A method for identifying maize haploid

Abstract

The invention belongs to the field of agriculture and forestry technology, and in particular to a method for identifying corn haploids, the method comprising the following steps: step one, selecting an identification object; step two, pretreatment: soaking the identification object in a distilled water solution containing sulfite for 30-50 min, then washing with water to obtain a pretreated sample; step three, fixing: fixing the pretreated sample in a modified fixing solution for 8-20 h; the modified fixing solution is mixed with anhydrous ethanol, glacial acetic acid, and sulfite solution in a volume ratio of 3:1:0.01-0.03; step four, staining, pressing and microscopy. The method for identifying corn haploids provided by the present invention can complete identification within one day, is time-consuming, has high identification efficiency and high accuracy.

Description

Technical Field

[0001] The invention belongs to the technical field of agriculture and forestry, and particularly relates to a method for identifying corn haploids. Background Art

[0002] Haploid breeding can obtain homozygous individuals of the desired shape, and the self-pollinated offspring will not separate the traits, so homozygous individuals can be obtained quickly, shortening the breeding period and accelerating the breeding process. Traditional breeding methods usually require 6-10 generations to obtain stable inbred lines of corn, which takes a long time, so the rapid identification and development of haploid corn plants is of great significance to accelerate the corn breeding process.

[0003] In the prior art, there are many methods for identifying haploid corn, including: cytogenetic methods, which use dyes to stain plant cells and examine them under a microscope to distinguish haploids based on the staining results. The advantage is that the detection results are highly accurate, but the disadvantage is that the staining time is long; plant morphological identification, which requires accurate grasp of the morphology of haploids and other plants. For example, haploid corn plants are usually smaller than standard parent plants. The advantage is that no chemical reagents are required, but the disadvantage is that this method needs to distinguish haploids with poor growth in a large number of samples, which is labor-intensive and has poor accuracy; genetic marker methods, which mark corn haploids with special molecular markers and then identify them. Although this method has high accuracy, the molecular marker process also takes a long time, and the molecular markers are highly selective and are not suitable for most corn varieties.

[0004] In summary, the problems existing in the prior art are poor accuracy, or the method with high haploid identification accuracy is time-consuming. Therefore, it is necessary to develop a method for identifying corn haploids that is short in time and has good accuracy. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a method for identifying corn haploids.

[0006] The object of the present invention is to provide a method for identifying maize haploids, comprising the following steps:

[0007] Step 1: Select the identification object;

[0008] Step 2, pretreatment: soak the identification object in a distilled water solution containing sulfite for 30-50 minutes, and then wash with water for 15-30 minutes to obtain a pretreated sample;

[0009] Step 3, fixation: fix the pretreated sample in a modified fixative for 8-20 hours;

[0010] The improved fixative is prepared by mixing anhydrous ethanol, glacial acetic acid, and sulfite solution in a volume ratio of 3:1:0.01-0.03;

[0011] Step 4: staining, pressing and microscopic examination.

[0012] Preferably, in the above method for identifying corn haploids, the identification object is the pollen of the male parent and the pistil of the female parent; or the young embryo of corn after pollination; or the root tip of corn.

[0013] Preferably, in the above method for identifying corn haploids, the identification object is pollen and pistil 1-2 days before pollination; or corn embryos 5-10 mm in length; or corn root tips 1-2 mm in length.

[0014] Preferably, in the above method for identifying haploid maize, when the identification object is pollen and pistil, or maize immature embryo, the male parent of the identification object is Nongda Gaoyu No. 1, and the female parent is Zhengdan 958;

[0015] When the identification object is the root tip of corn, the plant body is the corn parthenogenetic induction line Nongda Gaoyu No. 1.

[0016] Preferably, in the above method for identifying corn haploids, the concentration of sulfite in the distilled water solution containing sulfite is 0.01-0.03 mol / L.

[0017] Preferably, in the above method for identifying corn haploids, the concentration of sulfite in the sulfite solution is 0.1-0.3 mol / L.

[0018] Preferably, in the above method for identifying corn haploids, the sulfite is derived from sodium sulfite.

[0019] Preferably, in the above method for identifying corn haploids, a non-oxidizing acid solution is added to the solution during soaking in step 2.

[0020] Preferably, in the above method for identifying corn haploids, the non-oxidizing acid solution is a 0.01-0.05 mol / L hydrochloric acid solution, and the added amount is 1% of the volume of distilled water containing sulfite, which is added once every 15 minutes, in two additions.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention utilizes the principle that sulfite ions are hydrolyzed into sulfurous acid and hydroxide ions in water, and sulfurous acid is unstable and can be decomposed into sulfur dioxide and water. A distilled water solution containing sulfite is used to pretreat the identification object. Sulfur dioxide can not only fade some pigments in the identification object, but also increase the cell permeability of the identification object, so that substances such as oil and fat that interfere with the staining result in the cell are dissolved out of the cell, thereby improving the staining effect of the subsequent staining step. Hydroxyl ions can also increase the permeability of the cell. When the cell permeability increases, it is also convenient for the fixative to enter the cell, maintain the integrity of the chromosome structure, and enhance the basophilic effect of the chromosome.

[0023] The present invention also adds a small amount of sulfite solution to the fixative, using sulfite and sulfur dioxide produced by its hydrolysis to improve fixation efficiency and reduce the time consumed by fixation. Note that the concentration of hydroxide ions produced by the hydrolysis of sulfite in the fixative is much lower than the concentration provided by glacial acetic acid, so the fixative is still acidic.

[0024] According to the experimental results of the present invention, the method for identifying corn haploids provided by the present invention can complete the identification within one day, which is time-saving and has high identification efficiency. Moreover, since the present invention directly stains chromosomes for identification, it has high accuracy. DETAILED DESCRIPTION

[0025] In order to enable those skilled in the art to better understand and implement the technical solution of the present invention, the present invention is further described below in conjunction with specific embodiments.

[0026] In the description of the present invention, unless otherwise specified, all reagents used are commercially available and all methods used are conventional techniques in the art.

[0027] In the following embodiments, the dyeing adopts the prior art iron alum-hematoxylin dyeing method: the identification object is cleaned with distilled water, placed in 4g / 100mL iron alum for mordanting for 20min, washed with running water for 5min to remove excess iron alum, washed with distilled water 3 times, placed in hematoxylin solution for dyeing for 1h, washed with running water to remove excess dye, separated with 2g / 100mL iron alum, washed with running water for 40min, then treated with 50%, 70%, 85%, 95% ethanol by volume in sequence, treated with anhydrous ethanol twice, treated with xylene twice, and sealed;

[0028] The hematoxylin solution is obtained by diluting a 10% by mass hematoxylin stock solution to a 0.5% by mass fraction.

[0029] In the following embodiments, tableting and microscopic examination can be performed according to the prior art.

[0030] In the following examples, the improved fixative was prepared and used immediately.

[0031] In the following embodiments, the chromosomes that can be clearly identified and counted are considered to be successfully stained; the chromosomes that are lightly stained, fuzzyly stained, and unable to be identified and counted are considered to be unstainable.

[0032] Example 1

[0033] A method for identifying corn haploids comprises the following steps:

[0034] Step 1: Select the identification object;

[0035] The identification object is the root tip of Nongda Gaoyou No. 1 with a height of 15-20 cm, and the root tip with a length of 1-2 mm is taken. One root tip sample is taken from each corn plant, and a total of 20 samples are taken for standby;

[0036] Step 2, pretreatment: soak the identification object in a distilled water solution containing sulfite for 30 minutes, and then rinse with running water for 15 minutes to obtain a pretreated sample;

[0037] The distilled water solution containing sulfite is a freshly prepared sodium sulfite solution with a concentration of 0.03 mol / L;

[0038] Step 3, fixation: fix the pretreated samples in the modified fixative for 10 hours;

[0039] The improved fixing liquid is prepared by mixing anhydrous ethanol, glacial acetic acid, and sulfite solution in a volume ratio of 3:1:0.03; the sulfite solution is a freshly prepared sodium sulfite solution with a concentration of 0.3 mol / L;

[0040] Step 4: staining, pressing and microscopic examination.

[0041] The staining success rate of the 20 samples was 100%, and the microscopic examination results were all haploid, with an identification accuracy of 100%. However, this method detected cells that were dividing, and the chromosomes of these cells were even, which caused some inconvenience in the statistical distinction of the microscopic examination results.

[0042] Example 2

[0043] A method for identifying corn haploids comprises the following steps:

[0044] Step 1: Select the identification object;

[0045] The method for obtaining the identification object is as follows: after the male parent Nongda Gaoyou No. 1 is hybridized with the female parent Zhengdan 958, the corn embryos are harvested, the length of the corn embryos is 5-10 mm, 2 mm corn embryos are randomly intercepted as the identification object, and the remaining part after interception is discarded;

[0046] A total of 20 samples of young maize embryos were collected for later use;

[0047] Step 2, pretreatment: soak the identification object in a distilled water solution containing sulfite for 30 minutes, and then rinse with running water for 15 minutes to obtain a pretreated sample;

[0048] The distilled water solution containing sulfite is a freshly prepared sodium sulfite solution with a concentration of 0.03 mol / L;

[0049] Step 3, fixation: fix the pretreated samples in the modified fixative for 10 hours;

[0050] The improved fixing liquid is prepared by mixing anhydrous ethanol, glacial acetic acid, and sulfite solution in a volume ratio of 3:1:0.03; the sulfite solution is a freshly prepared sodium sulfite solution with a concentration of 0.3 mol / L;

[0051] Step 4: staining, pressing and microscopic examination.

[0052] The staining success rate of the 20 samples was 100%, and the microscopic examination results were all haploid, with an identification accuracy of 100%. However, this method detected cells that were dividing, and the chromosomes of these cells were even, which caused inconvenience to the statistics of the microscopic examination results.

[0053] Example 3

[0054] A method for identifying corn haploids comprises the following steps:

[0055] Step 1: Select the identification object;

[0056] The identification object is the pollen and pistil of Agricultural University Gaoyu No. 1 one day before pollination, and a total of 20 plants are taken. The chromosome multiples of pollen and pistil are identified for each plant. The sum of the chromosome numbers of the two cells is the multiple of the hybrid corn plant. For example, when the chromosome number of pollen cells is odd and the chromosome number of pistil cells is even, the corn plant hybridized by the two is haploid. When the chromosome number of pollen cells is odd and the chromosome number of pistil cells is odd, the corn plant is non-haploid.

[0057] Step 2, pretreatment: soak the identification object in a distilled water solution containing sulfite for 30 minutes, and then wash it with running water for 15 minutes to obtain a pretreated sample;

[0058] The distilled water solution containing sulfite is a freshly prepared sodium sulfite solution with a concentration of 0.03 mol / L;

[0059] Step 3, fixation: fix the pretreated samples in the modified fixative for 10 hours;

[0060] The improved fixing liquid is prepared by mixing anhydrous ethanol, glacial acetic acid, and sulfite solution in a volume ratio of 3:1:0.03; the sulfite solution is a freshly prepared sodium sulfite solution with a concentration of 0.3 mol / L;

[0061] Step 4: staining, pressing and microscopic examination.

[0062] The staining success rate of 40 samples from 20 plants was 100%, and the identification results of 20 plants were all haploid, with an identification accuracy of 100%. Since pollen cells and pistil cells are obtained through meiosis, they will not divide again, and there will be no even chromosomes. This completely avoids the interference of dividing plant cells on the test results and improves the detection accuracy. This method is particularly suitable for the identification of haploid corn plants in flowering.

[0063] Example 4

[0064] A method for identifying corn haploids comprises the following steps:

[0065] Step 1: Select the identification object;

[0066] The identification object is the root tip of Nongda Gaoyou No. 1 with a height of 15-20 cm, and the root tip with a length of 1-2 mm is taken. One root tip sample is taken from each corn plant, and a total of 20 samples are taken for standby;

[0067] Step 2: pretreatment: soak the identification object in a distilled water solution containing sulfite for 50 minutes, and then wash it with running water for 30 minutes to obtain a pretreated sample;

[0068] The distilled water solution containing sulfite is a freshly prepared sodium sulfite solution with a concentration of 0.01 mol / L;

[0069] Step 3, fixation: fix the pretreated samples in the modified fixative for 20 hours;

[0070] The improved fixing liquid is prepared by mixing anhydrous ethanol, glacial acetic acid, and sulfite solution in a volume ratio of 3:1:0.01; the sulfite solution is a freshly prepared sodium sulfite solution with a concentration of 0.1 mol / L;

[0071] Step 4: staining, pressing and microscopic examination.

[0072] The staining success rate of the 20 samples was 100%, and the microscopic examination results were all haploid, with an identification accuracy of 100%.

[0073] Example 5

[0074] A method for identifying corn haploids comprises the following steps:

[0075] Step 1: Select the identification object;

[0076] The identification object is the root tip of Nongda Gaoyou No. 1 with a height of 15-20 cm, and the root tip with a length of 1-2 mm is taken. One root tip sample is taken from each corn plant, and a total of 20 samples are taken for standby;

[0077] Step 2, pretreatment: soak the identification object in a distilled water solution containing sulfite for 30 minutes, and during the soaking process, add 0.01 mol / L hydrochloric acid solution to the solution, the added amount is 1% of the volume of the distilled water containing sulfite, and add it once every 15 minutes, in two times;

[0078] Then rinse with running water for 15 min to obtain the pretreated sample;

[0079] The distilled water solution containing sulfite is a freshly prepared sodium sulfite solution with a concentration of 0.03 mol / L;

[0080] Step 3, fixation: fix the pretreated samples in the modified fixative for 8 hours;

[0081] The improved fixing liquid is prepared by mixing anhydrous ethanol, glacial acetic acid, and sulfite solution in a volume ratio of 3:1:0.03; the sulfite solution is a freshly prepared sodium sulfite solution with a concentration of 0.3 mol / L;

[0082] Step 4: staining, pressing and microscopic examination.

[0083] The staining success rate of the 20 samples was 100%, and the microscopic examination results were all haploid, with an identification accuracy of 100%. However, this method detected cells that were dividing, and the chromosomes of these cells were even, which caused inconvenience to the statistics of the microscopic examination results.

[0084] Control group 1

[0085] A method for identifying corn haploids comprises the following steps:

[0086] Step 1: Select the identification object;

[0087] The identification object is the root tip of Nongda Gaoyou No. 1 with a height of 15-20 cm, and the root tip with a length of 1-2 mm is taken. One root tip sample is taken from each corn plant, and a total of 20 samples are taken for standby;

[0088] Step 2: Fixation: fix the pretreated samples in a modified fixative for 10 hours;

[0089] The improved fixing liquid is prepared by mixing anhydrous ethanol, glacial acetic acid, and sulfite solution in a volume ratio of 3:1:0.03; the sulfite solution is a freshly prepared sodium sulfite solution with a concentration of 0.3 mol / L;

[0090] Step 2: staining, pressing and microscopic examination.

[0091] The staining success rate of the 20 samples was 65%, and the microscopic examination results were all haploid. Only the samples that were successfully stained were counted, and the identification accuracy was 100%, while the identification accuracy of the samples that failed to stain was 0. Compared with the control group 1, the staining efficiency increased after the sodium sulfite solution was added to the fixative.

[0092] Control group 2

[0093] A method for identifying corn haploids comprises the following steps:

[0094] Step 1: Select the identification object;

[0095] The identification object is the root tip of Nongda Gaoyou No. 1 with a height of 15-20 cm, and the root tip with a length of 1-2 mm is taken. One root tip sample is taken from each corn plant, and a total of 20 samples are taken for standby;

[0096] Step 2: Fixation: fix the pretreated samples in a modified fixative for 10 hours;

[0097] The improved fixative is prepared by mixing anhydrous ethanol and glacial acetic acid in a volume ratio of 3:1;

[0098] Step 2: staining, pressing and microscopic examination.

[0099] The staining success rate of the 20 samples was 40%, and the microscopic examination results were all haploid. Only the samples with successful staining were counted, and the identification accuracy was 100%, while the identification accuracy of the samples with failed staining was 0. The reason for the staining failure is considered to be that the fixation time of the fixative is too short, because the fixation time of the fixative commonly used in the prior art is 24 hours.

[0100] It should be noted that when the present invention involves a numerical range, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as those in the embodiments, in order to avoid redundancy, the present invention describes a preferred embodiment. Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they know the basic creative concept. Therefore, the attached claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0101] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for identifying maize haploids, characterized in that: The following steps are involved: Step 1: Select the identification object; Step 2, pretreatment: soak the identification object in a distilled water solution containing sulfite for 30-50 minutes, then wash with water to obtain a pretreated sample; Step 3, fixation: fix the pretreated sample in a modified fixative for 8-20 hours; The improved fixative is prepared by mixing anhydrous ethanol, glacial acetic acid, and sulfite solution in a volume ratio of 3:1:0.01-0.03; Step 4: staining, pressing and microscopic examination.

2. A method for identifying maize haploids according to claim 1, characterized in that: The identification object is the pollen of the male parent and the pistil of the female parent; or the young embryo of corn after pollination; or the root tip of corn.

3. A method for identifying maize haploids according to claim 2, characterized in that: The identification object is pollen and pistil 1-2 days before pollination; or corn embryo with a length of 5-10 mm; or corn root tip with a length of 1-2 mm.

4. A method for identifying maize haploids according to claim 3, characterized in that: When the identification object is pollen and pistil, or maize embryo, the male parent of the identification object is Nongda Gaoyu No. 1, and the female parent is Zhengdan 958; When the identification object is the root tip of corn, the plant body is Nongda Gaoyu No.

1.

5. The method for identifying maize haploids according to claim 1, characterized in that: The concentration of sulfite in the distilled water solution containing sulfite is 0.01-0.03 mol / L.

6. The method for identifying maize haploids according to claim 1, characterized in that: The concentration of sulfite in the sulfite solution is 0.1-0.3 mol / L.

7. A method for identifying maize haploids according to claim 5 or 6, characterized in that: The sulfite is derived from sodium sulfite.

8. The method for identifying maize haploids according to claim 7, characterized in that: During the soaking in step 2, a non-oxidizing acid solution is added to the solution.

9. The method for identifying maize haploids according to claim 8, characterized in that: The non-oxidizing acid solution is a 0.01-0.05 mol / L hydrochloric acid solution, and the added amount is 1% of the volume of the distilled water containing sulfite, and is added once every 15 minutes in two additions.