CN108513908B - Method for cultivating tetraploid loose-leaved Chinese cabbage - Google Patents
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Abstract
The invention discloses a method for cultivating tetraploid loose-leaved Chinese cabbage, which comprises the steps of dripping a cell microtubule inhibitor amisulin mixed aqueous solution into growth points of seedlings of the loose-leaved Chinese cabbage to double cell chromosomes, identifying stomata in the seedling stage, identifying pollen in the flowering stage, identifying by a flow cytometer, and screening to obtain tetraploid plants. Meanwhile, Tween 80 and Silwet L-77 are added into the aqueous solution of the asulam, the Tween 80 can enhance the solubility of the asulam and improve the stability of the aqueous solution of the asulam, and the Silwet L-77 can greatly reduce the surface tension of water and is beneficial to the adhesion of the solution and the leaf surface, thereby improving the tetraploid mutagenesis rate. The method utilizes the amisulpride mixed aqueous solution to inhibit the formation of spindle yarn at the later stage of cell mitosis, thereby realizing chromosome doubling and providing technical support for creating rich new germplasm of non-heading Chinese cabbage tetraploid and cultivating new variety of tetraploid.
Description
Technical Field
The invention belongs to the technical field of genetic breeding, and particularly relates to a method for cultivating tetraploid loose-leaved Chinese cabbage.
Background
Chinese cabbage without heading (Brassica cappestris ssp.chinensis) Also called pakchoi, is a brassica plant of the family brassicaceae, a primary or secondary herbaceous plant. The plants are short and small, have shallow root systems and developed fibrous roots. The non-heading Chinese cabbage is originated from China and is rich in minerals and vitamins. The ploidy breeding can make the plant genome multiply to obtain the variety with vigorous growth vigor, strong adaptability and good stress resistance. Tetraploid is a tetraploid plant which contains four chromosome groups in somatic cells and can obtain high-quality, high-yield and high-resistance tetraploid plants through artificial induction, breeding and cultivation.
The cell microtubule inhibitor Oryzalin as colchicine substitute can inhibit the formation of spindle yarn in the late mitosis, realize chromosome doubling, and make up the defect of high toxicity caused by colchicine induction. However, when used directly, sulfamethoxazole has low water solubility and is difficult to stay on the leaf surface. Therefore, a set of technical method for inducing the non-heading Chinese cabbage by using the amisulpride is necessary to be established, and abundant new tetraploid germplasm is provided for breeding and cultivating new tetraploid varieties.
Disclosure of Invention
The invention aims to provide a method for cultivating tetraploid loose-leaved Chinese cabbage, which adopts the cooperation of wetting dispersant and amisulide, and has the advantages of simple operation, low toxicity and high efficiency.
In order to solve the technical problems, the invention is realized by the following technical scheme:
a method for cultivating tetraploid loose-head Chinese cabbage comprises the following steps:
step 1, selecting full non-heading Chinese cabbage seeds harvested in the current year, and sowing and raising seedlings according to a conventional technology;
step 2, dripping seedling growing points by using a amisulpride mixed aqueous solution at each point in the morning and at each point in the evening when the cotyledon of the seedling is completely unfolded, continuously treating for 5 times, and taking dripped distilled water as a reference;
step 3, after the plants are grown, performing morphological identification, taking the lower epidermis of the 5 th to 10 th true leaves of the suspected plant, performing stomata identification, and selecting the plant with the significantly enlarged stomata as a tetraploid variation primary selection plant;
step 4, entering a flowering phase, taking the tetraploid variation primary selected plants for pollen identification, and further screening to obtain plants with obviously increased and deformed pollen as tetraploid variation suspected plants;
step 5, taking the tender leaves of the tetraploid variant suspected strain to perform nuclear DNA content flow cytometry analysis, and finally confirming to obtain the tetraploid plant;
the mixed aqueous solution of the amisulpride is a mixed aqueous solution of the amisulpride, Tween 80 and an organosilicon surfactant Silwet L-77.
Further, the concentration of the amisulpride in the amisulpride mixed water solution is 0.06-0.10 mmol.L-1。
Further, the concentration of Tween 80 in the amisulpride mixed water solution is 0.02% v/v, and the concentration of the organosilicon surfactant Silwet L-77 is 0.02% v/v. The concentration of the two is too low, the treatment effect is slight, and the concentration is too high, the plant can be poisoned.
Further, the preparation process of the amisulpride mixed aqueous solution is as follows: dissolving amisulben in 15ml ethanol, dripping the solution into distilled water, shaking the distilled water continuously during the dripping process to ensure that no medicament is separated out, and obtaining the concentration of 0.06-0.10 mmol.L-1Adding Tween 80 and Silwet L-77 into the aqueous solution of benazolin, and shaking up to obtain the final product.
Further, in the morphological identification in the step 3, the leaf length and width, whether the leaf surface is twisted, the stem thickness, the plant height and the crown width are used as indexes for screening and identification.
Further, the pore identification process in step 3 is as follows: measuring the length and width of the stomata under a microscope, calculating the circumference of the stomata according to an ellipse circumference formula, and measuring 30-50 stomata for each plant; and (3) determining whether the stomatal perimeter of the plant is different from the stomatal perimeter of a control plant by adopting a statistical T test method, wherein the plant with the stomatal significantly larger than the control plant is a tetraploid initially-selected variant plant.
Further, the process of identifying pollen in step 4 is as follows: comparing the size and shape of the pollen grains under a microscope, and determining whether the shape of the pollen grains of the plant is different from the shape of the pollen grains of a control plant, wherein the irregular shape of the pollen grains which is obviously different from that of the diploid plant is a tetraploid initially-selected variant suspected plant.
Tween 80 is a nonionic surfactant, is generally considered as a nontoxic and nonirritating material, can be used as a solubilizer, a wetting agent, a dispersant and a stabilizer, improves the solubility of the amisulpride in water, enhances the physiological activity, increases the absorption and improves the stability of an aqueous solution. Silwet-L77 can greatly reduce the surface tension of water, so that the solution can easily wet almost all kinds of leaf surfaces and has extremely strong water washing and infiltration resistance.
The method takes the non-heading Chinese cabbage seeds harvested by artificial cultivation as the material, adopts the cell microtubule inhibitor Oryzalin (Oryzalin) to drip the cotyledon growing point of the plant, improves the stability, the adhesive force and the permeability of the aqueous solution of the Oryzalin by compounding the Tween 80 and the Silwet-L77, better inhibits the formation of spindle yarn at the later mitosis stage, realizes chromosome doubling and generates tetraploid variant plants. The method provides technical support for creating abundant new tetraploid germplasm and new variety of non-heading Chinese cabbage.
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FIG. 1(a) is the stomata electron microscope image of diploid plant in example 1, and FIG. 1(b) is the stomata electron microscope image of tetraploid variant plant in example 1.
FIG. 2(a) is the pollen electron microscope image of diploid plant in example 1, and FIG. 2(b) is the pollen electron microscope image of tetraploid mutant plant in example 1.
FIG. 3 shows examples 1 of different ploidy Brassica campestris plants and their ploidy identification, wherein the upper left is a diploid plant photograph, the upper right is a diploid nuclear DNA flow cytometry, the lower left is a tetraploid plant photograph, and the lower right is a tetraploid variant plant nuclear DNA flow cytometry.
Detailed Description
Example 1
Step 1, 216 full seeds of the non-heading Chinese cabbage harvested in the current year are selected.
And 2, carrying out germination accelerating treatment on the seeds, and poking the seeds into a plug after white exposure. When the seedling cotyledon is completely unfolded, the amisulpride mixed aqueous solution (the amisulpride concentration is 0.06 mmol.L)-1Tween 80 concentration of 0.02% v/v, Silwet L-77 concentration of 0.02% v/v) the seedling growing point is dripped in the morning and evening, and the treatment is carried out for 5 times.
And 3, screening tetraploid plants according to the length and width of the 5 th to 10 th true leaves, the distortion degree of leaf surfaces, the thickness of main stems and the plant height after the plants are grown. And determining the tetraploid variation primary plant through the identification of the air holes in anatomy, wherein the variation rate reaches 4.63%.
And 4, entering a flowering phase, performing pollen identification on the tetraploid variation primary selected plants, and taking the plants with obviously increased and deformed pollen as tetraploid variation suspected plants, wherein the tetraploid variation suspected plants account for 50% of the number of the primary selected variation plants.
And 5, taking the tender leaves of the tetraploid variant suspected strain to perform nuclear DNA content flow cytometry analysis, and finally confirming to obtain the tetraploid plant.
Anatomical pores were identified as:
tearing off the epidermis on the back of the leaf by using a pointed forceps, placing the leaf on a glass slide, dropping 1-2 drops of clear water, covering the glass slide, measuring the length and the width of the air hole by using an eyepiece with a micro-micrometer scale under a 20-or 40-fold microscope, and selecting a plant with the length and the width which are obviously larger than those of a control diploid as a tetraploid variation primary plant.
As shown in FIG. 1, the stomata of the tetraploid mutant plants are larger than that of the control diploid plants.
The pollen is identified as:
taking full stamens of pollen, shaking the pollen on a glass slide uniformly, observing the shape and size of the pollen by using an eyepiece with a microscopic micrometer scale under a 20-fold or 40-fold microscope, and selecting plants with irregular pollen grains which are obviously different from diploid plants as tetraploid variant suspected plants.
As shown in FIG. 2, the pollen of the tetraploid mutant plant is irregular and is obviously different from the pollen of the diploid plant.
The agronomic characters are as follows:
the leaf color deepens, the leaf thickens, the length-width ratio of the leaf becomes smaller, and the growth potential is slow.
As shown in FIGS. 3, 1 and 3, the tetraploid mutant plants had a higher leaf color, a higher leaf thickness, a lower leaf length-width ratio and a slower growth potential than the control diploid plants.
The method for determining the DNA content by the flow cytometer comprises the following steps:
adding 50mg of non-heading Chinese cabbage leaves into 1mL of Tris-HCl buffer solution, quickly cutting the leaves with a blade, filtering the cut leaves with a 40-micron filter to a 1.5 mL centrifuge tube, and incubating the cut leaves on ice for 5-10 min at 1000 r.min-1Centrifuging at low temperature for 5min, discarding supernatant, and adding 1mL precooled Tris-HCl bufferFlushing, 1000 r.min-1Centrifugation was carried out at low temperature for 5min, the supernatant was discarded, 500. mu.L of a precooled Tris-HCl buffer was added, and ribonuclease and Propidium Iodide (PI) as a fluorescent dye were added. Adding 10 μ L (10mg/mL) of RNAse, adding 7 μ L (10mg/mL) of PI staining agent, mixing, keeping dark, staining for 15 min, and detecting.
As shown in 2 and 4 of FIG. 3, the abscissa peak position is shown as the fluorescence intensity of DNA of the tested plant, and the fluorescence intensity of DNA of the tetraploid mutant plant is about twice that of the diploid plant used as the control.
Example 2
Step 1, selecting 212 full seeds of the non-heading Chinese cabbage harvested in the current year.
And 2, carrying out germination accelerating treatment on the seeds, and poking the seeds into a plug after white exposure. When the seedling cotyledon is completely unfolded, the amisulpride mixed aqueous solution (the amisulpride concentration is 0.08 mmol.L)-1Tween 80 concentration of 0.02% v/v, Silwet L-77 concentration of 0.02% v/v) the seedling growing point is dripped in the morning and evening, and the treatment is carried out for 5 times.
And 3, screening tetraploid plants according to the length and width of the 5 th to 10 th true leaves, the distortion degree of leaf surfaces, the thickness of main stems and the plant height after the plants are grown. And determining the tetraploid variation primary plant through the identification of the air holes in anatomy, wherein the variation rate reaches 9.91%.
And 4, entering a flowering phase, performing pollen identification on the tetraploid variation primary selected plants, and taking the plants with obviously increased and deformed pollen as tetraploid variation suspected plants, wherein the tetraploid variation suspected plants account for 66.7 percent of the number of the primary selected variation plants.
And 5, taking the tender leaves of the tetraploid variant suspected strain to perform nuclear DNA content flow cytometry analysis, and finally confirming to obtain the tetraploid plant.
Anatomical pores were identified as:
tearing off the epidermis on the back of the leaf by using a pointed forceps, placing the leaf on a glass slide, dropping 1-2 drops of clear water, covering the glass slide, measuring the length and the width of the air hole by using an eyepiece with a micro-micrometer scale under a 20-or 40-fold microscope, and selecting a plant with the length and the width which are obviously larger than those of a control diploid as a tetraploid variation primary plant.
The pollen is identified as:
taking full stamens of pollen, shaking the pollen on a glass slide uniformly, observing the shape and size of the pollen by using an eyepiece with a microscopic micrometer scale under a 20-fold or 40-fold microscope, and selecting plants with irregular pollen grains which are obviously different from diploid plants as tetraploid variant suspected plants.
The agronomic characters are as follows:
the leaf color deepens, the leaf thickens, the length-width ratio of the leaf becomes smaller, and the growth potential is slow. Compared with a diploid plant for comparison, the tetraploid variant plant has the advantages of deepened leaf color, thickened leaf, reduced leaf length-width ratio and slow growth potential.
The DNA content is measured by a flow cytometer, and the fluorescence intensity of the DNA of the tetraploid mutant plant is about twice that of the diploid plant used for control.
Example 3
And step 1, selecting 199 full seeds of the non-heading Chinese cabbages harvested in the current year.
And 2, carrying out germination accelerating treatment on the seeds, and poking the seeds into a plug after white exposure. When the seedling cotyledon is completely unfolded, the amisulpride mixed aqueous solution (the amisulpride concentration is 0.1 mmol.L)-1Tween 80 concentration of 0.02% v/v, Silwet L-77 concentration of 0.02% v/v) the seedling growing point is dripped in the morning and evening, and the treatment is carried out for 5 times.
And 3, screening tetraploid plants according to the length and width of the 5 th to 10 th true leaves, the distortion degree of leaf surfaces, the thickness of main stems and the plant height after the plants are grown. And identifying pores in anatomy to determine the tetraploid variation primary plant, wherein the variation rate reaches 8.04%.
And 4, entering a flowering period, performing pollen identification on the tetraploid variation primary selected plants, and taking the plants with obviously increased and deformed pollen as tetraploid variation suspected plants, wherein the tetraploid variation suspected plants account for 62.5% of the number of the primary selected variation plants.
And 5, taking the tender leaves of the tetraploid variant suspected strain to perform nuclear DNA content flow cytometry analysis, and finally confirming to obtain the tetraploid plant.
Anatomical pores were identified as:
tearing off the epidermis on the back of the leaf by using a pointed forceps, placing the leaf on a glass slide, dropping 1-2 drops of clear water, covering the glass slide, measuring the length and the width of the air hole by using an eyepiece with a micro-micrometer scale under a 20-or 40-fold microscope, and selecting a plant with the length and the width which are obviously larger than those of a control diploid as a tetraploid variation primary plant.
The pollen is identified as:
taking full stamens of pollen, shaking the pollen on a glass slide uniformly, observing the shape and size of the pollen by using an eyepiece with a microscopic micrometer scale under a 20-fold or 40-fold microscope, and selecting plants with irregular pollen grains which are obviously different from diploid plants as tetraploid variant suspected plants.
The agronomic characters are as follows:
the leaf color deepens, the leaf thickens, the length-width ratio of the leaf becomes smaller, and the growth potential is slow. Compared with a diploid plant for comparison, the tetraploid variant plant has the advantages of deepened leaf color, thickened leaf, reduced leaf length-width ratio and slow growth potential.
The DNA content is measured by a flow cytometer, and the fluorescence intensity of the DNA of the tetraploid mutant plant is about twice that of the diploid plant used for control.
Claims (5)
1. A method for cultivating tetraploid loose-head Chinese cabbage is characterized in that: the method comprises the following steps:
step 1, selecting full non-heading Chinese cabbage seeds harvested in the current year, and sowing and raising seedlings according to a conventional technology;
step 2, dripping seedling growing points by using a amisulpride mixed aqueous solution at each point in the morning and at each point in the evening when the cotyledon of the seedling is completely unfolded, continuously treating for 5 times, and taking dripped distilled water as a reference;
step 3, after the plants are grown, performing morphological identification, taking the lower epidermis of the 5 th to 10 th true leaves of the suspected plant, performing stomata identification, and selecting the plant with the significantly enlarged stomata as a tetraploid variation primary selection plant;
step 4, entering a flowering phase, taking the tetraploid variation primary selected plants for pollen identification, and further screening to obtain plants with obviously increased and deformed pollen as tetraploid variation suspected plants;
step 5, taking the tender leaves of the tetraploid variant suspected strain to perform nuclear DNA content flow cytometry analysis, and finally confirming to obtain the tetraploid plant;
the mixed aqueous solution of the amisulpride is a mixed aqueous solution of the amisulpride, Tween 80 and an organosilicon surfactant Silwet L-77, and the concentration of the amisulpride is 0.08-0.10 mmol.L-1The concentration of Tween 80 was 0.02% v/v, and the concentration of silicone surfactant Silwet L-77 was 0.02% v/v.
2. The method of claim 1, wherein the method comprises the steps of: the preparation process of the amisulpride mixed aqueous solution is as follows: dissolving amisulben in 15mL of ethanol, dripping the solution into distilled water, and shaking the distilled water continuously during the dripping process to ensure that no medicament is separated out to obtain the amisulben with the concentration of 0.08-0.10 mmol.L-1Adding Tween 80 and Silwet L-77 into the aqueous solution of benazolin, and shaking up to obtain the final product.
3. The method of claim 1, wherein the method comprises the steps of: and 3, performing morphological identification, namely screening and identifying by taking the length and width of the leaves, whether the leaves are twisted, the stem thickness, the plant height and the crown width as indexes.
4. The method of claim 1, wherein the method comprises the steps of: the stomata identification process in step 3 is as follows: measuring the length and width of the stomata under a microscope, calculating the circumference of the stomata according to an ellipse circumference formula, and measuring 30-50 stomata for each plant; and (3) determining whether the stomatal perimeter of the plant is different from the stomatal perimeter of a control plant by adopting a statistical T test method, wherein the plant with the stomatal significantly larger than the control plant is a tetraploid initially-selected variant plant.
5. The method of claim 1, wherein the method comprises the steps of: the process of identifying pollen in step 4 is as follows: comparing the size and shape of the pollen grains under a microscope, and determining whether the shape of the pollen grains of the plant is different from the shape of the pollen grains of a control plant, wherein the irregular shape of the pollen grains which is obviously different from that of the diploid plant is a tetraploid initially-selected variant suspected plant.
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