CN111837944A - Method for doubling corn haploid - Google Patents
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- CN111837944A CN111837944A CN202010760287.3A CN202010760287A CN111837944A CN 111837944 A CN111837944 A CN 111837944A CN 202010760287 A CN202010760287 A CN 202010760287A CN 111837944 A CN111837944 A CN 111837944A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/06—Processes for producing mutations, e.g. treatment with chemicals or with radiation
- A01H1/08—Methods for producing changes in chromosome number
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01C—PLANTING; SOWING; FERTILISING
- A01C1/00—Apparatus, or methods of use thereof, for testing or treating seed, roots, or the like, prior to sowing or planting
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G24/00—Growth substrates; Culture media; Apparatus or methods therefor
- A01G24/10—Growth substrates; Culture media; Apparatus or methods therefor based on or containing inorganic material
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
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- A01G24/20—Growth substrates; Culture media; Apparatus or methods therefor based on or containing natural organic material
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G24/00—Growth substrates; Culture media; Apparatus or methods therefor
- A01G24/20—Growth substrates; Culture media; Apparatus or methods therefor based on or containing natural organic material
- A01G24/22—Growth substrates; Culture media; Apparatus or methods therefor based on or containing natural organic material containing plant material
- A01G24/25—Dry fruit hulls or husks, e.g. chaff or coir
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/02—Methods or apparatus for hybridisation; Artificial pollination ; Fertility
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01N33/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic nitrogen compounds
- A01N33/16—Biocides, pest repellants or attractants, or plant growth regulators containing organic nitrogen compounds containing nitrogen-to-oxygen bonds
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N41/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a sulfur atom bound to a hetero atom
- A01N41/02—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a sulfur atom bound to a hetero atom containing a sulfur-to-oxygen double bond
- A01N41/04—Sulfonic acids; Derivatives thereof
- A01N41/06—Sulfonic acid amides
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Abstract
The invention provides a method for doubling corn haploid, which comprises the following steps: soaking haploid seeds in a germination accelerating period by using a mixed solution prepared from fomesafen and prodiamine, and washing to obtain pretreated haploid seeds; wherein the soaking time is 6-12 hours. The haploid seeds in the germination accelerating period are soaked in the mixed solution prepared from fomesafen and prodiamine, so that the pollution of an inducer to the environment is reduced.
Description
Technical Field
The invention relates to the technical field of agricultural production, in particular to a method for doubling corn haploid.
Background
China is the second major corn producing and consuming countries in the world, 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 plant parthenogenesis to form a haploid, then doubling the chromosome of the haploid by a natural or artificial mode to form a homozygous diploid (namely a pure line), and then breeding a new variety (line) from the homozygous diploid.
Compared with the conventional breeding means, the haploid induction breeding has obvious advantages: firstly, the homozygous diploid can be obtained by controlling hybrid segregation and 2 generations, so that the breeding of a selfing line is accelerated, and the breeding period is obviously shortened; since the diploid formed by the haploid is highly homozygous and has no covering of dominant characters, the recessive characters can be screened visually through the plant, so that the misselection rate is reduced, the breeding efficiency is effectively improved, and the breeding cost is saved; the formed homozygous diploid can be used as an excellent material for molecular marker assisted breeding and transgenic breeding.
At present, the corn haploid doubling agent is mainly colchicine. On one hand, colchicine is extremely toxic, seriously harms the physical health of operating personnel and pollutes the operating environment; on the other hand, colchicine easily causes the haploidy dead seedlings, malformation and other conditions to appear, and is not beneficial to the breeding of corn plants. Therefore, a nontoxic and environmentally friendly method for doubling corn haploids is imperative.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a method for doubling the haploid of the corn, which solves at least one of the technical problems.
The invention provides a method for doubling corn haploid, which comprises the following steps: soaking haploid seeds in a germination accelerating period by using a mixed solution prepared from fomesafen and prodiamine, and washing to obtain pretreated haploid seeds; wherein the soaking time is 6-12 hours.
Optionally, the mass ratio of the fomesafen to the prodiamine in the mixed solution is 5-15: 1.5 to 6.
Optionally, the specific steps of soaking and washing haploid seeds in a germination accelerating period by using a mixed solution prepared from fomesafen and prodiamine include:
1 to 3 in terms of volume ratio: 1-3, weighing 0.05mg/mL fomesafen and 0.0192mg/mL prodiamine according to a proportion, and uniformly mixing; dilution with pure water 102～106Preparing the mixed solution;
and (3) soaking the haploid seeds in the germination accelerating period in the mixed solution for 6-12 hours, and washing for 1-2 hours to obtain the pretreated haploid seeds.
Optionally, the haploid seed at the germination stage is a haploid seed at the germination stage after removal of 1/3 radicles and embryos.
Optionally, the method for doubling the corn haploid further comprises the following steps after the step of obtaining the pretreated haploid seeds:
seedling culture and hardening: sowing the obtained pretreated haploid seeds in nutrient soil, watering, and culturing in a sun-shading mode until the three-leaf one-heart period is reached;
seedling stage management: transplanting the seedlings in the three-leaf one-heart stage to a greenhouse, watering and culturing; removing non-haploid plants in the seedling stage, and recording scattered powder plants; and (5) managing and culturing water and fertilizer to obtain the haploid corn plant.
Optionally, the nutrient soil is carbonized chaff with a mass ratio of 2-6: 3-7: 0.5-1.5: vegetable garden soil: a mixture of dry rotten pig manure.
Optionally, maintaining light intensity less than 1000lx in said sun-shading culture.
Optionally, the corn haploid doubling method further comprises the following steps of soaking and washing haploid seeds in a germination accelerating period by using a mixed solution prepared from fomesafen and prodiamine:
induction of haploid seeds: taking Fengnong 88 maize hybrid as a female parent and taking an induction line Stock6 as a male parent; sowing in a staggered period to synchronize the flowering period of the male ear of the male parent and the silking period of the female ear of the female parent; in the pollen stage, female parent ears are removed, and pollen of male parent ears is pollinated to female parents; harvesting the fruit ears of the female parent in the mature period;
selecting haploid seeds: and (3) after the fruit ears of the female parent are threshed, selecting the seeds with purple tops and colorless embryos to obtain haploid seeds.
Optionally, the method for removing female parent ears is bagging the female parent ears before spinning.
Optionally, the conditions for preparing haploid seeds in the germination accelerating stage are as follows: the preparation method is carried out in an environment with the temperature of 20-25 ℃ and the humidity of 80-90% RH.
Compared with the prior art, the invention has the following beneficial effects:
in the technology of the invention, haploid seeds in the germination accelerating stage are soaked by a mixed solution prepared from fomesafen and prodiamine so as to induce the haploid seeds in the germination accelerating stage. By washing the soaked haploid seeds in the germination accelerating period, the influence of a mixed solution prepared from fomesafen and prodiamine on the germination of the haploid seeds is reduced. Thus, colchicine is avoided, and harm to operators is reduced; the whole induction process is carried out in the germination accelerating stage of the seeds, and the haploid seeds are directly induced, so that the pollution to the environment by the induction operation such as spraying and the like in the later stage is avoided; meanwhile, the germination rate and the survival rate of the haploid seed seedlings are improved, and the method is favorable for breeding the haploid corn plants.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more clearly apparent, the technical solutions of the present invention are further described below with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The invention provides a method for doubling corn haploid, which comprises the following steps: soaking haploid seeds in a germination accelerating period by using a mixed solution prepared from fomesafen and prodiamine, and washing to obtain pretreated haploid seeds; wherein the soaking time is 6-12 hours.
In this embodiment, by washing the haploid seeds in the germination accelerating period soaked in the mixed solution prepared from fomesafen and prodiamine, the influence of the mixed solution prepared from fomesafen and prodiamine on the germination of the haploid seeds is reduced, and the survival rate of the seeds is increased.
Optionally, the mass ratio of the fomesafen to the prodiamine in the mixed solution is 5-15: 1.5 to 6.
Optionally, the specific steps of soaking and washing haploid seeds in a germination accelerating period by using a mixed solution prepared from fomesafen and prodiamine include:
1 to 3 in terms of volume ratio: 1-3, weighing 0.05mg/mL fomesafen and 0.0192mg/mL prodiamine according to a proportion, and uniformly mixing; dilution with pure water 102～106Preparing the mixed solution;
and (3) soaking the haploid seeds in the germination accelerating period in the mixed solution for 6-12 hours, and washing for 1-2 hours to obtain the pretreated haploid seeds.
Optionally, the haploid seed at the germination stage is a haploid seed at the germination stage after removal of 1/3 radicles and embryos.
Optionally, the method for doubling the corn haploid further comprises the following steps after the step of obtaining the pretreated haploid seeds:
seedling culture and hardening: sowing the obtained pretreated haploid seeds in nutrient soil, watering, and culturing in a sun-shading mode until the three-leaf one-heart period is reached;
seedling stage management: transplanting the seedlings in the three-leaf one-heart stage to a greenhouse, watering and culturing; removing non-haploid plants in the seedling stage, and recording scattered powder plants; and (5) managing and culturing water and fertilizer to obtain the haploid corn plant.
In this example, it is understood that maize haploid plants and non-haploid plants can be identified morphologically because of the significant difference in the length of the main root and the coleoptile at the early stage of seedling growth. Specifically, in the seedling stage, the first leaf of the corn haploid is shorter, the growth is slow in the jointing stage, the plant is short, the leaf is narrow and upright, the color is light, the tassel and branch are few in the flowering stage, and the anther is small.
Optionally, the nutrient soil is carbonized chaff with a mass ratio of 2-6: 3-7: 0.5-1.5: vegetable garden soil: a mixture of dry rotten pig manure.
For example, but not limited to, the mass ratio of 4: 5: 1 of the carbonized chaff: the vegetable garden soil: the dry rotten pig manure is nutrient soil.
Optionally, maintaining light intensity less than 1000lx in said sun-shading culture.
Optionally, the corn haploid doubling method further comprises the following steps of soaking and washing haploid seeds in a germination accelerating period by using a mixed solution prepared from fomesafen and prodiamine:
induction of haploid seeds: taking Fengnong 88 maize hybrid as a female parent and taking an induction line Stock6 as a male parent; sowing in a staggered period to synchronize the flowering period of the male ear of the male parent and the silking period of the female ear of the female parent; in the pollen stage, female parent ears are removed, and pollen of male parent ears is pollinated to female parents; harvesting the fruit ears of the female parent in the mature period;
selecting haploid seeds: and (3) after the fruit ears of the female parent are threshed, selecting the seeds with purple tops and colorless embryos to obtain haploid seeds.
In this embodiment, in order to meet the flowering time of the male ear of the male parent and the female ear of the female parent, the male parent and the female parent are sown in a staggered way. In order to ensure the purity of haploid seeds, it is necessary to remove the female parent ear at the flowering stage of the female parent.
Optionally, the method for removing female parent ears is bagging the female parent ears before spinning.
Optionally, the conditions for preparing haploid seeds in the germination accelerating stage are as follows: the preparation method is carried out in a closed environment with the temperature of 20-25 ℃ and the humidity of 80-90% RH.
To better illustrate the doubling effect of the corn haploid doubling method of the present invention, the following example sets were chosen for further detailed description. It should be understood that the following example sets are only used to illustrate the effect of haploid doubling in the present invention and do not limit the method of haploid doubling in the present invention.
EXAMPLES haploid doubling of maize without treatment
1. The experimental process comprises the following steps:
1.1 Induction of haploid seeds: taking Fengnong 88 maize hybrid as a female parent and taking an induction line Stock6 as a male parent; sowing the female parent and the male parent in a greenhouse in a staggered period so as to synchronize the flowering period of the male ear of the male parent and the silking period of the female ear of the female parent; in the flowering phase, bagging is carried out on female parent ears before silking so as to remove the female parent ears, and pollen of male parent ears is pollinated to the female parent ears; and harvesting the fruit ears of the female parent in the mature period.
1.2 selection of haploid seeds: and (3) after the fruit ears of the female parent are threshed individually, selecting the seeds with purple tops and colorless embryos to obtain haploid seeds.
1.3 pregermination of haploid seeds: wrapping the obtained haploid seeds with a wet tissue, then putting the wrapped haploid seeds into a warm box with the temperature of 25 ℃ and the humidity of 95% RH for accelerating germination until radicles and germs are exposed, and cutting 1/3 of the radicles and the germs to obtain the haploid seeds in the accelerating germination period.
1.4 pretreatment: soaking haploid seeds in a germination accelerating period by using a mixed solution prepared from fomesafen and prodiamine, wherein the volume preparation ratio of the fomesafen and the prodiamine, the dilution times of the mixed solution and the soaking time are shown in table 1; and washing the soaked haploid seeds in the germination accelerating period for 2 hours by using clear water to obtain the pretreated haploid seeds.
1.5 seedling raising and hardening: sowing the obtained pretreated haploid seeds in a seed sowing machine with a mass ratio of 4: 5: 1 of the carbonized chaff: the vegetable garden soil: watering and irrigating thoroughly in the nutrient soil mixed with the dry rotten pig manure, then placing under a sunshade net, and carrying out sunshade culture until the three-leaf one-heart period is reached;
1.6 seedling management: transplanting the seedlings in the three-leaf one-heart stage to a greenhouse, watering and irrigating thoroughly, and culturing by a conventional method; removing non-haploid plants in the seedling stage, and recording the pollen scattering plants in the pollen scattering stage so as to measure the haploid doubling effect of the corn through the pollen scattering plants; and performing conventional water and fertilizer management, culturing and selfing to obtain haploid corn plant grains. The corn haploid gain (%), (loose powder/treated corn haploid) was 100%, and the data shown in table 1 were obtained.
TABLE 1 corn haploid multiplying power statistical table under different treatment conditions (wherein, the concentration of fomesafen is 0.05 mg/mL; the concentration of prodiamine is 0.0192mg/mL)
2. The experimental results are as follows: as can be seen from Table 1, the mixed solution of fomesafen and prodiamine has doubling effect on corn haploid, and the corn haploid plant pollinated and fructified grains which are successfully doubled have consistency with corn haploid plants obtained by natural doubling or colchicine doubling through planting identification. And the preparation ratio of fomesafen and prodiamine, the dilution multiple of the mixed solution and the soaking time of haploid seeds in the germination accelerating period influence the doubling rate.
Specifically, in a certain range, the number of loose powder plants in the maize plant is increased and the doubling rate of the maize haploid is increased along with the increase of the content of fomesafen in the mixed solution of the maize plant cultivated by the maize haploid doubling method provided by the application; after a certain range of value is exceeded (fomesafen: prodiamine ═ 2: 3), the number of loose powder plants in the plants is reduced and the doubling rate of corn haploids is reduced along with the increase of the content of fomesafen in the mixed liquor. Within a certain dilution range, the number of loose powder plants in the plants is increased along with the increase of the dilution multiple of the mixed solution, and the doubling rate of the corn haploid is increased; over a certain dilution range (dilution factor of 10)4) Then, as the dilution multiple of the mixed solution is increased, the number of loose powder plants in the plants is reduced, and the doubling rate of the corn haploids is reduced. Within a certain soaking time, along with the increase of the soaking time of haploid seeds in the germination accelerating period, the number of loose powder plants in the plants is increased, and the doubling rate of corn haploid is increased; after a certain soaking time (soaking for 8 hours), along with the increase of the soaking time of the haploid seeds in the germination accelerating period, the number of loose powder plants in the plants is reduced, and the doubling rate of the corn haploid is reduced. The volume ratio of fomesafen to prodiamine is 2:3, and the dilution multiple of the mixed solution is 104Doubled, haploid species in the pregermination stageAfter 8 hours of immersion of the seeds, an optimal doubling rate was obtained, which reached 27%.
Finally, the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all of them should be covered in the claims of the present invention.
Claims (10)
1. A method for doubling a corn haploid, wherein the method for doubling a corn haploid comprises the following steps: soaking haploid seeds in a germination accelerating period by using a mixed solution prepared from fomesafen and prodiamine, and washing to obtain pretreated haploid seeds; wherein the soaking time is 6-12 hours.
2. The corn haploid doubling method of claim 1, wherein the mass ratio of fomesafen to prodiamine in the mixed solution is 5-15: 1.5 to 6.
3. The method for haploid doubling of maize as claimed in claim 2, wherein the specific steps of soaking and washing haploid seeds in a germination accelerating period by using a mixed solution prepared from fomesafen and prodiamine comprise:
1 to 3 in terms of volume ratio: 1-3, weighing 0.05mg/mL fomesafen and 0.0192mg/mL prodiamine according to a proportion, and uniformly mixing; dilution with pure water 102～106Preparing the mixed solution;
and (3) soaking the haploid seeds in the germination accelerating period in the mixed solution for 6-12 hours, and washing for 1-2 hours to obtain the pretreated haploid seeds.
4. The method for haploid doubling of maize as claimed in claim 3, wherein the haploid seed at the germination stage is a haploid seed at the germination stage after removal of 1/3 radicles and embryos.
5. The method for corn haploid doubling as claimed in any one of claims 1 to 4, wherein the method for corn haploid doubling after the step of obtaining the pretreated haploid seeds further comprises:
seedling culture and hardening: sowing the obtained pretreated haploid seeds in nutrient soil, watering, and culturing in a sun-shading mode until the three-leaf one-heart period is reached;
seedling stage management: transplanting the seedlings in the three-leaf one-heart stage to a greenhouse, watering and culturing; removing non-haploid plants in the seedling stage, and recording scattered powder plants; and (5) managing and culturing water and fertilizer to obtain the haploid corn plant.
6. The method for doubling haploid corn as claimed in claim 5, wherein the nutrient soil is carbonized chaff with a mass ratio of 2-6: 3-7: 0.5-1.5: vegetable garden soil: a mixture of dry rotten pig manure.
7. The method of haploid doubling of maize as described in claim 6, wherein light intensity of less than 1000lx is maintained in the shaded culture.
8. The method for haploid doubling of maize as claimed in any one of claims 1 to 4, wherein the method for haploid doubling of maize further comprises the following steps before the step of soaking and washing haploid seeds in a germination accelerating period by using a mixed solution prepared from fomesafen and prodiamine:
induction of haploid seeds: taking Fengnong 88 maize hybrid as a female parent and taking an induction line Stock6 as a male parent; sowing in a staggered period to synchronize the flowering period of the male ear of the male parent and the silking period of the female ear of the female parent; in the pollen stage, female parent ears are removed, and pollen of male parent ears is pollinated to female parents; harvesting the fruit ears of the female parent in the mature period;
selecting haploid seeds: and (3) after the fruit ears of the female parent are threshed, selecting the seeds with purple tops and colorless embryos to obtain haploid seeds.
9. The method for haploid doubling of maize as claimed in claim 8, wherein the method for removing female parent ears is by bagging the female parent ears before silking.
10. The method for haploid doubling of maize as claimed in any one of claims 1 to 4, wherein haploid seed in the pregermination stage is produced under the following conditions: the preparation method is carried out in an environment with the temperature of 20-25 ℃ and the humidity of 80-90% RH.
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2020
- 2020-07-31 CN CN202010760287.3A patent/CN111837944A/en active Pending
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| CN105104182A (en) * | 2015-08-17 | 2015-12-02 | 中国农业大学 | Method for doubling young embryos of maize haploids |
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| 慈佳宾等: "抗微管除草剂对玉米单倍体加倍效果研究", 《玉米科学》 * |
| 林霞等: "基于不同诱导群体和除草剂对玉米单倍体加倍效果的研究", 《新疆农业大学学报》 * |
| 程江等: "不同生态条件下玉米单倍体自然加倍率及除草剂加倍效果", 《安徽农业大学学报》 * |
| 陈宝建等: "玉米单倍体幼胚加倍效果观察", 《中国农业大学学报》 * |
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