CN117099681B - Method for doubling corn haploid by using taxol - Google Patents

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

A method for doubling corn haploid using paclitaxel

Technical Field

The invention relates to a method for doubling corn haploid by utilizing paclitaxel, and belongs to the technical field of plant breeding.

Background technique

Common crop breeding methods include hybrid breeding, mutation breeding, haploid breeding, polyploid breeding, transgenic breeding, etc. Among them, haploid breeding has the advantages of shortening the breeding period and improving breeding efficiency, and is a breeding method that has attracted extensive attention or application by breeders in recent years. In corn, it is easy to obtain more haploid grains by using "haploid induction lines" to cross single crosses or other maternal materials. Therefore, in recent years, domestic and foreign corn breeding units have widely used haploid breeding methods to select new corn varieties.

Corn haploid breeding technology is a breeding method that uses haploid induction lines to produce haploid grains and then doubles its chromosomes to obtain homozygous inbred lines. Corn haploid induction lines are a special type of corn inbred line, and their genome contains genes that promote parthenogenesis, such as MATL, ZmDMP, and ZmPLD3, and the grain color marker gene R-nj. Usually, the top of the grain (endosperm crown) and the embryo tip (scutellum) are both obviously purple-red. When it is used as a male parent to pollinate a certain female parent material, a few percent of the offspring plants are maternal haploids, that is, they only contain one set of chromosomes from the mother parent. This maternal haploid becomes a homozygous diploid after "natural chromosome doubling" or "artificial chromosome doubling", with no more trait separation, and becomes a new pure line (inbred line). Because these pure lines are formed by doubling the chromosomes of haploids, this highly homozygous inbred line is academically called a double haploid. The homozygosity of its genetic loci reaches 100%, and it is commonly known as the DH line (doublehaploid line).

The haploid breeding technology of corn based on haploid induction lines has become one of the core technologies of modern corn breeding. This technology breaks through the shortcomings of the traditional inbred line breeding method (labor-intensive, time-consuming, long cycle, slow progress, etc.), and transforms the traditional method of "continuous multi-generation self-pollination" (more than 7 generations of bagging self-pollination) into "induction and doubling haploids". The inbred line breeding work can be completed in 2 generations, and a high-purity DH line is obtained, which greatly accelerates the breeding process. Moreover, the genetic loci of the DH line are 100% pure, while the purity of the traditional inbred line (F8 generation inbred line) is only 99.2%. Currently, haploid breeding technology has become one of the commonly used technologies in corn breeding. Its technical process is not complicated. Its basic process includes seven major technical links: preparation of basic materials for breeding materials, induction of haploids (pollinating maternal materials with pollen from the induced line), selection of haploid grains, chromosome doubling of haploid grains, reproduction of DH lines, preparation of hybrid combinations, and yield measurement tests (yield identification tests, regional tests, and production demonstrations). Among them, "induction of haploids" 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 for doubling haploid chromosomes that have been reported, which can be generally divided into two categories: natural doubling and artificial doubling (chemical doubling). Since the haploid corn has only one set of chromosomes in its cells, its pollen mother cells cannot undergo normal reduction division, and anthers/pollen cannot be formed normally, so the haploid corn plants are all male sterile. However, the chromosomes of some haploid plants will double in field cultivation to form diploid cells. When the male ear fertility is restored or partially restored during flowering, the female ear can be pollinated to obtain seeds. This phenomenon is called the natural doubling of haploids. The natural doubling percentage of haploids varies depending on the source of the basic material. Some have a higher doubling rate, while others have a very low doubling rate. In general, the natural doubling rate is generally 1%-2%. The natural doubling rate of materials that are easy to naturally double can reach 5%. Artificial doubling of haploid chromosomes mainly involves treating (immersing) haploid seedling individuals or organs with some anti-microtubule drugs, so that the mitosis of the stem apical meristem cells is disturbed, and then chromosome doubling occurs. The use of colchicine as a drug for doubling plant chromosomes began in the 1830s, and it has been used for nearly a hundred years, and its application range is very wide. In recent years, many scholars have reported on the use of tissue culture to double the chromosomes of corn haploids. Due to the use of haploid embryonic callus tissue and the addition of colchicine to the culture medium, the doubling efficiency is greatly improved. However, the doubling of tissue culture has obvious disadvantages: high cost, slow speed, and inability to process haploid grains on a large scale. At present, the most commonly used method is to treat haploid seedlings that have germinated for 2-3 days with colchicine (bud dipping method), generally using 0.06% (equal to 1.5mM) or higher concentrations. Such a high concentration of colchicine will cause great toxicity to the plant materials being treated, resulting in a high rate of seedling death or deformed seedlings, a low survival rate of general sprouts (about 50%), and a total doubling success rate of only about 10%. In addition, the operation procedure is complicated and cumbersome, time-consuming and costly, and this drug has a large toxic effect on the human body and is also harmful to the environment. In recent years, some researchers have replaced colchicine with anti-microtubule herbicides to double the chromosomes of haploid grains or seedlings, and the processing cost has decreased, but the herbicide is more toxic to plants. When processing sprouts or seedlings, the survival rate of the plants is very low (less than 50%), and the herbicide has a certain toxicity to the human body and is also harmful to the environment.

Paclitaxel is a natural secondary metabolite separated and purified from the bark of the gymnosperm yew. It is basically harmless to the human body and has little harm to the environment. Clinical studies have shown that paclitaxel has a good anti-tumor effect and can be used for the treatment of various cancers such as esophageal cancer, bladder cancer, prostate cancer, gastric cancer, ovarian cancer, non-small cell lung cancer, head and neck cancer, and breast cancer. The effect of paclitaxel in inhibiting cancer cells is achieved by inhibiting cell mitosis and inducing apoptosis of cancer cells. The present invention intends to use paclitaxel to treat corn haploids to force their chromosomes to double. There is currently no report on paclitaxel being used for plant chromosome doubling.

Summary of the invention

The purpose of the present invention is to solve the shortcomings of the existing method for doubling the haploid chromosome of corn, and to provide a method for doubling the haploid chromosome of corn by using paclitaxel. The method is simple to operate, has a high doubling success rate, a high survival rate of sprouts, is harmless to plants, does not produce dead and deformed seedlings, and is non-toxic and harmless to humans and the environment.

Technical solutions

A method for doubling corn haploid using paclitaxel comprises the following steps:

(1) Seed disinfection:

The haploid corn kernels are rinsed and dried, then soaked in sterile water, taken out and cleaned with sterile water to obtain the sterilized haploid corn kernels;

(2) Seed germination:

Place a sterilized and cleaned seed germination box on the bottom, place sterilized haploid corn kernels in it, cover it with a layer of moist sterile germination paper, and place it in a 28°C incubator for 2-6 days;

(3) Collection of sprouts:

After culturing in the incubator for 36 hours, the seedlings with coleoptiles longer than 10 mm were selected every 5-6 hours, collected in a seed germination box lined with moist germination paper, covered with a layer of moist sterile germination paper, and placed in a refrigerator at 10-12°C for standby use. The seedlings collected after 3-5 times were regarded as the first batch of seedlings, and the seedlings collected later were regarded as the second batch of seedlings.

(4) Cutting sprouts:

Cutting the first batch of sprouts to obtain cut sprout seeds;

(5) Paclitaxel treatment:

The cut seeds are immersed in a paclitaxel emulsion with a paclitaxel concentration of 100-400 μM, sealed and placed in the dark at 20-25° C. for 8-24 hours, and then taken out and washed with water;

(6) Transplanting of sprouts and seedlings:

The cleaned seedlings are transplanted into seedling trays, grown in a greenhouse to the three-leaf stage, and then transplanted to the field for cultivation;

(7) Repeat steps (4), (5) and (6) with the second batch of sprouts.

Furthermore, in step (1), the disinfectant water is composed of 84 disinfectant and water in a volume ratio of 1:4.

Furthermore, in step (2), during the culture process, the moisture content in the germination box is checked every 12 hours, and water is sprayed to keep the germination paper moist.

Furthermore, in step (3), before the bud cutting process, pseudohaploid seedlings need to be selected, and those with purple-red roots and bud tips are pseudohaploids.

Further, in step (4), the method for cutting the buds is: pinching the seeds by hand, cutting off 1-2 mm of the top of the coleoptile with scissors, and cutting off 8-10 mm of the primary radicle, without cutting the secondary radicle.

Furthermore, in step (5), the preparation method of the paclitaxel emulsion is as follows: first, paclitaxel powder is added to dimethyl sulfoxide to dissolve to obtain a paclitaxel solution, the paclitaxel solution is added dropwise to a Tween-80 solution with a mass concentration of 0.025%-0.1% while stirring, and finally the volume is fixed with a Tween-80 solution with a mass concentration of 0.025%-0.1% to obtain the paclitaxel emulsion; the volume concentration of dimethyl sulfoxide in the paclitaxel emulsion is 2%.

Beneficial effects of the present invention:

1) The present invention provides a method for doubling corn haploids using paclitaxel. Compared with the current chemical treatment (colchicine or anti-microtubule herbicide treatment) for doubling corn haploids, the method of the present invention is harmless to the treated plants, does not produce dead and deformed seedlings, and has a high survival rate (>90%) after transplanting the treated seedlings, thereby saving a large amount of precious haploid seeds for breeding, improving breeding efficiency, and also improving the success rate of haploid chromosome doubling.

2) The present invention uses paclitaxel to double the haploid of corn. Paclitaxel is an orally administrable drug, which is basically harmless to the human body and non-toxic to humans and the environment. Compared with the current chemical treatment, it reduces the harm to the operator's body and the environment, reduces the labor and reduces the breeding cost.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a test result of plant survival rate of Variety 1 and Variety 2 after being treated with the method of doubling corn haploid using paclitaxel of the present invention;

FIG. 2 is a test result of the percentage of pollen-shedding plants of Variety 1 and Variety 2 after being treated with the method of doubling corn haploid with paclitaxel according to the present invention.

Detailed ways

The technical solution of the present invention is clearly and completely explained below in conjunction with specific experiments.

In the following experiment, the method of doubling maize haploid using paclitaxel of the present invention comprises the following steps:

(1) Seed disinfection:

The corn haploid kernels are rinsed and dried, and then immersed in disinfectant water (composed of 84 disinfectant and water in a volume ratio of 1:4), and then taken out and washed with sterile water to obtain the disinfected corn haploid kernels;

(2) Seed germination:

Place a sterilized and cleaned seed germination box on the bottom, place the sterilized haploid corn kernels in it, cover it with a layer of moist sterile germination paper, and place it in a 28°C incubator for 2-6 days. Check the moisture in the germination box every 12 hours, and spray water to keep the germination paper moist.

(3) Collection of sprouts:

After culturing in the incubator for 36 hours, the seedlings with coleoptiles longer than 10 mm were selected every 5-6 hours, collected in a seed germination box lined with moist germination paper, covered with a layer of moist sterile germination paper, and placed in a refrigerator at 10-12°C for standby use. The seedlings collected after 3-5 times were regarded as the first batch of seedlings, and the seedlings collected later were regarded as the second batch of seedlings.

(4) Cutting sprouts:

The first batch of seedlings are cut into buds. Before the cutting process, pseudohaploid seedlings need to be selected. Those with purple-red roots and bud tips are pseudohaploids. When cutting the buds, pinch the seeds with your hands, cut off 1-2 mm of the top of the coleoptile with scissors, and cut off 8-10 mm of the primary radicle, without cutting the secondary radicle to obtain the cut seeds.

(5) Paclitaxel treatment:

The cut seeds are immersed in a paclitaxel emulsion with a paclitaxel concentration of 100-400 μM, sealed and placed in the dark at 20-25° C. for 8-24 hours, and then taken out and washed with water;

The preparation method of the paclitaxel emulsion comprises the following steps: firstly, adding paclitaxel powder into dimethyl sulfoxide to dissolve to obtain a paclitaxel solution; then, adding the paclitaxel solution dropwise into a Tween-80 solution with a mass concentration of 0.025%-0.1% while stirring; and finally, fixing the volume with the Tween-80 solution with a mass concentration of 0.025%-0.1% to obtain the paclitaxel emulsion; the volume concentration of the dimethyl sulfoxide in the paclitaxel emulsion is 2%.

(6) Transplanting of sprouts and seedlings:

The cleaned seedlings are transplanted into seedling trays, grown in a greenhouse to the three-leaf stage, and then transplanted to the field for cultivation;

(7) Repeat steps (4), (5) and (6) with the second batch of sprouts.

The current colchicine bud dipping method for maize haploids includes the following steps:

1) Seed disinfection

Weigh the haploid maize seeds that have been manually selected twice or count them with a grain counter, pour them into a washing basin and rinse with tap water. Take them out, dry them slightly, pour them into bleach disinfectant water (bleaching powder solution containing 1-2% sodium hypochlorite or 15% 84 disinfectant), the disinfectant water needs to cover the seeds by 3-5cm, and place them on a shaker and shake for 5-10 minutes. After pouring out the bleach, wash them with sterile water 3-5 times, each time for 3-5 minutes.

2) Seed germination (optional)

Pour the disinfected and washed seeds into an appropriate amount of 45°C sterile warm water for 5-10 minutes, transfer to 28°C sterile warm water, and soak in a 28°C light incubator for 4-7 hours for germination.

3) Seed germination

Soak the sterilized towel in sterile warm water and wring it dry (keep a certain amount of moisture), spread it flat on a sterilized stainless steel square plate, and spread the sterilized and cleaned seeds on the towel. The seed density should not be too large, generally about 1 seed/ ^cm2 . Cover it with the same wet towel and cover it with a stainless steel square plate of the same size (to reduce the evaporation of water from the towel), and then place it in an intelligent light incubator (28℃) for about 24 hours. When the seeds in the stainless steel plate germinate to white (about 30% of the seeds expose 0.5-1mm of the radicle), pour them into a clean basin and wash them with running water, and then transfer them to the germination box to continue germination. Put 2 layers of germination paper in the germination box (if there is no germination paper, it can be replaced with stronger paper towels), soak the germination paper with water, transfer the seeds to the germination paper, and control the density within 0.5 seeds/ ^cm2 . Cover the seeds with a layer of wet thin square towel (handkerchief) or paper towel. Keep the germination paper in the box moist and twist the handkerchief gently until no water drips (no water accumulates in the box). After the germination box is covered (with ventilation holes), place it in a germination incubator (28°C) and continue to cultivate in the dark. Check the moisture content in the germination box every 12 hours. Spray to replenish water when water loss is obvious.

4) Collection and cleaning of sprouts

Usually, the germination rate of haploid maize seeds is very inconsistent, so it is necessary to sort out the germinated seeds several times. When the embryo (mesocotyl + coleoptile) of the seeds is higher than about 8mm, it needs to be sorted out in time for use. The sorted seedlings are placed in a new germination box with 2 layers of moist germination paper. After the germination time is greater than 36h, the germination seedlings with embryos longer than 8mm are sorted every 5-6 hours. The germination seedlings sorted out each time are placed in a refrigerator (dark conditions) at 10-12℃ for use. After each sorting, those that do not reach 8mm are put back into the germination box to continue germination, but each sorting needs to remove moldy kernels and rinse with 28℃ sterile water for 1-2 minutes. The germination seedlings collected after 3-5 sortings are used as the first batch of double-treated germination seedlings for incision and colchicine treatment. The germination seedlings sorted out 3 days later are used for the second batch of double treatment. Germination seedlings that germinate after 6 days are generally no longer used.

5) First bud cutting

When the number of seeds in the sorted and collected seedlings reaches 50% (generally, the total germination time is about 72 hours), the first incision is made. Before incision, select the pseudohaploid seedlings (the roots and bud tips are purple-red, which are pseudohaploids). When cutting the buds, pinch the seeds with your hands, cut off the top 1-2mm of the coleoptile with scissors, and cut off the primary radicle 8-10mm, without cutting the secondary radicle. Carefully put the cut seeds into a plastic box and cover with a wet towel to keep them moist.

6) Colchicine treatment of sprouts

Wash the cut sprouts with running water in a plastic box (, and pour out the water. Add the prepared colchicine solution (0.06% colchicine + 0.5% DMSO) into the plastic box, and the solution should submerge all the sprouts. Then seal the container mouth with plastic film, treat it in the dark at 20-25℃, and soak it for 8 hours. The colchicine solution can be reused 3 times, and after 3 times, it can be recycled as toxic and hazardous waste liquid.

7) Wash the processed sprouts

The sprouts treated with colchicine were immediately soaked in clean water for 30 minutes, shaken from time to time, and then washed with running water for 3 times. Avoid damaging the sprouts and roots during the washing process. Wear double rubber gloves and a gas mask during operation to prevent poisoning.

8) Transplanting of sprouts

The cleaned seedlings are sent to the greenhouse for transplanting on the same day. If they cannot be transplanted on the same day, they can be stored in a 10℃ refrigerator for several hours to keep them moist. When the seedlings grow to the three-leaf stage in the plug tray, they are transplanted to the field for cultivation.

9) Repeat the treatment process of the first batch of collected sprouts: steps 5), 6), 7), and 8).

Haploid grain chromosome doubling experiment:

Obtaining haploid corn grains for the experiment: The following two hybrid _F1s : Kang 273×HD645 and (Zheng 58×SD65)×SD375 were used as female parents and crossed with inductive stock6, and a large number of seeds of two _IF1s were obtained, each weighing more than 20 kg. According to the color of the grain top and embryo tip, haploid grains of the above two female materials, about 1 kg each, were selected, respectively called variety 1 (V1) and variety 2 (V2), for this haploid grain chromosome doubling experiment.

1. The method of doubling corn haploids using paclitaxel of the present invention was used to treat variety 1 (V1), and the paclitaxel treatment groups M1 to M9 were divided according to the different concentrations of paclitaxel emulsions and the different treatment times. The specific treatment conditions are shown in Table 1. At the same time, the current chromosome doubling method "colchicine bud soaking method" in the field was used as the colchicine treatment group for comparison, and the seedlings with normal germination of 2-3 grains were soaked in sterile water for 8 hours as the control group of the whole experiment. When the seedlings grew to the one-leaf stage, the number of surviving seedlings was counted, and the number of surviving plants, the number of doubled plants, and the number of plants with anther hanging were counted during the flowering period, and the percentage of plant survival, the percentage of doubled plants, and the percentage of plants with anther hanging were calculated.

Among them, 1) The percentage of doubled plants is equal to the percentage of plants shedding pollen, which is equal to the percentage of plants shedding a lot of pollen and a little pollen to the surviving plants. When the number of shed pollen spikelets is less than 25% of the total number of spikelets in the tassel, it is called a plant shedding a little pollen, and when the number of spikelets that can shed pollen is greater than 25%, it is called a plant shedding a lot of pollen. 2) The percentage of anther hanging refers to the percentage of plants with "anther hanging" phenomenon to the number of surviving plants. Plants with "anther hanging" include three types of plants: anther hanging without shedding pollen, anther hanging with a little shedding pollen, and anther hanging with a lot of shedding pollen. The percentage of anther hanging is the percentage of the sum of the above three types of plants to all surviving plants.

The experimental results are shown in Table 2.

Table 1 Treatment conditions of paclitaxel-treated groups M1 to M9

Processing Name Processing time Paclitaxel emulsion ingredients Paclitaxel-treated M1 group 8h Paclitaxel 100 μM + 0.025% Tween-80 + 2% DMSO Paclitaxel-treated M2 group 8h Paclitaxel 200 μM + 0.050% Tween-80 + 2% DMSO Paclitaxel-treated M3 group 8h Paclitaxel 400 μM + 0.1% Tween-80 + 2% DMSO Paclitaxel-treated M4 group 16h Paclitaxel 100 μM + 0.025% Tween-80 + 2% DMSO Paclitaxel-treated M5 group 16h Paclitaxel 200 μM + 0.050% Tween-80 + 2% DMSO Paclitaxel-treated M6 group 16h Paclitaxel 400 μM + 0.1% Tween-80 + 2% DMSO Paclitaxel-treated M7 group 24h Paclitaxel 100 μM + 0.025% Tween-80 + 2% DMSO Paclitaxel-treated M8 group 24h Paclitaxel 200 μM + 0.050% Tween-80 + 2% DMSO Paclitaxel-treated M9 group 24h Paclitaxel 400 μM + 0.1% Tween-80 + 2% DMSO

Table 2 The doubling effect test results of maize haploid seedlings in different paclitaxel treatment groups and colchicine treatment groups

2. The method of doubling corn haploid by using paclitaxel of the present invention is used to treat Variety 1 and Variety 2 as the paclitaxel treatment group, wherein the composition of the paclitaxel treatment solution is: paclitaxel 400 μM + 0.1% Tween-80 + 2% DMSO, and the treatment time is 24 hours; at the same time, the current chromosome doubling method "colchicine bud soaking method" in the art is used as the colchicine treatment group for comparison, and the seedlings with normal germination of 2-3 grains are soaked in sterile water for 8 hours as a control, and the survival rate of the seedlings is counted 14 days after transplanting to the field, and the number of anthers that do not shed powder, the number of anthers that shed a small amount of powder, and the number of anthers that shed a large amount of powder are counted during the flowering period, and the three are added together and compared with the total number of surviving plants to calculate the percentage of plants with anthers shed powder, and the latter two are added together and compared with the total number of surviving plants to calculate the percentage of plants that can shed powder.

Seedling survival test results are shown in Figure 1, and the test results of the percentage of plants that can shed pollen are shown in Figure 2. As can be seen from Figure 1, the survival rate of maize haploid kernels after paclitaxel treatment is about 70%, while only about 50% of the plants treated with colchicine survive, compared with the survival rate of the control group of more than 95%. The results show that colchicine is highly toxic to seedlings. Two weeks after transplanting, only about 50% survived, that is, about 50% of the dead plants, while paclitaxel treatment is much less toxic to seedlings. Two weeks after transplanting, more than 70% of the plants can survive. As can be seen from Figure 2, only about 3% of the chromosomes in the control group have doubled plants, which is caused by natural chromosome doubling. Both paclitaxel treatment and colchicine treatment can significantly increase the chromosome doubling rate. For variety 1, the doubling effect of paclitaxel is greater than that of colchicine. For variety 2, the effect of paclitaxel treatment is similar to that of colchicine, indicating that the chromosome doubling caused by paclitaxel treatment varies from variety to variety. In conclusion, the doubling effect of paclitaxel on maize chromosomes was better than or equal to that of colchicine. Therefore, paclitaxel is an excellent substitute for colchicine for maize chromosome doubling.

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.