | 79.1 | 58.8 | 43.4 |
The antidote used in the invention can also be added into other inorganic and organic nutrient solutions for matching use, including the well-known MS culture solution, Hoagland nutrient solution and various water culture nutrient solutions with various changed compositions, but the core composition is 0.1-2% (W/V, mass percentage content) of potassium phosphate and/or dipotassium hydrogen phosphate and/or potassium dihydrogen phosphate.
The method can effectively double plant chromosomes, but the method still has the potential of improvement and optimization for more plant species, and the improvement and optimization of the method can further improve the doubling effect; modifications to the present method may also be useful for chromosome doubling in animals and fungi that have mitotic and meiotic abilities; and it will be apparent to those skilled in the art that, in light of the teachings of the present invention, modifications and variations can be made in the foregoing teachings and all such modifications and variations are within the purview of the scope of the appended claims.
Claims (1)
1. A composition for doubling chromosome of corn haploid seedling, which consists of a composite doubling agent, an auxiliary agent, an antidote and a growth regulator, and is characterized in that: the compound doubling agent single product is nocodazole, glufosinate-methyl, benazolin, chlorpropham, trifluralin, propyzamide, metolachlor and pendimethalin, and the concentration of the compound doubling agent single product in a final solution is as follows: nocodazole 50 μ M, glufosinate-methyl 50 μ M, benazolin 30 μ M, chlorpropham 30 μ M, trifluralin 20 μ M, propyzamide 10 μ M, metolachlor 10 μ M, pendimethalin 10 μ M; the auxiliary agent is dimethyl sulfoxide and ethylene glycol; the antidote is prepared from 0.2g of monopotassium phosphate and 0.5g of urea in every 100ml of distilled water; the growth regulator is ABT rooting powder No. 6, and the concentration of the ABT rooting powder No. 6 in the final solution is 20 ppm;
the method for doubling the chromosome of the corn haploid seedling by using the composition comprises the following steps: (1) preparing a reagent, namely dissolving a single raw pesticide of the compound preparation in a small amount of dimethyl sulfoxide, adding polyethylene glycol to a constant volume to prepare a high-concentration mother solution, and diluting the mother solution to a final concentration by using water as a solvent when the compound preparation is used; (2) preparation and pretreatment of plant materials to be doubled, (3) doubling and post-doubling: when the haploidy seeds under sowing emerge and the seedlings grow to 1 leaf and 1 core period, uniformly spraying the haploidy seeds downwards from the upper parts of the seedlings by using a sprayer, manpower or machinery before sunset every evening in a sunny day, and requiring that the corn cores are filled with liquid medicine and flow downwards along with the seedlings; secondly, spraying an antidote and rooting powder by a sprayer in the morning of the next day of spraying the doubling agent; thirdly, spraying doubling agent every night, spraying antidote and rooting powder the next morning, and repeating the operation for 1 leaf and 1 core of the seedling until the 6 leaf stage is finished; (4) double effect detection and verification.
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