CN117099681B - Method for doubling corn haploid by using taxol - Google Patents
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- 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
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/06—Processes for producing mutations, e.g. treatment with chemicals or with radiation
- A01H1/08—Methods for producing changes in chromosome number
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/20—Reduction of greenhouse gas [GHG] emissions in agriculture, e.g. CO2
- Y02P60/21—Dinitrogen oxide [N2O], e.g. using aquaponics, hydroponics or efficiency measures
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Abstract
The invention discloses a method for doubling corn haploid by using taxol, which comprises the steps of seed disinfection, seed germination, bud cutting, taxol treatment, bud seedling transplanting and the like, and belongs to the technical field of plant breeding. The invention adopts taxol to double the haploid of corn, the taxol is an orally taken medicine, is basically harmless to human body, and is nontoxic and harmless to human and environment. Compared with the existing chemical method, the method is harmless to the treated plants, dead seedlings and abnormal seedlings are not generated, the survival rate of the treated seedlings after transplanting is very high, a large number of precious haploid seeds are saved for breeding, the breeding efficiency is improved, the doubling success rate of haploid chromosomes is also improved, the harm to the body and the environment of operators is reduced, the labor amount is reduced, and the breeding cost is reduced.
Description
Technical Field
The invention relates to a method for doubling corn haploid by using taxol, belonging to the technical field of plant breeding.
Background
Common crop breeding methods include crossbreeding, mutation breeding, haploid breeding, polyploid breeding, transgenic breeding and the like, wherein haploid breeding has the advantages of shortening the breeding period, improving the breeding efficiency and the like, and is a breeding method which is widely focused or applied by breeding workers in recent years. On corn, a haploid induction line is used for hybridization of a single cross or other female parent materials, so that a relatively large number of haploid grains can be easily obtained, and a haploid breeding method is widely used for breeding new corn varieties by domestic and foreign corn breeding units in recent years.
The corn haploid breeding technology is a breeding method for producing haploid grains by using a haploid induction system and then doubling the chromosomes of the haploid grains to obtain a homozygous inbred line. The corn haploid inducing line is a special corn inbred line, the genome of which contains parthenogenesis promoting genes MATL, zmDMP, zmPLD and the like and kernel chromogenic marker genes R-nj, and the top of the kernel (endosperm crown) and the tip of the embryo (scutellum) are all obviously purplish red, and the corn haploid inducing line is used as a male parent to pollinate a female parent material, and a few percent of individuals in a offspring plant are maternal haploids, namely the corn inbred inducing line only contains a set of chromosomes of the female parent. The maternal haploid becomes homozygous diploid after 'natural chromosome doubling' or 'artificial chromosome doubling', no character separation exists, and a new pure line (inbred line) is formed, and the pure lines are formed by doubling the haploid chromosome, so that the highly homozygous inbred line is technically called doubled haploid (double haploid), the degree of homozygosity of genetic loci reaches 100%, and the inbred line is called DH line (double haploid line).
Haploid inducer-based maize haploid breeding technology has become one of the core technologies for modern maize breeding. The technology breaks through the defects (labor and time consumption, long period, slow progress and the like) of the traditional inbred line breeding method, converts continuous multi-generation inbreeding (bagging inbreeding of more than 7 generations) of the traditional method into induction and doubling of haploid for 2 generations, and can finish the inbred line breeding work, obtain a DH line with high homozygosity and greatly accelerate the breeding process. And the homozygosity of the genetic locus of the DH line reaches 100%, and the homozygosity of the traditional inbred line (F8 generation inbred line) is only 99.2%. The current haploid breeding technology is one of common technologies for corn breeding, the technical process is not complex, the basic process comprises 7 main technical links of breeding material base material preparation, haploid induction (pollinating female parent material with pollen of an induction line), haploid grain selection, chromosome doubling of haploid grains, propagation of DH line, hybrid combination preparation, yield determination test (yield identification test, region test and production demonstration) and the like, wherein 'haploid induction' and 'chromosome doubling of haploid grains' are the two most important technical links and are the core of haploid breeding technology.
There are many methods of chromosome doubling of haploids reported so far, which can be generally divided into two categories, one is natural doubling and the other is artificial doubling (chemical doubling). The haploid plant of the corn is male sterile because the haploid of the corn has only one set of chromosome in the cell, the pollen mother cell can not normally reduce division, and anther/pollen can not normally form. However, the chromosome of part of plants in the field cultivation of haploids can be doubled to form diploid cells, and when flowers are opened, the fertility of tassels is restored or partially restored, female ears can be pollinated to obtain seeds, and the phenomenon is called natural doubling of haploids. The natural doubling percentage of haploids is different according to the sources of the base materials, some doubling rates are high, some doubling rates are low, and the natural doubling rate is generally 1% -2% in general, and the natural doubling rate of materials easy to naturally double can reach 5%. The artificial doubling of haploid chromosomes mainly comprises the steps of treating (soaking) haploid seedling individuals or organs with anti-microtubule medicines, so that the mitosis of stem tip meristematic cells is disturbed, and chromosome doubling occurs. Colchicine was used as a drug for plant chromosome doubling, beginning in the 30 s of the 19 th century, and has been used for nearly one hundred years, and has a very wide range of applications. In recent years, several students reported chromosome doubling of maize haploids by tissue culture. The doubling efficiency is greatly improved due to the use of haploid immature embryo embryogenic callus and the addition of colchicine to the culture medium. But tissue culture doubling has significant drawbacks: high cost, low speed and inability of large-scale treatment of haploid kernels. Haploid seedlings (budding) germinated with colchicine for 2-3 days are currently the most commonly used method, typically using concentrations of 0.06% (equal to 1.5 mM) or higher. Colchicine with high concentration can generate larger toxicity to the processed plant materials, generate higher death rate or abnormal seedling rate, generally has very low survival rate (about 50 percent), has total doubling success rate of about 10 percent, has complex and tedious operation procedure, takes time and labor, has high cost, has larger toxic action to human bodies and is harmful to the environment. In recent years, some researchers use microtubule-resistant herbicide to replace colchicine to double the chromosome of single seed or seedling, the treatment cost is reduced, but the herbicide has higher toxicity to plants, the survival rate of plants is very low (less than 50 percent) when the seedlings or sprouts are treated, and the herbicide has certain toxicity to human bodies and is harmful to the environment.
Paclitaxel is a natural secondary metabolite separated and purified from the bark of the gymnosperm yew, is basically harmless to human body and has little harm to environment. Clinical researches prove that the taxol has good anti-tumor effect and can be used for treating various cancers such as esophagus cancer, bladder cancer, prostate cancer, gastric cancer, ovarian cancer, non-small cell lung cancer, head and neck cancer, breast cancer and the like. Paclitaxel inhibits cancer cells by inhibiting cell mitosis and inducing apoptosis. The invention aims to treat corn haploid by using taxol to force chromosome doubling, and no report of taxol for plant chromosome doubling is currently seen.
Disclosure of Invention
The invention aims to solve the defects of the existing corn haploid chromosome doubling method, and provides a method for doubling corn haploid by using taxol, which has the advantages of simple operation, high doubling success rate, high bud seedling survival rate, no harm to plants, no generation of dead seedlings and abnormal seedlings, and no toxicity or harm to human bodies and the environment.
Technical proposal
A method for doubling corn haploids with paclitaxel comprising the steps of:
(1) Seed disinfection:
Washing and airing the corn haploid grains, soaking the corn haploid grains in sterile water, taking out the corn haploid grains, and cleaning the corn haploid grains with sterile water to obtain sterilized corn haploid grains;
(2) Seed germination:
Taking a sterilized and washed seed germination box, filling wet sterile germination paper on the bottom of the box, filling sterilized corn haploid grains, covering a layer of wet sterile germination paper, and culturing in an incubator at 28 ℃ for 2-6 days;
(3) And (3) sprout collection:
Culturing in incubator for 36 hr, selecting bud seedlings with coleoptile longer than 10mm every 5-6 hr, collecting in seed germination box filled with wet germination paper, covering with a layer of wet sterile germination paper, placing in refrigerator at 10-12deg.C for use, collecting bud seedlings after 3-5 times as first batch of bud seedlings, and collecting later as second batch of bud seedlings;
(4) Cutting buds:
Cutting buds of the first batch of buds to obtain seeds after bud cutting;
(5) Paclitaxel treatment:
Immersing the seeds after bud cutting in taxol emulsion with taxol concentration of 100-400 mu M, sealing, placing in dark at 20-25 ℃ for 8-24h, taking out after finishing and washing with water;
(6) Transplanting the sprouts:
transplanting the cleaned sprouts into seedling raising plug trays, growing to a three-leaf period in a greenhouse, and transplanting to a field for cultivation;
(7) Repeating the steps (4), (5) and (6) on the second batch of sprouts.
Further, in the step (1), the disinfectant is composed of 84 disinfectant and water in a volume ratio of 1:4.
Further, in the step (2), during the cultivation, the moisture in the germination box is checked every 12 hours, and the germination paper is kept wet by spraying and supplementing water.
In the step (3), before the bud cutting treatment, false haploid buds need to be selected, and the roots and the bud tips of the false haploid buds have mauve color.
Further, in the step (4), the bud cutting treatment method comprises the following steps: pinching the seeds by hand, cutting the top end of the coleoptile by a pair of scissors for 1-2mm, and cutting the primary radicle for 8-10mm without cutting the secondary radicle.
Further, in step (5), the paclitaxel emulsion is prepared by the following steps: firstly, adding taxol powder into dimethyl sulfoxide for dissolution to obtain taxol solution, dropwise adding the taxol solution into tween-80 solution with the mass concentration of 0.025% -0.1% while stirring, and finally, fixing the volume by using tween-80 solution with the mass concentration of 0.025% -0.1% to obtain the taxol-containing taxol composite; the volume concentration of dimethyl sulfoxide in paclitaxel emulsion is 2%.
The invention has the beneficial effects that:
1) Compared with the existing chemical method treatment (colchicine or microtubule herbicide resistant treatment), the method provided by the invention is harmless to the treated plants, does not generate dead seedlings and abnormal seedlings, has high survival rate (more than 90%) after transplanting the treated seedlings, saves a large amount of precious haploid seeds for breeding, improves breeding efficiency, and also improves the doubling success rate of haploid chromosomes.
2) The invention adopts the taxol to double the corn haploid, the taxol is an orally taken medicine, is basically harmless to human bodies, is nontoxic and harmless to human bodies and the environment, reduces harm to the bodies and the environment of operators, reduces labor amount and lowers breeding cost compared with the existing chemical treatment.
Drawings
FIG. 1 shows the plant survival rate test results after variety 1 and variety 2 are treated by using the method for doubling corn haploids with paclitaxel;
FIG. 2 shows the results of the percent test of the stimulable plants after treatment of variety 1 and variety 2 using the method of the present invention for doubling maize haploids with paclitaxel.
Detailed Description
The technical scheme of the invention is clearly and completely described below in connection with specific experiments.
In the following experiments, the method for doubling corn haploid by using paclitaxel comprises the following steps:
(1) Seed disinfection:
Washing and airing the corn haploid grains, soaking the corn haploid grains in sterilizing water (consisting of 84 sterilizing liquid and water in a volume ratio of 1:4), taking out the corn haploid grains, and cleaning the corn haploid grains with sterile water to obtain sterilized corn haploid grains;
(2) Seed germination:
Taking a sterilized and washed seed germination box, filling wet sterile germination paper on the bottom of the box, filling sterilized corn haploid grains, covering a layer of wet sterile germination paper, culturing in an incubator at 28 ℃ for 2-6 days, checking the water content in the germination box every 12 hours, and spraying water to keep the germination paper wet;
(3) And (3) sprout collection:
Culturing in incubator for 36 hr, selecting bud seedlings with coleoptile longer than 10mm every 5-6 hr, collecting in seed germination box filled with wet germination paper, covering with a layer of wet sterile germination paper, placing in refrigerator at 10-12deg.C for use, collecting bud seedlings after 3-5 times as first batch of bud seedlings, and collecting later as second batch of bud seedlings;
(4) Cutting buds:
cutting buds of the first batch of buds, and selecting false haploid buds before cutting buds, wherein the false haploid buds are purple at roots and buds tips; when the bud is cut, the seeds are pinched by hands, the top end of the coleoptile is cut by a pair of scissors for 1-2mm, the primary radicle is cut for 8-10mm, and the secondary radicle is not cut to obtain the seeds after bud cutting;
(5) Paclitaxel treatment:
Immersing the seeds after bud cutting in taxol emulsion with taxol concentration of 100-400 mu M, sealing, placing in dark at 20-25 ℃ for 8-24h, taking out after finishing and washing with water;
The preparation method of the paclitaxel emulsion comprises the following steps: firstly, adding taxol powder into dimethyl sulfoxide for dissolution to obtain taxol solution, dropwise adding the taxol solution into tween-80 solution with the mass concentration of 0.025% -0.1% while stirring, and finally, fixing the volume by using tween-80 solution with the mass concentration of 0.025% -0.1% to obtain the taxol-containing taxol composite; the volume concentration of dimethyl sulfoxide in paclitaxel emulsion is 2%.
(6) Transplanting the sprouts:
transplanting the cleaned sprouts into seedling raising plug trays, growing to a three-leaf period in a greenhouse, and transplanting to a field for cultivation;
(7) Repeating the steps (4), (5) and (6) on the second batch of sprouts.
The current colchicine bud leaching method of the corn haploid comprises the following steps:
1) Seed disinfection
And weighing the corn haploid seeds subjected to 2 times of manual selection or counting grains by a grain counter, pouring the corn haploid seeds into a cleaning basin, and flushing with tap water. Taking out, air drying, and pouring into bleaching disinfectant (bleaching powder solution containing 1-2% sodium hypochlorite or 84 disinfectant solution containing 15%) which needs to spread over 3-5cm of seeds, and shaking on a shaking table for 5-10min. After pouring out the bleaching water, washing with sterile water for 3-5 times, each for 3-5min.
2) Seed germination accelerating (optional)
The sterilized and cleaned seeds are poured into proper amount of sterile warm water with the temperature of 45 ℃ for 5-10min, transferred into sterile warm water with the temperature of 28 ℃, and placed into an illumination incubator with the temperature of 28 ℃ for soaking for 4-7h for accelerating germination.
3) Seed germination
The sterilized towel is soaked and wrung (certain moisture is required to be kept) in sterile warm water, the sterilized and washed seeds are spread in sterilized stainless steel square trays, the sterilized and washed seeds are spread on the towel in a dispersing way, the density of the seeds is not too large, the seeds are generally about 1 grain/cm 2, the seeds are covered by the same wet towel, the stainless steel square trays with the same size are covered (moisture evaporation of the towel is reduced), and the towel is placed in an intelligent illumination incubator (28 ℃) for about 24 hours. When the seeds in the stainless steel tray germinate to be exposed (about 30% of the seeds are exposed out of radicle by 0.5-1 mm), the seeds are poured into a clean basin and washed by running water, and then transferred into a germination box for continuous germination. The germination box is internally filled with 2 layers of germination paper (the germination paper can be replaced by paper towel with higher strength when no germination paper exists), the germination paper is soaked with water, seeds are transferred onto the germination paper, and the density is controlled within 0.5 grains/cm 2. The seeds are covered with a layer of wet tissue or towel. The sprouting paper in the box is kept moist, and handkerchief is screwed lightly until no water drops (no water is accumulated in the box). After the germination box is covered (vent hole is reserved), the germination box is placed in a germination incubator (28 ℃) and is continuously cultivated under dark conditions, and the moisture content in the germination box is checked every 12 hours. When the water content is obviously lost, the spray can supplement water.
4) Bud seedling collecting and cleaning
Usually the germination rates of maize haploid seeds are very inconsistent, so it is necessary to sort out the already germinated seeds multiple times. When the embryo (mesocotyl+coleoptile) of the seed is higher than about 8mm, the embryo is picked up in time for standby. The separated seedlings are put into a new sprouting box, and 2 layers of moist sprouting paper are filled in the box. And after the germination time is more than 36 hours, the seedlings with germs longer than 8mm are separated every 5-6 hours. The bud seedlings sorted out each time are placed in a refrigerator (dark condition) with the temperature of 10-12 ℃ for standby. After each sorting, the seeds which do not reach 8mm are put back into a germination box for continuous germination, but each sorting needs to remove the mildewed seeds and is sprayed with sterile water at 28 ℃ for 1-2 minutes. Sorting the collected sprouts 3-5 times as the first bud seedling of doubling treatment, and performing incisor and colchicine treatment. And the bud seedlings sorted out in the following 3 days are subjected to second batch doubling treatment. Sprouts which germinate after 6 days are generally no longer used.
5) First bud cutting
When the bud seedlings collected by sorting reach 50% of seed grain number (generally about 72 hours of total germination time), first incisors are made. False haploid sprouts (purple red at roots and tips is false haploid) are selected before incisors. When cutting buds, the seeds are pinched by hands, the top ends of coleoptile are cut by scissors for 1-2mm, primary radicle is cut for 8-10mm, and secondary radicle is not cut. Carefully placing the seeds with the buds cut into a plastic box, and covering a wet shawl for moisturizing.
6) Colchicine treated sprouts
Washing the cut sprouts with running water in a plastic box, and pouring out water, adding the prepared colchicine solution (0.06% colchicine+0.5% DMSO) into the plastic box, submerging all the sprouts, sealing the container mouth with a plastic film, and performing dark treatment at 20-25 ℃ for 8 hours, wherein the colchicine solution can be reused for 3 times and recovered as toxic and harmful waste liquid after 3 times.
7) Cleaning the treated sprouts
The colchicine-treated sprouts were immediately soaked with clear water for 30min, shaken from time to time, and rinsed 3 times with running water to avoid damaging the sprouts and roots during rinsing, and the Dai Shuangceng rubber glove and gas mask were used to prevent poisoning.
8) Bud seedling transplanting
And (3) conveying the cleaned sprouts to a greenhouse for plug transplanting on the same day, wherein the sprouts can not be transplanted on the same day, and the sprouts can be stored for a plurality of hours after being moisturized by a refrigerator at 10 ℃. Transplanting the seedlings to a field for cultivation after the seedlings grow to a trefoil period in the plug tray.
9) The collected second set of sprouts repeats the first set of treatment: step 5), 6), 7), 8).
Haploid grain chromosome doubling experiments:
Obtaining experimental corn haploid grains: the following 2 hybrids F 1 were used: the anti 273×hd645, (zheng 58×sd 65) ×sd375 is the female parent hybridized with the inducible stock6, 20 kg each of a large number of seeds of 2 IF 1 are obtained, about 1kg each of the haploid seeds of the above 2 female parent materials are picked out according to the top end and tip colors of the seeds), and are respectively called as variety 1 (V1) and variety 2 (V2) for the chromosome doubling experiment of the haploid seeds of the present time.
1. The method for doubling corn haploid by using paclitaxel is adopted to treat variety 1 (V1), and the variety is divided into groups M1-M9 of paclitaxel treatment according to the concentration of paclitaxel emulsion and the treatment time, and the specific treatment conditions are shown in Table 1. Meanwhile, the current chromosome doubling method 'colchicine germination method' in the field is used as a colchicine treatment group for comparison, and seedlings with 2-3 normal seeds germinating are germinated by sterile water for 8 hours as a control group of a full experiment. When the seedlings grow to a leaf period, counting the number of the survival seedlings, counting the number of the survival plants, the number of the double plants and the number of the anther plug-in plants in the flowering period, and calculating the survival percentage of the plants, the percentage of the double plants and the percentage of the anther plug-in plants.
Wherein, 1) the doubling plant percentage is equal to the scattered powder plant percentage, which is equal to the percentage of the large scattered powder plants and the small scattered powder plants in the survival plants. When the number of loose powder spikelets is less than 25% of the total number of tassels, it is called a small number of loose powder plants, and when the number of loose powder spikelets is greater than 25%, it is called a large number of loose powder plants. 2) The percent of anther plug-in refers to the percentage of plants that exhibit the "anther plug-in" phenomenon relative to the number of surviving plants. Plants with "anther plug-in" include three plant types, namely anther plug-in non-loose powder, anther plug-in small amount of loose powder and anther plug-in large amount of loose powder. The percent anther plug-in is the percentage of the sum of the three plants to the total surviving plants.
The experimental results are shown in Table 2.
TABLE 1 treatment conditions for paclitaxel treatment M1 to M9 groups
| Process name | Processing time | Paclitaxel emulsion composition |
| Paclitaxel treatment M1 group | 8h | Paclitaxel 100. Mu.M+0.025% Tween-80+2% DMSO |
| Paclitaxel treatment group M2 | 8h | Paclitaxel 200 μM+0.050% Tween-80+2% DMSO |
| Paclitaxel treatment M3 group | 8h | Paclitaxel 400. Mu.M+0.1% Tween-80+2% DMSO |
| Paclitaxel treatment M4 group | 16h | Paclitaxel 100. Mu.M+0.025% Tween-80+2% DMSO |
| Paclitaxel treatment group M5 | 16h | Paclitaxel 200 μM+0.050% Tween-80+2% DMSO |
| Paclitaxel treatment M6 group | 16h | Paclitaxel 400. Mu.M+0.1% Tween-80+2% DMSO |
| Paclitaxel treatment M7 group | 24h | Paclitaxel 100. Mu.M+0.025% Tween-80+2% DMSO |
| Paclitaxel treatment group M8 | 24h | Paclitaxel 200 μM+0.050% Tween-80+2% DMSO |
| Paclitaxel treatment M9 group | 24h | Paclitaxel 400. Mu.M+0.1% Tween-80+2% DMSO |
TABLE 2 results of doubling effect test of maize haploid seedlings from different paclitaxel-treated groups and colchicine-treated groups
2. The method for doubling corn haploids by using the taxol is adopted to treat the variety 1 and the variety 2 as taxol treatment groups, wherein the taxol treatment solution comprises the following components: paclitaxel 400 μM+0.1% Tween-80+2% DMSO for 24h; meanwhile, the current chromosome doubling method 'colchicine bud leaching method' in the field is used as a colchicine treatment group for comparison, the seedlings with 2-3 normal sprouting of seeds are used as a comparison, the seedlings are transplanted to a field for 14 days by using sterile water bud leaching for 8 hours, the survival rate of the seedlings is counted, the number of the anther which is not scattered, the number of the anther which is slightly scattered and the number of the anther which is greatly scattered are counted in the flowering period, the three are added together to be compared with the total survival plant number, the anther plant percentage is calculated, the two are added together to be compared with the total survival plant number, and the scattered plant percentage is calculated.
The results of the seedling survival rate test are shown in FIG. 1, and the results of the scattered plant percentage test are shown in FIG. 2. As can be seen from FIG. 1, the survival rate of the maize haploid grain after being treated by the taxol is about 70%, and only about 50% of the plants treated by the colchicine survive, and the survival rate of the maize haploid grain is more than 95% compared with that of the control group, so that the maize haploid grain shows that the colchicine has great toxicity to seedlings, and about 50% of the plants survive after being transplanted, namely the dead plants reach about 50%, and the taxol-treated plants have much less toxicity to the seedlings, and more than 70% of the plants survive after being transplanted for 2 weeks. As can be seen from FIG. 2, the control group had only about 3% of chromosome doubling plants, which were caused by natural chromosome doubling. The chromosome doubling rate can be obviously improved by the paclitaxel treatment and the colchicine treatment, the doubling effect of the paclitaxel is larger than that of the colchicine for the variety 1, and the effect of the paclitaxel treatment is almost equal to that of the colchicine for the variety 2, so that the chromosome doubling caused by the paclitaxel treatment is different from variety to variety. In summary, the doubling effect of taxol treatment on corn chromosomes is better than or equal to that of colchicine treatment, so that the corn chromosome doubling treatment is carried out, and taxol is an excellent substitute of colchicine.
Claims (5)
1. A method for doubling corn haploid by using paclitaxel, which is characterized by comprising the following steps:
(1) Seed disinfection:
Washing and airing the corn haploid grains, soaking the corn haploid grains in sterile water, taking out the corn haploid grains, and cleaning the corn haploid grains with sterile water to obtain sterilized corn haploid grains;
(2) Seed germination:
Taking a sterilized and washed seed germination box, filling wet sterile germination paper on the bottom of the box, filling sterilized corn haploid grains, covering a layer of wet sterile germination paper, and culturing in an incubator at 28 ℃ for 2-6 days;
(3) And (3) sprout collection:
Culturing in incubator for 36 hr, selecting bud seedlings with coleoptile longer than 10mm every 5-6 hr, collecting in seed germination box filled with wet germination paper, covering with a layer of wet sterile germination paper, placing in refrigerator at 10-12deg.C for use, collecting bud seedlings after 3-5 times as first batch of bud seedlings, and collecting later as second batch of bud seedlings;
(4) Cutting buds:
Cutting buds of the first batch of buds to obtain seeds after bud cutting;
(5) Paclitaxel treatment:
immersing the seeds after bud cutting in taxol emulsion with taxol concentration of 100-400 mu M, sealing, placing in dark at 20-25 ℃ for 16-24h, taking out after finishing and washing with water;
(6) Transplanting the sprouts:
transplanting the cleaned sprouts into seedling raising plug trays, growing to a three-leaf period in a greenhouse, and transplanting to a field for cultivation;
(7) Repeating the steps (4), (5) and (6) on the second batch of sprouts;
In the step (5), the preparation method of the paclitaxel emulsion comprises the following steps: firstly, adding taxol powder into dimethyl sulfoxide for dissolution to obtain taxol solution, dropwise adding the taxol solution into tween-80 solution with the mass concentration of 0.025% -0.1% while stirring, and finally, fixing the volume by using tween-80 solution with the mass concentration of 0.025% -0.1% to obtain the taxol-containing taxol composite; the volume concentration of dimethyl sulfoxide in paclitaxel emulsion is 2%.
2. The method of doubling corn haploid with paclitaxel according to claim 1, characterized in that in step (1), the sterilizing water consists of 84 sterilizing liquid and water in a volume ratio of 1:4.
3. The method for doubling corn haploid by paclitaxel according to claim 1, characterized in that in step (2), the moisture in the germination box is checked every 12 hours during the cultivation, and the germination paper is kept wet by spraying and moisturizing.
4. The method for doubling corn haploid by paclitaxel according to claim 1, characterized in that in step (3), false haploid sprouts are selected before the bud cutting treatment, and the roots and the tips of the sprouts have mauve color and are false haploids.
5. The method for doubling corn haploids with paclitaxel according to claim 1, wherein in step (4), the method for cutting buds is as follows: pinching the seeds by hand, cutting the coleoptile tip by scissors to 1-2 mm, and cutting the primary radicle by 8-10mm without cutting the secondary radicle.
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