阅读说明：本技术 一种提高双单倍体成活率的方法 (Method for improving survival rate of double haploids ) 是由 刘艳红 王建民 付建征 韩福祥 付俊清 李娜 张泽伟 于 2021-09-02 设计创作，主要内容包括：本发明公开了一种提高双单倍体成活率的方法,包括以下步骤：步骤一、玉米单倍体种子的筛选；步骤二发芽纸的准备：将所购滤纸折成大小合适的扇形,大小要一致；步骤三、消毒剂的配制；步骤四、叠好的扇形滤纸放入配好的溶液中,适量水分后取出放到托盘上；步骤六、将上述获得的预处理后的单倍体种子播种于营养土中、浇水,遮阳培养至三叶一心；步骤七、染色体加倍的玉米幼苗移栽及管理。本发明所提供的玉米双单倍体幼芽成活率方法能显著地提高玉米单倍体的加倍过程中双单倍体幼芽的成活率,有利于双单倍体植株的选育,解决了单倍体育种过程中单倍体萌发过程中长菌影响单倍体幼芽成活率的技术难题,从而大大提高了双单倍体成活率。(The invention discloses a method for improving the survival rate of a doubled haploid, which comprises the following steps: step one, screening corn haploid seeds; step two, preparation of the germinating paper: folding the purchased filter paper into a fan shape with proper size, wherein the size is consistent; step three, preparation of a disinfectant; step four, putting the stacked fan-shaped filter paper into the prepared solution, taking out the filter paper after a proper amount of water is contained, and putting the filter paper on a tray; step six, sowing the obtained pretreated haploid seeds in nutrient soil, watering, and culturing in a sun-shading mode until three leaves and one heart are obtained; and seventhly, transplanting and managing the maize seedlings with doubled chromosomes. The method for survival rate of the doubled haploid sprouts of the corn can obviously improve the survival rate of the doubled haploid sprouts in the doubling process of the corn haploid, is beneficial to breeding of doubled haploid plants, solves the technical problem that the survival rate of the doubled haploid sprouts is influenced by growth bacteria in the monoploid germination process in the haploid breeding process, and greatly improves the survival rate of the doubled haploid.)

1. A method for improving the survival rate of double haploids is characterized by comprising the following steps: step one, screening corn haploid seeds;

step two, preparation of the germinating paper: folding the purchased filter paper into a fan shape with proper size, wherein the size is consistent;

step three, preparation of the disinfectant: the mass concentration of the prepared disinfectant is about 0.1-0.5% of chlorine dioxide, the dosage of the medicament and distilled water is calculated according to the above concentration, the chlorine dioxide is firstly added into a beaker, the distilled water is added into the beaker until the chlorine dioxide is completely dissolved, and the chlorine dioxide is shaken up and then stored in a plastic container;

fourthly, putting the stacked fan-shaped filter paper into the prepared solution, taking out the stacked fan-shaped filter paper after a proper amount of water is removed, putting the stacked fan-shaped filter paper on a tray, washing haploid seeds clean, uniformly putting the seeds at the bottom of the fan-shaped filter paper with a proper amount of water at intervals of 1 cm-1.5 cm, sealing the seeds with tin foil paper, and putting the seeds in an incubator for culture;

step five, processing corn haploid seeds:

(1) cutting off the coleoptile tip of the haploid bud of the corn;

(2) treating the sprouts with colchicine mixture;

pouring the prepared colchicine mixed solution into a container, completely immersing all cut buds, sealing the opening of the container, and treating for 12 hours at 23 ℃ in a dark place to obtain maize buds with doubled chromosomes;

(3) washing the soaked haploid seeds with clear water for 1-2 hours to obtain pretreated haploid seeds;

step six, sowing the obtained pretreated haploid seeds in nutrient soil, watering, and culturing in a sun-shading mode until three leaves and one heart are obtained;

seventhly, transplanting and managing the maize seedlings with doubled chromosomes:

transplanting the Wang Lai seedlings with doubled chromoplasts cultivated in the sixth step to a testing station of Hebei Ji crop species company Limited responsibility, shading the seedlings for 5 days by using a shading net, finely managing the seedlings in the field to ensure the normal growth of the seedlings, removing non-haploid plants in the seedling stage, and carrying out artificial pollination to obtain the haploid corn plants.

2. The method for improving doubled haploid survival rate of claim 1, wherein in step one, the selection of maize haploid seeds is to select seeds with purple top colorless embryos to obtain haploid seeds.

3. The method for improving doubled haploid survival rate of claim 1, wherein in step two, the disinfectant is formulated with chlorine dioxide at a mass concentration of about 0.1-0.5%, preferably at a mass concentration of about 0.4% and 0.5%.

4. The method for improving doubled haploid survival rate of claim 1, wherein in step four, the cultivation temperature in the incubator is 25-26 degrees, and the cultivation time is 4 days.

Technical Field

The invention relates to the technical field of breeding, in particular to a method for improving the survival rate of doubled haploids.

Background

China is the second largest corn producing country and consuming country, and the improvement of the corn yield and quality requires continuous improvement and development of breeding means. The breeding of the inbred line with high yield, high resistance and high combining ability is a basic and core link in the corn breeding. Haploid breeding refers to a method of utilizing parthenogenesis of a plant to form but replace the haploid, and doubling the chromosome of the plant in a natural or artificial manner to form a homozygous diploid (i.e., a pure line), and then breeding a new variety (line) from the homozygous diploid.

The cultivation of excellent pure lines is a core link of corn breeding. Using traditional breeding methods, it is generally necessary to self-breed 4-6 generations to make the line Write homozygous. And theoretically, even though selfing n generations, the genotype cannot reach a complete homozygous state of 100%. Therefore, it usually takes several decades to breed a variety, consuming a lot of manpower, material resources and financial resources, and therefore shortening the breeding process. The improvement of the breeding efficiency is a target which is always pursued by breeders, and the haploid breeding technology can obtain a homozygous diploid by controlling the hybrid separation and 2 generations, so that the breeding of a selfing line is accelerated, and the breeding period is obviously shortened.

The haploid doubling method is commonly used and comprises a natural doubling method and a drug doubling method. The drug doubling is a currently commonly used haploid doubling method, and the mainly used drugs are colchicine and herbicide. In the colchicine bud soaking method, the growth of fungus is serious in the process of haploid bud germination, so that the survival rate of the doubled haploid is low, and therefore, the improvement of the survival rate of the doubled haploid has important significance for doubling the haploid.

Disclosure of Invention

The present invention aims to provide a method for increasing the survival rate of doubled haploids, so as to solve the problems in the background technology.

In order to achieve the purpose, the invention provides the following technical scheme:

a method for improving the survival rate of double haploids comprises the following steps: step one, screening corn haploid seeds;

step two, preparation of the germinating paper: folding the purchased filter paper into a fan shape with proper size, wherein the size is consistent;

step three, preparation of the disinfectant: the mass concentration of the prepared disinfectant is about 0.1-0.5% of chlorine dioxide, the dosage of the medicament and distilled water is calculated according to the above concentration, the chlorine dioxide is firstly added into a beaker, the distilled water is added into the beaker until the chlorine dioxide is completely dissolved, and the chlorine dioxide is shaken up and then stored in a plastic container;

fourthly, putting the stacked fan-shaped filter paper into the prepared solution, taking out the stacked fan-shaped filter paper after a proper amount of water is removed, putting the stacked fan-shaped filter paper on a tray, washing haploid seeds clean, uniformly putting the seeds at the bottom of the fan-shaped filter paper with a proper amount of water at intervals of 1 cm-1.5 cm, sealing the seeds with tin foil paper, and putting the seeds in an incubator for culture;

step five, processing corn haploid seeds:

(1) cutting off the coleoptile tip of the haploid bud of the corn;

(2) treating the sprouts with colchicine mixture;

pouring the prepared colchicine mixed solution into a container, completely immersing all cut buds, sealing the opening of the container, and treating for 12 hours at 23 ℃ in a dark place to obtain maize buds with doubled chromosomes;

(3) washing the soaked haploid seeds with clear water for 1-2 hours to obtain pretreated haploid seeds;

step six, sowing the obtained pretreated haploid seeds in nutrient soil, watering, and culturing in a sun-shading mode until three leaves and one heart are obtained;

seventhly, transplanting and managing the maize seedlings with doubled chromosomes:

transplanting the Wang Lai seedlings with doubled chromoplasts cultivated in the sixth step to a testing station of Hebei Ji crop species company Limited responsibility, shading the seedlings for 5 days by using a shading net, finely managing the seedlings in the field to ensure the normal growth of the seedlings, removing non-haploid plants in the seedling stage, and carrying out artificial pollination to obtain the haploid corn plants.

As a further scheme of the invention, in the step one, the screening of the corn haploid seeds is to select seeds of purple top colorless embryos to obtain haploid seeds;

as a further aspect of the invention, in step two, the sanitizing agent is formulated to have a concentration of chlorine dioxide of about 0.1 to 0.5% by mass, preferably about 0.4% and 0.5% by mass.

As a further scheme of the invention, in the fourth step, the culture temperature in the incubator is 25-26 ℃ and the culture time is 4 days.

Compared with the prior art, the invention has the beneficial effects that: the method for survival rate of doubled haploid sprouts of corn provided by the invention can obviously improve the survival rate of doubled haploid sprouts in the doubling process of corn haploid, is beneficial to breeding of doubled haploid plants, and solves the technical problem that the survival rate of haploid sprouts is influenced by growth bacteria in the monoploid germination process in the haploid breeding process, thereby greatly improving the survival rate of doubled haploid and making the large-scale application of haploid doubling technology more likely.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1 a method for increasing the survival rate of doubled haploids, comprising the steps of: step one, screening corn haploid seeds:

selecting seeds with purple tops and colorless embryos to obtain haploid seeds;

step two, preparation of the germinating paper: folding the purchased filter paper into a fan shape with proper size, wherein the size is consistent;

step three, preparation of the disinfectant: the mass concentration of the prepared disinfectant is about 0.5 percent of chlorine dioxide, the dosage of the medicament and distilled water is calculated according to the above concentration, the chlorine dioxide is firstly added into a beaker, the distilled water is added into the beaker until the chlorine dioxide is completely dissolved, and the chlorine dioxide is uniformly shaken and then stored in a plastic container;

fourthly, putting the stacked fan-shaped filter paper into the prepared solution, taking out the stacked fan-shaped filter paper after a proper amount of water is removed, putting the stacked fan-shaped filter paper on a tray, washing haploid seeds cleanly, uniformly putting the seeds at the bottom of the fan-shaped filter paper with a proper amount of water at intervals of 1 cm-1.5 cm, sealing the seeds with tinfoil paper, putting the seeds in an incubator, and culturing for 4 days at the temperature of 25-26 ℃;

step five, processing corn haploid seeds:

(1) coleoptile tip for cutting haploid young bud of corn

(2) Treating the sprouts with colchicine mixture

Pouring the prepared colchicine mixed solution into a container, completely immersing all cut buds, sealing the opening of the container, and treating for 12 hours at 23 ℃ in a dark place to obtain maize buds with doubled chromosomes;

(3) washing the soaked haploid seeds with clear water for 1-2 hours to obtain pretreated haploid seeds;

step six, sowing the obtained pretreated haploid seeds in nutrient soil, watering, and culturing in a sun-shading mode until three leaves and one heart are obtained;

seventhly, transplanting and managing the maize seedlings with doubled chromosomes:

transplanting the Wang Lai seedlings with doubled chromoplasts cultivated in the sixth step to a testing station of Hebei Ji crop species company Limited responsibility, shading the seedlings for 5 days by using a shading net, finely managing the seedlings in the field to ensure the normal growth of the seedlings, removing non-haploid plants in the seedling stage, and carrying out artificial pollination to obtain the haploid corn plants.

Example 2 a method for increasing the survival rate of doubled haploids, comprising the steps of: step one, screening corn haploid seeds:

selecting seeds with purple tops and colorless embryos to obtain haploid seeds;

step two, preparation of the germinating paper: folding the purchased filter paper into a fan shape with proper size, wherein the size is consistent;

step three, preparation of the disinfectant: the mass concentration of the prepared disinfectant is about 0.4 percent of chlorine dioxide, the dosage of the medicament and distilled water is calculated according to the above concentration, the chlorine dioxide is firstly added into a beaker, the distilled water is added into the beaker until the chlorine dioxide is completely dissolved, and the chlorine dioxide is uniformly shaken and then stored in a plastic container;

fourthly, putting the stacked fan-shaped filter paper into the prepared solution, taking out the stacked fan-shaped filter paper after a proper amount of water is removed, putting the stacked fan-shaped filter paper on a tray, washing haploid seeds cleanly, uniformly putting the seeds at the bottom of the fan-shaped filter paper with a proper amount of water at intervals of 1 cm-1.5 cm, sealing the seeds with tinfoil paper, putting the seeds in an incubator, and culturing for 4 days at the temperature of 25-26 ℃;

step five, processing corn haploid seeds:

(1) coleoptile tip for cutting haploid young bud of corn

(2) Treating the sprouts with colchicine mixture

Pouring the prepared colchicine mixed solution into a container, completely immersing all cut buds, sealing the opening of the container, and treating for 12 hours at 23 ℃ in a dark place to obtain maize buds with doubled chromosomes;

(3) washing the soaked haploid seeds with clear water for 1-2 hours to obtain pretreated haploid seeds;

step six, sowing the obtained pretreated haploid seeds in nutrient soil, watering, and culturing in a sun-shading mode until three leaves and one heart are obtained;

seventhly, transplanting and managing the maize seedlings with doubled chromosomes:

transplanting the Wang Lai seedlings with doubled chromoplasts cultivated in the sixth step to a testing station of Hebei Ji crop species company Limited responsibility, shading the seedlings for 5 days by using a shading net, finely managing the seedlings in the field to ensure the normal growth of the seedlings, removing non-haploid plants in the seedling stage, and carrying out artificial pollination to obtain the haploid corn plants.

Example 3 a method for increasing the survival rate of doubled haploids, comprising the steps of: step one, screening corn haploid seeds:

selecting seeds with purple tops and colorless embryos to obtain haploid seeds;

step two, preparation of the germinating paper: folding the purchased filter paper into a fan shape with proper size, wherein the size is consistent;

step three, preparation of the disinfectant: the mass concentration of the prepared disinfectant is about 0.3 percent of chlorine dioxide, the dosage of the medicament and distilled water is calculated according to the above concentration, the chlorine dioxide is firstly added into a beaker, the distilled water is added into the beaker until the chlorine dioxide is completely dissolved, and the chlorine dioxide is uniformly shaken and then stored in a plastic container;

fourthly, putting the stacked fan-shaped filter paper into the prepared solution, taking out the stacked fan-shaped filter paper after a proper amount of water is removed, putting the stacked fan-shaped filter paper on a tray, washing haploid seeds cleanly, uniformly putting the seeds at the bottom of the fan-shaped filter paper with a proper amount of water at intervals of 1 cm-1.5 cm, sealing the seeds with tinfoil paper, putting the seeds in an incubator, and culturing for 4 days at the temperature of 25-26 ℃;

step five, processing corn haploid seeds:

(1) coleoptile tip for cutting haploid young bud of corn

(2) Treating the sprouts with colchicine mixture

Pouring the prepared colchicine mixed solution into a container, completely immersing all cut buds, sealing the opening of the container, and treating for 12 hours at 23 ℃ in a dark place to obtain maize buds with doubled chromosomes;

(3) washing the soaked haploid seeds with clear water for 1-2 hours to obtain pretreated haploid seeds;

step six, sowing the obtained pretreated haploid seeds in nutrient soil, watering, and culturing in a sun-shading mode until three leaves and one heart are obtained;

seventhly, transplanting and managing the maize seedlings with doubled chromosomes:

transplanting the Wang Lai seedlings with doubled chromoplasts cultivated in the sixth step to a testing station of Hebei Ji crop species company Limited responsibility, shading the seedlings for 5 days by using a shading net, finely managing the seedlings in the field to ensure the normal growth of the seedlings, removing non-haploid plants in the seedling stage, and carrying out artificial pollination to obtain the haploid corn plants.

Example 4 a method for increasing the survival rate of doubled haploids, comprising the steps of: step one, screening corn haploid seeds:

selecting seeds with purple tops and colorless embryos to obtain haploid seeds;

step two, preparation of the germinating paper: folding the purchased filter paper into a fan shape with proper size, wherein the size is consistent;

step three, preparation of the disinfectant: the mass concentration of the prepared disinfectant is about 0.2 percent of chlorine dioxide, the dosage of the medicament and distilled water is calculated according to the above concentration, the chlorine dioxide is firstly added into a beaker, the distilled water is added into the beaker until the chlorine dioxide is completely dissolved, and the chlorine dioxide is uniformly shaken and then stored in a plastic container;

fourthly, putting the stacked fan-shaped filter paper into the prepared solution, taking out the stacked fan-shaped filter paper after a proper amount of water is removed, putting the stacked fan-shaped filter paper on a tray, washing haploid seeds cleanly, uniformly putting the seeds at the bottom of the fan-shaped filter paper with a proper amount of water at intervals of 1 cm-1.5 cm, sealing the seeds with tinfoil paper, putting the seeds in an incubator, and culturing for 4 days at the temperature of 25-26 ℃;

step five, processing corn haploid seeds:

(1) coleoptile tip for cutting haploid young bud of corn

(2) Treating the sprouts with colchicine mixture

Pouring the prepared colchicine mixed solution into a container, completely immersing all cut buds, sealing the opening of the container, and treating for 12 hours at 23 ℃ in a dark place to obtain maize buds with doubled chromosomes;

(3) washing the soaked haploid seeds with clear water for 1-2 hours to obtain pretreated haploid seeds;

step six, sowing the obtained pretreated haploid seeds in nutrient soil, watering, and culturing in a sun-shading mode until three leaves and one heart are obtained;

seventhly, transplanting and managing the maize seedlings with doubled chromosomes:

transplanting the Wang Lai seedlings with doubled chromoplasts cultivated in the sixth step to a testing station of Hebei Ji crop species company Limited responsibility, shading the seedlings for 5 days by using a shading net, finely managing the seedlings in the field to ensure the normal growth of the seedlings, removing non-haploid plants in the seedling stage, and carrying out artificial pollination to obtain the haploid corn plants.

Example 5 a method for increasing the survival rate of doubled haploids, comprising the steps of: step one, screening corn haploid seeds:

selecting seeds with purple tops and colorless embryos to obtain haploid seeds;

step two, preparation of the germinating paper: folding the purchased filter paper into a fan shape with proper size, wherein the size is consistent;

step three, preparation of the disinfectant: the mass concentration of the prepared disinfectant is about 0.1 percent of chlorine dioxide, the dosage of the medicament and distilled water is calculated according to the above concentration, the chlorine dioxide is firstly added into a beaker, the distilled water is added into the beaker until the chlorine dioxide is completely dissolved, and the chlorine dioxide is uniformly shaken and then stored in a plastic container;

fourthly, putting the stacked fan-shaped filter paper into the prepared solution, taking out the stacked fan-shaped filter paper after a proper amount of water is removed, putting the stacked fan-shaped filter paper on a tray, washing haploid seeds cleanly, uniformly putting the seeds at the bottom of the fan-shaped filter paper with a proper amount of water at intervals of 1 cm-1.5 cm, sealing the seeds with tinfoil paper, putting the seeds in an incubator, and culturing for 4 days at the temperature of 25-26 ℃;

step five, processing corn haploid seeds:

(1) coleoptile tip for cutting haploid young bud of corn

(2) Treating the sprouts with colchicine mixture

Pouring the prepared colchicine mixed solution into a container, completely immersing all cut buds, sealing the opening of the container, and treating for 12 hours at 23 ℃ in a dark place to obtain maize buds with doubled chromosomes;

(3) washing the soaked haploid seeds with clear water for 1-2 hours to obtain pretreated haploid seeds;

step six, sowing the obtained pretreated haploid seeds in nutrient soil, watering, and culturing in a sun-shading mode until three leaves and one heart are obtained;

seventhly, transplanting and managing the maize seedlings with doubled chromosomes:

transplanting the Wang Lai seedlings with doubled chromoplasts cultivated in the sixth step to a testing station of Hebei Ji crop species company Limited responsibility, shading the seedlings for 5 days by using a shading net, finely managing the seedlings in the field to ensure the normal growth of the seedlings, removing non-haploid plants in the seedling stage, and carrying out artificial pollination to obtain the haploid corn plants.

Control group: the procedure is exactly the same as in the example except that distilled water is used instead of the disinfectant in the third step.

The results of the survival rate of the haploids germinated by the haploid seeds after the disinfectant solution with different concentrations and the water treatment are as follows:

table 1 shows the survival rate of the haploids germinated on the seeds treated by disinfectant solutions of different concentrations

Experimental group	Total number of haploid seeds	Concentration of disinfectant solution%	Survival rate of sprout%
				Example 1	600	0.5	93
Example 2	600	0.4	92
				Example 3	600	0.3	80
Example 4	600	0.2	70
				Example 5	600	0.1	65
Control group	600	Water (W)	58

As can be seen from Table 1, the disinfectant solution has the effect of inhibiting the growth of fungi in the germination process of the corn haploid seeds, so that the survival rate of the sprouts is increased, and the survival rate of the doubled haploid is increased. The disinfectant solutions vary in concentration to inhibit fungal growth, with concentrations of 0.5% or 0.4% being most effective. The method for survival rate of doubled haploid sprouts of corn provided by the invention can obviously improve the survival rate of doubled haploid sprouts in the doubling process of corn haploid, is beneficial to breeding of doubled haploid plants, and solves the technical problem that the survival rate of haploid sprouts is influenced by growth bacteria in the monoploid germination process in the haploid breeding process, thereby greatly improving the survival rate of doubled haploid and making the large-scale application of haploid doubling technology more likely.

It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.