CN102763506A - Method for increasing purity of plant variety - Google Patents
Method for increasing purity of plant variety Download PDFInfo
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- CN102763506A CN102763506A CN2012102140873A CN201210214087A CN102763506A CN 102763506 A CN102763506 A CN 102763506A CN 2012102140873 A CN2012102140873 A CN 2012102140873A CN 201210214087 A CN201210214087 A CN 201210214087A CN 102763506 A CN102763506 A CN 102763506A
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Abstract
The present invention relates to a method for increasing purity of a plant variety. The method comprises step of: providing a plurality of plant seeds, wherein at least a portion of the plant seeds express an acetolactate synthase mutant and have herbicide resistance; and cultivating the seeds to obtain plants with herbicide resistance. Before the seed cultivation or in the cultivation process, at least one of the following treatments is carried out: before cultivation, a herbicide is utilized to treat the seeds, in order to remove seeds without herbicide resistance; and during the cultivation, herbicide is utilized to treat seedlings, so as to remove the seedlings without herbicide resistance. The method can improve and guarantee purity of hybrid varieties in the field and endow the hybrid seeds and seedlings with herbicide resistance activity.
Description
Technical Field
The present invention relates to the field of plant genetic breeding. Specifically, the invention relates to a method for improving the purity of plant varieties, a method for preparing plant hybrid seeds and a method for improving and ensuring the purity of plant field hybrid varieties.
Background
Crossbreeding is the world recognized most effective way to improve the comprehensive productivity of crops. The hybrid breeding can cross strains and varieties with different ecotypes and relatively distant relationships, and the produced hybrid seeds show the characteristics of surpassing the parents, namely the heterosis. In recent decades, cross breeding has been widely used as a means for increasing crop yield, improving crop quality, and improving insect resistance, disease resistance and stress resistance of crops, and has become a main breeding method for many crops.
The key to producing high-purity hybrid seeds is to effectively control self-pollination and fertilization of crops. At present, methods for controlling self-pollination and fertilization of crops comprise an artificial emasculation method, a chemical emasculation method and the utilization of a male sterile line. The artificial emasculation method is to remove male organs of plants through mechanical or manual operation, the chemical emasculation method is to achieve the emasculation effect by utilizing the inhibition effect of chemical reagents on the development of the male organs of the plants, and both methods require a large amount of labor force and are not thorough in emasculation, so that the self-pollination of crops is difficult to control effectively, and the purity of hybrid seeds is influenced. The use of the male sterile line can not only omit the step of manual emasculation, but also reduce the cost of hybrid seed production, and is a main approach of crop hybrid breeding.
The male sterile lines currently used in cross breeding include cytoplasmic sterility and nuclear sterility. Cytoplasmic sterility (i.e., tripling) requires a male sterile line (female parent), a restorer line (male parent), and a maintainer line (to maintain the sterile line). The genic male sterile line is controlled by recessive or dominant genic male sterile gene. At present, most of the adopted cytogenic sterile lines are photo-thermo-sensitive sterile lines, and fertility of the cytogenic sterile lines is influenced by environmental temperature and illumination, namely, under a certain temperature or illumination condition, the mutant is sterile, but fertility is restored due to change of the temperature or illumination condition, so that the sterile lines are maintained. Because the instability of environmental factors can directly influence the stability of the sterile line, the sterile line generates selfing seeds, therefore, in the years of unstable environmental conditions, the purity of hybrid seeds can be difficult to control by using the photo-thermo-sensitive sterile line, and the seed production risk is increased. In addition, both cytoplasmic and nuclear sterile lines produce spurious hybrid seeds from non-male parent pollen that wander in the environment. On one hand, the generation of pseudo hybrid seeds affects the seed quality due to the reduction of the purity of the hybrid seeds, and causes direct economic loss; on the other hand, the grain yield is reduced due to low purity of the hybrid seeds, which causes indirect economic loss. Therefore, a method capable of effectively eliminating the false hybrid seeds and improving the purity of plant varieties has important significance for improving the grain production, however, the current method still needs to be improved.
Disclosure of Invention
The present invention is directed to solving at least one of the problems of the prior art. To this end, it is an object of the present invention to propose a method for increasing the purity of plant varieties.
According to one aspect of the invention, a method of increasing the purity of a plant variety is provided. According to an embodiment of the invention, the method comprises: providing seeds of a plurality of plants, at least a portion of the plurality of seeds expressing an acetolactate synthase mutant and having herbicide resistance; cultivating the seed to obtain a herbicide resistant plant, wherein at least one of the following treatments is performed before or during the cultivation of the seed: treating the plurality of seeds with a herbicide to remove seeds that do not have herbicide resistance prior to the growing; and treating the seedlings with herbicide during the cultivation process to remove the seedlings having no herbicide resistance. Therefore, the 'false hybrid seeds' can be effectively removed, so that the purity of the plant seeds produced by the hybrid seeds can be improved and guaranteed, the application of hybrid breeding and heterosis in the breeding and production of crops and other plants, such as grain production, can be effectively promoted, and the grain yield can be improved. Herein, the expression "pseudo hybrid seed" used herein refers to a hybrid seed produced from a sterile plant by selfing resulting from unstable fertility or by pollen from a non-restorer plant (male parent) drifting in the receiving environment.
The inventors have found that acetolactate synthase mutants are not sensitive to herbicides, in particular imidazolinone herbicides. Therefore, the above-mentioned "acetolactate synthase mutant-expressing and herbicide-resistant" seeds have imidazolinone-type herbicide resistance, and thus, in the method for improving the purity of a plant variety of the present invention, the seeds can be treated with the imidazolinone-type herbicide before or during the cultivation of the seeds to remove the seeds or seedlings that do not have herbicide resistance. According to an embodiment of the present invention, in the method for improving the purity of a plant variety of the present invention, the herbicide is an imidazolinone herbicide, and the acetolactate synthase mutant has an amino acid sequence selected from the group consisting of SEQ ID NO: 1-8, or a pharmaceutically acceptable salt thereof. Further, according to an embodiment of the present invention, in the method for improving the purity of a plant variety of the present invention, the acetolactate synthase mutant is represented by a nucleotide sequence having a sequence selected from the group consisting of SEQ ID NO: 9-16. As used herein, the term "nucleic acid" can be any polymer comprising at least one of ribonucleotides and deoxyribonucleotides, including but not limited to modified or unmodified nucleic acids.
According to an embodiment of the present invention, in the method of improving the purity of a plant variety of the present invention, providing seeds of a plurality of plants is achieved by crossing a restorer line plant having herbicide resistance with a sterile line plant not having herbicide resistance, wherein the restorer line plant is obtained by at least one of:
(a) causing the restorer line plant to comprise a restorer line having an amino acid sequence selected from the group consisting of SEQ ID NO: 9-16;
(b) causing the restorer line plant to express a gene having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-8 in the amino acid sequence shown in any one of the formulas.
Wherein the polypeptide has an amino acid sequence selected from SEQ ID NO: 1-8 is the acetolactate synthase mutant as described above, and is a mutant of acetolactate synthase represented by a protein having an amino acid sequence selected from the group consisting of SEQ id nos: 9-16, or a nucleic acid encoding a nucleotide sequence set forth in any one of seq id nos.
Specifically, according to an embodiment of the present invention, in the method of improving the purity of a plant variety of the present invention, the restorer line plant is obtained through at least one of crossing, backcrossing and asexual propagation. Thus, it is effective to have a restorer line plant comprising a restorer line having a nucleotide sequence selected from the group consisting of SEQ ID NO: 9-16, or expressing a nucleic acid having a nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 8, namely the acetolactate synthase mutant as described above.
According to an embodiment of the present invention, the method for improving the purity of a plant variety of the present invention may further comprise identifying a restorer line plant. It will be appreciated by those skilled in the art that the method of identifying restorer plants is not particularly limited. According to some embodiments of the invention, in the method of improving the purity of a plant variety of the invention, the identifying may further comprise: determining whether the restorer line plant comprises a nucleic acid having an amino acid sequence selected from the group consisting of SEQ ID NO: 9-16; and/or determining whether the restorer line plant expresses the acetolactate synthase mutant.
According to an embodiment of the present invention, in the method of improving the purity of a plant variety of the present invention, treating a plurality of seeds with a herbicide to remove seeds having no herbicide resistance before cultivation may further include: soaking the hybrid seeds with at least one of the following imidazolinone herbicides in the following concentration ranges: 0.1-2.5% by weight of imazapyr; 0.012-0.2 wt% imazethapyr; 0.012-0.2 wt% of Bai Ri Tong; 0.016-0.2 wt% of imazaquin. Therefore, seeds without herbicide resistance can be effectively removed, and the purity of planted plant seeds can be effectively improved.
According to an embodiment of the present invention, in the method of improving plant variety purity of the present invention, treating the seedlings with herbicide during cultivation to remove seedlings that do not have herbicide resistance further comprises: the hybrid seed seedlings are subjected to spray treatment with imidazolinone herbicides in the following concentration ranges, wherein the seedlings are preferably seedlings in two-leaf stage: 0.1-2.5% by weight of imazapyr; 0.012-0.2 wt% imazethapyr; 0.012-0.2 wt% of Bai Ri Tong; 0.016-0.2 wt% of imazaquin. Therefore, seedlings without herbicide resistance can be effectively removed, and the purity of the planted plant species can be effectively improved.
According to the embodiment of the present invention, the plant species to which the method for improving the purity of a plant variety of the present invention can be applied is not particularly limited. According to some specific examples of the present invention, the plant to which the method of the present invention can be applied may be at least one selected from the group consisting of crops and pasture grasses, preferably at least one of rice, wheat, oilseed rape, corn, cotton, millet and alfalfa.
According to another aspect of the present invention, there is also provided a method of preparing plant hybrid seed. According to an embodiment of the invention, the method comprises: crossing a restorer line plant having herbicide resistance with a sterile line plant not having herbicide resistance to obtain a plant hybrid seed, wherein the restorer line plant expresses an acetolactate synthase mutant and the sterile line plant does not express the acetolactate synthase mutant. According to the embodiment of the invention, the method can be used for efficiently preparing the hybrid seeds, and the hybrid seeds are further treated by the herbicide, so that the 'false hybrid seeds' which are not resistant to the herbicide can be effectively removed, the hybrid seeds with herbicide resistance are reserved, the purity of the hybrid seeds can be greatly improved, the application of hybrid breeding and heterosis in the breeding and production of crops and other plants, such as grain production, can be effectively promoted, and the grain productivity is improved. The inventors have surprisingly found that the plant hybrid seeds obtained by the method for preparing plant hybrid seeds of the present invention have high purity, excellent quality, and imidazolinone herbicide resistance.
According to the embodiments of the present invention, the kind of plant to which the method for producing plant hybrid seeds of the present invention can be applied is not particularly limited as long as cross breeding can be performed using the restorer line and the sterile line. According to some embodiments of the present invention, the plant to which the method of the present invention can be applied may be at least one of a crop and a pasture, preferably at least one of rice, wheat, canola, corn, cotton, millet and alfalfa. Therefore, the hybrid breeding of rice, wheat, corn and rape can be effectively carried out, and the obtained hybrid seed has good quality and high purity.
According to an embodiment of the present invention, in the method for preparing plant hybrid seeds of the present invention, the herbicide is an imidazolinone herbicide, and the acetolactate synthase mutant has an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-8, or a pharmaceutically acceptable salt thereof. Further, according to an embodiment of the present invention, in the method for preparing plant hybrid seeds of the present invention, the acetolactate synthase mutant is represented by a nucleotide sequence having a sequence selected from the group consisting of SEQ ID NO: 9-16. As used herein, the term "nucleic acid" can be any polymer comprising at least one of ribonucleotides and deoxyribonucleotides, including but not limited to modified or unmodified nucleic acids.
According to an embodiment of the invention, in the method of preparing plant hybrid seed of the invention, the restorer line plant is obtained by at least one of:
(a) causing the restorer line plant to comprise a restorer line having an amino acid sequence selected from the group consisting of SEQ ID NO: 9-16;
(b) causing the restorer line plant to express a gene having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-8 in the amino acid sequence shown in any one of the formulas.
Wherein the polypeptide has an amino acid sequence selected from SEQ ID NO: 1-8 is the acetolactate synthase mutant as described above, and is a mutant of acetolactate synthase represented by a protein having an amino acid sequence selected from the group consisting of SEQ id nos: 9-16, or a nucleic acid encoding a nucleotide sequence set forth in any one of seq id nos.
Specifically, according to an embodiment of the present invention, in the method for preparing hybrid plant seeds of the present invention, the restorer line plant is obtained by at least one of crossing, backcrossing and asexual propagation. Thus, it is effective to have a restorer line plant comprising a restorer line having a nucleotide sequence selected from the group consisting of SEQ ID NO: 9-16, or expressing a nucleic acid having a nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 8, namely the acetolactate synthase mutant as described above.
According to an embodiment of the present invention, the method for preparing plant hybrid seed of the present invention may further comprise identifying the restorer line plant. It will be appreciated by those skilled in the art that the method of identifying restorer plants is not particularly limited. According to some embodiments of the invention, in the method of improving the purity of a plant variety of the invention, the identifying may further comprise: determining whether the restorer line plant comprises a nucleic acid having an amino acid sequence selected from the group consisting of SEQ ID NO: 9-16; and/or determining whether the restorer line plant expresses the acetolactate synthase mutant.
According to another aspect of the invention, the invention also provides a method for improving and ensuring the purity of the plant field hybrid variety. According to an embodiment of the invention, the method comprises: according to the method for preparing the plant hybrid seeds, the plant hybrid seeds are obtained; and growing the seed to obtain a herbicide resistant plant, at least one of the following treatments being performed prior to or during growing the seed: treating the plurality of seeds with a herbicide to remove seeds that do not have herbicide resistance prior to the growing; and treating the seedlings with herbicide during the cultivation process to remove the seedlings having no herbicide resistance. Therefore, the false hybrid seeds can be effectively removed, so that the purity of the hybrid seeds in the plant field can be improved and guaranteed, the application of the hybrid breeding and the heterosis in the breeding and production of crops and other plants, such as grain production, can be effectively promoted, and the grain yield can be improved.
According to an embodiment of the present invention, in the method for improving and ensuring the purity of a field hybrid of a plant of the present invention, the treating a plurality of seeds with a herbicide to remove the seeds having no herbicide resistance may further comprise, before the cultivating: soaking the hybrid seeds with at least one of the following imidazolinone herbicides in the following concentration ranges: 0.1-2.5% by weight of imazapyr; 0.012-0.2 wt% imazethapyr; 0.012-0.2 wt% of Bai Ri Tong; 0.016-0.2 wt% of imazaquin. Therefore, seeds without herbicide resistance can be effectively removed, and the purity of the hybrid varieties in the plant field can be effectively improved and guaranteed.
According to an embodiment of the present invention, in the method of improving and ensuring the purity of a field hybrid of a plant of the present invention, treating the seedling with a herbicide during cultivation so as to remove the seedling having no herbicide resistance further comprises: the hybrid seed seedlings are subjected to spray treatment with imidazolinone herbicides in the following concentration ranges, wherein the seedlings are preferably seedlings in two-leaf stage: 0.1-2.5% by weight of imazapyr; 0.012-0.2 wt% imazethapyr; 0.012-0.2 wt% of Bai Ri Tong; 0.016-0.2 wt% of imazaquin. Therefore, seedlings without herbicide resistance can be effectively removed, and the purity of the hybrid varieties in plant fields can be effectively improved and guaranteed.
According to the embodiment of the invention, the plant species to which the method for improving and ensuring the purity of the field hybrid variety of a plant can be applied is not particularly limited. According to some specific examples of the present invention, the plant to which the method of the present invention can be applied may be at least one selected from the group consisting of crops and pasture grasses, preferably at least one of rice, wheat, oilseed rape, corn, cotton, millet and alfalfa.
It should be noted that the method for improving the purity of a plant variety of the present invention is completed by the inventors of the present application through hard creative work and optimization work.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Drawings
The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:
FIG. 1 shows the germination of Ningchun No. 4 treated with 0.036 wt% imazethapyr according to one embodiment of the present invention;
FIG. 2 shows the germination of homozygous herbicide-resistant Ningchun No. 4 wheat seeds treated with 0.036 wt% imazethapyr, according to one embodiment of the present invention; and
FIG. 3 shows the germination of 0.036 wt% imazethapyr-treated seeds obtained after selfing of hybrid progeny of homozygous herbicide-resistant Ningchun No. 4 wheat seeds and herbicide-non-resistant ordinary Ningchun No. 4 wheat seeds, according to one embodiment of the present invention.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the accompanying drawings are illustrative only for the purpose of explaining the present invention, and are not to be construed as limiting the present invention.
Example 1: rice imidazolinone herbicide-resistant mutant genetic separation analysis
First, a plant of a mutant of oryza sativa resistant to imazapyr-aqua herbicide was selfed (DNA sequence analysis showed that the acetolactate synthase (ALS) gene of the plant existed in a hybrid state, the ALS mutant gene was mutated AT positions 1642 and 1643, TG to AT, resulting in tryptophan to methionine AT position 548 of the corresponding encoded protein, i.e., the acetolactate synthase had Trp548Cys mutation relative to the wild-type acetolactate synthase), so as to obtain selfed seeds. Then, the selfing seeds obtained by the method are respectively subjected to a seed soaking experiment and a seedling spraying experiment by using a imazapyr water agent (purchased from Shandong Xiandao chemical Co., Ltd.), and the method comprises the following specific steps:
1. seed soaking experiment
Seed soaking experiments were carried out with the aqueous imazapyr (0.12 wt%, 0.36 wt%, 0.6 wt%, 2.5 wt%) according to the following procedure: and soaking the obtained selfing seeds in a imazapyr aqueous solution for 24 hours, then placing the selfing seeds in an incubator at 32 ℃ for accelerating germination, after 24 hours, germinating in an incubator at 25 ℃, and observing the germination and growth conditions of the small buds after 5 days.
Counting the length of the buds of the seeds shows that the length of the buds of the seeds resistant to herbicide reaches 1.5-2cm, and the length of the buds of the seeds not resistant to herbicide stops at about 0.2 cm. Then, the number of resistant individuals and non-resistant individuals treated with the aqueous imazapyr agent at each concentration, and the segregation ratio of resistance and non-resistance were calculated, and the results are shown in table 1 below. The data in table 1 show that selfed seed exhibits resistance to herbicides in the experimental concentration range: non-resistant segregation ratio was close to 3: 1, the herbicide-resistant character is expressed as dominant inheritance, and accords with the expectation, which shows that the seeds can be distinguished from the non-resistant seeds by carrying out herbicide soaking treatment, and the concentration range of the herbicide can be 0.12-2.5%.
Randomly selecting 30 shoots with resistance phenotype, respectively extracting DNA, and detecting the genotypes of the shoots by using a high-resolution melting curve (HRM) method. The detection result shows that 12 strains are homozygous mutants, 18 strains are heterozygotes and are homozygotes: the hybrid was close to 1: 2. the results further indicate that resistant seeds (including homozygotes and heterozygotes) and non-resistant seeds can be distinguished by the herbicide soaking treatment, and that non-resistant seeds are sensitive to herbicides and can be eliminated by the herbicide soaking treatment.
2. Seedling spraying experiment
Randomly selecting 0.36 wt% of imazapyr aqueous solution to carry out field seedling spraying experiments on the selfing seeds, and specifically, carrying out spraying treatment on 2-leaf stage seedlings, wherein 87 milliliters of the seedlings are sprayed per square meter, and counting survival conditions of the seedlings 2-3 weeks after the spraying treatment. The results showed that 62 seedlings grew normally and 20 seedlings turned yellow and gradually died, indicating that 62 seedlings were herbicide resistant, 20 seedlings were not herbicide resistant, and seedlings were herbicide resistant: non-resistant segregation ratio was close to 3: 1 (as shown in table 1 below), consistent with the expected dominant inheritance. The above results indicate that resistant seeds and non-resistant seeds can be distinguished by herbicide spray treatment of seedlings of the seeds, whereby the purity of the crop hybrid seeds in the field use can be ensured by utilizing the herbicide resistance.
DNA was extracted from 30 phenotypically resistant seedlings and their genotypes were examined by high resolution melting curve (HRM) method. The detection result shows that 11 strains are homozygous mutants, 19 strains are heterozygotes, and the homozygote is as follows: the hybrid was close to 1: 2. the results further indicate that non-resistant plantlets can be eliminated by herbicide spray treatment.
TABLE 1 aqueous solution of imazapyr for soaking self-bred seeds or spraying seedlings in two-leaf period
| Concentration of herbicide | Resistant individuals | Non-resistant individuals | Separation ratio |
| a1x | 62 | 18 | 3.44 |
| a3x | 61 | 19 | 3.21 |
| a5x | 59 | 21 | 2.80 |
| a10x | 58 | 22 | 2.64 |
| b3x | 62 | 20 | 3.1 |
Note:athe experiment of soaking selfing seed-setting seeds by the imazapyr aqueous solution is shown;
bthe experiment that the imazapyr water agent is sprayed on seedlings in a two-leaf period is shown.
Example 2: rice seed soaking experiment by utilizing imidazolinone herbicide
Imidazolinone herbicides currently produced and sold in china include imazapyr, imazethapyr, and imazaquin. The rice hybrid selfing seed set in example 1 is soaked for 24 hours by using the herbicide solution, the seed is taken out, and is subjected to pregermination for 24 hours in an incubator at 32 ℃, then is subjected to germination in an incubator at 25 ℃, and the germination and growth conditions of the small bud are observed after 5 days.
Counting the length of the buds of the seeds shows that the length of the herbicide-resistant buds reaches 1.5-2cm, and the length of the herbicide-resistant buds stops at about 0.1-0.2 cm. Then, the numbers of resistant individuals and non-resistant individuals in the seeds after each herbicide soaking treatment, and the segregation ratio of the resistance and the non-resistance were calculated, and the results are shown in table 2 below. The data in table 2 show that the selfed seeds all show resistance to these herbicides: non-resistant segregation ratio was close to 3: 1, indicating that the herbicides can be used for soaking and cleaning non-resistant seeds, thereby ensuring the purity of the crop hybrid seeds in the field using process by utilizing the herbicide resistant characteristic.
TABLE 2 soaking of selfing set seeds with four imidazolinone herbicides
| Herbicide | Resistant individuals | Is notResistant individuals | Separation ratio |
| 0.36% by weight of imazapyr | 58 | 22 | 2.64 |
| 0.036% of Bai Ri Tong | 61 | 21 | 2.90 |
| 0.036% w/w imazethapyr | 59 | 20 | 2.95 |
| 0.048% by weight imazaquin | 57 | 21 | 2.71 |
Example 3: rice seedling spraying experiment by utilizing imidazolinone herbicide
The field seedling spraying experiment is carried out on the rice hybrid selfing seed set in the embodiment 1 by utilizing imazapyr, bermuda, imazethapyr and imazaquin respectively, specifically, spraying 87 milliliters of seedlings in the 2-leaf stage in 2 weeks, and counting the survival conditions of the seedlings after 2-3 weeks of spraying treatment, wherein the results are shown in the following table 3. As can be seen from Table 3, the seedlings were resistant to various herbicides: the non-resistant segregation ratios were all close to 3: 1, dominant inheritance as expected. The results further indicate that non-resistant plantlets can be cleaned by herbicide spray treatment, thereby utilizing herbicide resistance characteristics to ensure purity of crop hybrid seeds during field use.
TABLE 3 four imidazolinone herbicides sprayed two-leaf stage seedlings
| Herbicide | Resistant individuals | Non-resistant individuals | Separation ratio |
| 0.36% by weight of imazapyr | 58 | 18 | 3.22 |
| 0.036 wt% Bai Ri Tong | 61 | 21 | 2.90 |
| 0.036 wt% imazethapyr | 59 | 20 | 2.95 |
| 0.048% by weight imazaquin | 57 | 21 | 2.71 |
Example 4: experiment for soaking wheat seeds by using imidazolinone herbicide
With 0.036 wt% imazethapyr, seeds obtained after selfing of hybrid progeny of the ordinary niuchun No. 4 (herbicide-free) wheat seeds, homozygote herbicide-resistant niuchun No. 4 wheat seeds, and homozygote herbicide-resistant niuchun No. 4 wheat seeds and the ordinary niuchun No. 4 (herbicide-free) wheat seeds, respectively, were subjected to herbicide soaking treatment to investigate whether germination of herbicide-free resistant seeds could be prevented by the herbicide treatment. After 24 hours, the seeds were taken out, and pregermination was carried out in an incubator at 25 ℃ for 24 hours, then germination was carried out in an incubator at 25 ℃, germination and growth of the budlet were observed after 5 days, and photographs were taken, and the results are shown in FIGS. 1 to 3. As shown in the figure, after the soaking treatment of 0.036 wt% imazethapyr, the germination and seedling growth of the ordinary nikken 4 (herbicide-free) wheat seeds are both significantly inhibited (fig. 1), but the germination and seedling growth of homozygote herbicide-resistant wheat seeds are normal (fig. 2), while the seeds obtained after the selfing of the hybrid progeny of the homozygous herbicide-resistant nikken 4 wheat seeds and the ordinary nikken 4 (herbicide-free) wheat seeds are, because of the herbicide-resistant seeds and the herbicide-free seeds, the germination and seedling growth of a part of the seeds after the treatment of 0.036 wt% imazethapyr are normal and the seedling growth of another part of the seeds are inhibited, and the proportion of the normal and inhibited seeds is about 3: 1 (fig. 3). Thus, it was further shown that resistant and non-resistant seeds can be distinguished by subjecting the seeds to a herbicide soak treatment.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
While embodiments of the invention have been shown and described, it will be understood by those of ordinary skill in the art that: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.
Claims (10)
1. A method of increasing the purity of a plant variety, comprising:
providing a plurality of seeds of said plant, at least a portion of said plurality of seeds expressing an acetolactate synthase mutant and being herbicide resistant;
cultivating said seeds to obtain plants having herbicide resistance,
wherein,
before or during the cultivation of the seeds, at least one of the following treatments is carried out:
treating the plurality of seeds with a herbicide to remove seeds that do not have herbicide resistance prior to growing; and
during the cultivation, the seedlings were treated with herbicides to remove seedlings that did not have herbicide resistance.
2. The method of claim 1, wherein said herbicide is an imidazolinone herbicide and said acetolactate synthase mutant has an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-8, or a pharmaceutically acceptable salt thereof,
optionally, the acetolactate synthase mutant consists of a polypeptide having an amino acid sequence selected from SEQ ID NOs: 9-16.
3. The method of claim 2 wherein providing a plurality of seeds of said plant is effected by crossing a restorer line plant which is herbicide resistant with a sterile line plant which is not herbicide resistant,
wherein,
said restorer line plant is obtained from at least one of:
(a) causing said restorer plant to comprise a transgenic plant having an amino acid sequence selected from the group consisting of SEQ ID NO: 9-16;
(b) causing said restorer plant to express a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-8 in the amino acid sequence shown in any one of the formulas.
4. The method of claim 3, wherein said restorer line plant is obtained by at least one of crossing, backcrossing and asexual propagation,
optionally, further comprising identifying said restorer line plant,
optionally, the identifying further comprises:
determining whether said restorer plant comprises a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 9-16; and/or
Determining whether said restorer plant expresses said acetolactate synthase mutant.
5. The method of claim 1, wherein treating the plurality of seeds with a herbicide to remove seeds that are not herbicide resistant prior to incubating further comprises:
soaking the hybrid seed with at least one of the following imidazolinone herbicides in the following concentration ranges:
0.1-2.5% by weight of imazapyr; 0.012-0.2 wt% imazethapyr; 0.012-0.2 wt% of Bai Ri Tong; 0.016-0.2 wt% of imazaquin,
optionally, treating the seedlings with a herbicide during the growing process to remove seedlings that do not have herbicide resistance further comprises:
spraying the hybrid seed seedlings with an imidazolinone herbicide in the following concentration ranges, wherein the seedlings are preferably seedlings in two-leaf stage:
0.1-2.5% by weight of imazapyr; 0.012-0.2 wt% imazethapyr; 0.012-0.2 wt% of Bai Ri Tong; 0.016-0.2 wt% of imazaquin.
6. The method according to claim 1, wherein the plant is at least one selected from the group consisting of crops and pasture grasses, preferably at least one of rice, wheat, canola, corn, cotton, millet and alfalfa.
7. A method of producing plant hybrid seed comprising:
crossing restorer line plants with herbicide resistance with sterile line plants without herbicide resistance to obtain said plant hybrid seeds,
wherein the restorer line plant expresses acetolactate synthase mutant, the sterile line plant does not express acetolactate synthase mutant,
optionally, the plant is at least one of a crop and a pasture grass, preferably at least one of rice, wheat, canola, corn, cotton, millet, and alfalfa.
8. The method of claim 7, wherein said herbicide is selected from the group consisting of imidazolinone herbicides, and said acetolactate synthase mutant has an amino acid sequence selected from the group consisting of SEQ ID NO: 1-8, or a pharmaceutically acceptable salt thereof,
optionally, the acetolactate synthase mutant consists of a polypeptide having an amino acid sequence selected from SEQ ID NOs: 9-16, or a nucleic acid encoding a nucleotide sequence set forth in any one of SEQ ID NOs,
optionally, said restorer line plant is obtained from at least one of:
(a) causing said restorer plant to comprise a transgenic plant having an amino acid sequence selected from the group consisting of SEQ ID NO: 9-16;
(b) causing said restorer plant to express a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to 8 in the sequence of any one of the amino acids,
optionally, the restorer line plant is obtained by at least one of crossing, backcrossing and asexual propagation,
optionally, further comprising identifying said restorer line plant,
optionally, the identifying further comprises:
determining whether said restorer plant comprises a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 9-16; and/or
Determining whether said restorer plant expresses said acetolactate synthase mutant.
9. A method for improving and guaranteeing the purity of a plant field hybrid variety is characterized by comprising the following steps:
obtaining plant hybrid seed according to the method of claim 7 or 8;
and
cultivating said seeds to obtain plants having herbicide resistance,
before or during the cultivation of the seeds, at least one of the following treatments is carried out:
treating the plurality of seeds with a herbicide to remove seeds that do not have herbicide resistance prior to growing; and
during the cultivation, the seedlings were treated with herbicides to remove seedlings that did not have herbicide resistance.
10. The method of claim 9, wherein treating the plurality of seeds with a herbicide to remove seeds that are not herbicide resistant prior to incubating further comprises:
soaking the hybrid seed with at least one of the following imidazolinone herbicides in the following concentration ranges:
0.1-2.5% by weight of imazapyr; 0.012-0.2 wt% imazethapyr; 0.012-0.2 wt% of Bai Ri Tong; 0.016-0.2 wt% imazaquin;
optionally, treating the seedlings with a herbicide during the growing process to remove seedlings that do not have herbicide resistance further comprises:
spraying the hybrid seed seedlings with an imidazolinone herbicide in the following concentration ranges, wherein the seedlings are preferably seedlings in two-leaf stage:
0.1-2.5% by weight of imazapyr; 0.012-0.2 wt% imazethapyr; 0.012-0.2 wt% of Bai Ri Tong; 0.016-0.2 wt% of imazaquin,
optionally, the plant is at least one of a crop and a pasture grass, preferably at least one of rice, wheat, canola, corn, cotton, millet, and alfalfa.
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