CN110810229A - Identification method of glyphosate-resistant rape seeds - Google Patents
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Abstract
The invention discloses an identification method of glyphosate-resistant rape seeds, which relates to the field of glyphosate-resistant rape seeds and comprises the following steps: s1, placing the rape seeds to be detected in a solution with glyphosate resistance concentration of 0.1-1.5 (g/L) for soaking for 8-10 h; s2, carrying out germination dark culture on the soaked seeds in the S1 for 48-60 h; s3, judging that the rape variety is glyphosate-resistant rape when the embryo radicle of the seed cultured in the dark has root hair, and judging that the embryo radicle of the seed has no root hair, namely the non-glyphosate-resistant rape. The detection method disclosed by the invention is convenient, simple to operate, low in cost, rapid and effective, can obviously reduce the workload of resistance detection of glyphosate transgenic seeds, is significant in plant transgenic research, has a good application prospect in purity identification of the glyphosate-resistant transgenic rape hybrids, and has practical value in large-scale variety breeding and hybrid purity identification of the glyphosate-resistant rape hybrids.
Description
Technical Field
The invention relates to the field of glyphosate-resistant rape seeds, in particular to an identification method of glyphosate-resistant rape seeds.
Background
At present, glyphosate is one of the most widely applied herbicides in the world, and rapidly occupies the leading position of the herbicides in the world due to the characteristics of broad spectrum, low toxicity, safety and no soil residue. The action mechanism is mainly that the activity of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in a shikimate pathway is inhibited by hindering the biosynthesis of aromatic amino acids, so that shikimate is accumulated in organisms, and the plant amino acid metabolism is interfered to kill plants. At present, the glyphosate-resistant gene EPSPS has been successfully transferred to various crops, such as glyphosate-resistant rice, glyphosate-resistant soybean, glyphosate-resistant rape and the like. The gene can block the interference of glyphosate on a biosynthesis way by encoding glyphosate-resistant enzyme 5-enolpyruvylshikimate-3-phosphate synthase, so that plants are not killed by the glyphosate. Therefore, the gene transferred into crops can only act on weeds when the glyphosate applied to farmlands, and does not affect the normal growth of crops.
In 1985, Comai transferred the aroA mutant gene of Salmonella typhimurium to tobacco, and obtained a glyphosate-resistant transgenic plant for the first time, and a herbicide-resistant rape variety was obtained by using the transgenic means. At present, more transgenic glyphosate-resistant rapes are mainly used in production and are also transgenic glyphosate-resistant rapes with the greatest influence. Furthermore, countries such as Canada have registered the release of a large number of glyphosate resistant rape varieties such as RT73 (1995), Quest (1996), LG3295 (1996), Hysyn101RR (1997), 45A50 (1998), 45A51 (1998), SWArrow (1998), ZCT7076 (1999), PR5374 (2000) and the like. Therefore, the identification of transgenic glyphosate resistant oilseed rape is needed in the production and research of oilseed rape. In the prior breeding and application of glyphosate-resistant transgenic rape, firstly, a specific primer of a target gene is utilized to carry out Polymerase Chain Reaction (PCR) to identify whether a target gene DNA fragment exists, such as the Pangeh and the like, and PCR is utilized to detect the transgenic glyphosate-resistant rape; secondly, spraying a glyphosate solution to screen transgenic glyphosate-resistant rapes in the seedling stage of the rapes; and thirdly, after the rape seeds or tissues are ground, detecting by using a transgenic test strip. The first two methods have complicated steps, high cost, time and labor waste, and limitation in the mass identification of rape germplasm purity, and the non-resistant materials die when glyphosate solution is sprayed in the seedling stage. The third method is rapid and low in cost, but the detected seeds cannot be reserved; if individual trophozoites are detected, the identification period must be prolonged. Therefore, it is very important to establish a method for rapidly identifying glyphosate-resistant rape seeds.
The invention establishes a method for rapidly identifying the glyphosate-resistant rape seeds by exploring the optimum glyphosate concentration which can effectively distinguish the resistant rape seeds from the non-resistant rape seeds, and provides a new rapid detection means for screening the glyphosate-resistant rape seeds.
Disclosure of Invention
In view of the above, the present invention provides a method for identifying glyphosate resistant oilseed rape seeds, which overcomes at least to some extent one or more of the technical problems of the related art.
The embodiment of the application provides an identification method of glyphosate-resistant rape seeds, which comprises the following steps:
s1, placing the rape seeds to be detected in a solution with glyphosate resistance concentration of 0.1-1.5 (g/L) for soaking for 8-10 h;
s2, dark culturing the soaked seeds in the S1;
s3, judging the rape variety to be resistant rape when the embryo root of the young embryo of the seed has root hair; the embryonic roots of the young embryos have no root hairs, and the young embryos are the non-glyphosate-resistant rape.
In some embodiments, the glyphosate resistant concentration is 0.5-1.0 (g/L).
In some embodiments, the dark culture time is 48-60 h.
In some embodiments, the method further comprises determining the rape variety to be resistant or non-resistant rape based on whether the radicle reaches a predetermined length.
In some embodiments, the method further comprises determining the rapeseed variety to be resistant rape when the radicle reaches a predetermined length.
In some embodiments, the method further comprises transplanting the detected rape immature embryos into a substrate for culture, and performing resistance identification verification according to seedlings.
The invention has the advantages that:
according to the method provided by the embodiment of the application, the rape seeds are soaked in the solution with the glyphosate resistance concentration of 0.1-1.5 (g/L) for culture, and whether the rape varieties are resistant rape seeds is judged according to the existence of the root hairs of the young radicles of the seeds.
In addition to the objects, features and advantages described above, other objects, features and advantages of the present invention are also provided. The present invention will be described in further detail below with reference to the drawings.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the invention and, together with the description, serve to explain the invention and not to limit the invention.
FIG. 1 is the germination status of the radicles of rape at 1.0g/L in the method for identifying glyphosate resistant rape seeds of example 7 of the present invention;
FIG. 2 is the germination status of the rape radicles at 1.0g/L in the method for identifying glyphosate resistant rape seeds of example 8 of the present invention;
FIG. 3 shows the results of the group co-segregation verification of different rape varieties when the glyphosate concentration of the method for identifying glyphosate-resistant rape seeds of example 7 of the present invention is 1.0 g/L;
FIG. 4 shows the results of the group co-segregation verification of different rape varieties when the glyphosate concentration of the method for identifying glyphosate-resistant rape seeds of example 5 of the present invention is 0.5 g/L;
FIG. 5 shows the results of the coseparation analysis of the positive expression and the presence of immature embryo root hair for different rape varieties by PCR amplification with different primers in the method for identifying glyphosate-resistant rape seeds of the embodiment of the present invention;
FIG. 6 shows the results of the co-segregation analysis of the seedling resistance expression and the presence of immature embryo root hair in the method for identifying glyphosate-resistant oilseed rape seeds according to the embodiment of the invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and 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 an identification method of glyphosate-resistant rape seeds, which comprises the following steps:
s1, placing the rape seeds to be detected in a solution with glyphosate resistance concentration of 0.1-1.5 (g/L) to soak for 8-10 h.
In the embodiment of the application, the rape seed varieties to be detected comprise: wherein, BC1 (Zhongshuang) (note: Glyphosate-resistant transgenic rape transferred by taking Zhongshuang No. 11 as a receptor), F2 (103469-4) (note: Glyphosate-resistant transgenic rape transferred by taking SC4 as a receptor), SC4 (note: SC4 as a male parent of the oil Fense 682 of the hybrid rape variety), 103421-2 and 103426-6 (note: the homozygous resistant strain of the glyphosate-resistant transgenic rape transferred by taking SC4 as a receptor) and 20A as a female parent of the oil Feng 827 of the hybrid rape variety; glyphosate is a non-selective herbicide, and the action mechanism of the glyphosate is mainly competitive inhibition of the activity of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in the shikimate pathway. The synthetase is a key enzyme in the biosynthesis process of aromatic amino acid in fungi, bacteria, algae and higher plants; glyphosate resistant groups occur naturally in a variety of species, and vary in length and position, but collectively encode the glyphosate resistant enzyme 5-enolpyruvylshikimate-3-phosphate synthase. By the action of the enzyme, the interference of glyphosate on a biosynthesis pathway can be blocked, so that the strain or plant is not killed by the glyphosate. Therefore, the gene transferred into crops can enable glyphosate applied to farmlands to only act on weeds without influencing the normal growth of the crops, thereby being beneficial to improving the crop yield, reducing the labor capacity and improving the labor efficiency.
In some embodiments, in step S1, the rape seed to be detected is soaked in a solution with glyphosate resistance concentration of 0.1-1.5 (g/L) for 8-10 hours, which can be realized by the following steps:
step S11, preparing 0.1-1.5 g/L glyphosate solution;
step S12, preparing filter paper, and soaking the filter paper in 0.1-1.5 g/L glyphosate solution.
In the embodiment of the application, the soaking is carried out at room temperature, the temperature of the room temperature is 25-30 ℃, and the moisture retention time of the seeds on the filter paper is 8-10 h.
Illustratively, the rape seeds to be tested are placed on filter paper soaked with a solution with a glyphosate-resistant concentration of 1.0 (g/L) and are moisturized on the filter paper for 9h at 25 ℃.
S2, dark culturing the soaked seeds in the S1.
In the examples of the present application, dark culture was: culturing rape seeds in a dark environment with proper temperature, humidity, ventilation and the like. Because the dedifferentiation stage in plant tissue culture requires culture in the absence of light. Light can hinder tissue dedifferentiation, and callus grows faster in the absence of light. When the plant organs, tissues and cells are cultured in vitro, the callus can grow under proper conditions. The generation process is as follows: the viable cells in the explant are induced to restore their potential totipotency, and are converted into meristematic cells, and the derived cells are then differentiated into parenchyma to form callus. After the callus is formed, under the conditions of proper illumination, temperature, certain nutrient substances, hormone and the like, the callus begins to differentiate to generate various organs and tissues of the plant, and then the plant is developed into a complete plant; dark culture is carried out at the temperature of 25-28 ℃; the dark culture time is 48-60 h.
S3, judging the rape variety to be glyphosate-resistant rape when the embryo root of the young embryo of the seed has root hair; the embryo radicle of the young embryo is smooth and has no root hair, thus being the non-glyphosate-resistant rape.
In the embodiment of the present application, radicle: the lower, immature root of the embryo; root hair: the root hair is a tubular structure with the outwards protruded epidermal cells and a closed top end, is easy to cling to soil grains and effectively absorbs the soil grains.
According to the method provided by the embodiment of the application, the rape seeds are soaked in the solution with the glyphosate resistance concentration of 0.1-1.5 (g/L) for culture, and whether the rape varieties are resistant rape seeds is judged according to the existence of the root hairs of the young radicles of the seeds.
In some embodiments, the glyphosate resistant concentration is 0.5-1.0 (g/L).
In some embodiments, the dark culture time is 48-60 h.
In some embodiments, the method further comprises determining the rape variety to be resistant or non-resistant rape based on whether the radicle reaches a predetermined length.
In the embodiment of the application, the rape variety is judged to be resistant or non-resistant rape according to whether the radicle can reach the preset length, and can be used as an auxiliary index to efficiently distinguish the resistant or non-resistant rape variety, wherein the preset length is as follows: and when the length of the radicle reaches 25mm and exceeds 25mm, the rape variety can be judged to be resistant rape, and the length of the radicle of the immature embryo is used as an auxiliary index, so that double insurance is provided for the identification of the rape variety, and the accuracy of the identification result is further determined.
In some embodiments, the method further comprises determining the rapeseed variety to be resistant rape when the radicle reaches a predetermined length.
Illustratively, when the length of the radicle exceeds 25mm, the rape variety is judged to be resistant rape.
In some embodiments, the method further comprises transplanting the detected rape immature embryos into a substrate for culture, and performing resistance identification verification according to seedlings.
In the examples of the present application, the substrate: comprises mushroom residue, vinegar residue, manioc waste mixed fermentation material, coconut husk, carbon bran or plant ash, grass carbon, vermiculite and perlite which are subjected to fermentation and decomposition treatment; and (3) the detected rape immature embryos do not lose activity, the identified resistant immature embryos are transplanted into matrix soil for culture, and resistance identification can be further carried out according to the surviving seedlings. The method for identifying the glyphosate-resistant rape seeds is generally applicable to all rape varieties through multiple tests.
Example 1
S1, placing the rape seeds to be detected on filter paper soaked in 0.1g/L glyphosate solution, and keeping moisture for 8 hours at room temperature;
s2, placing the soaked seeds of S1 at 28 ℃ for germination dark culture for 60 h;
and S3, observing whether the root hair of the embryo radicle of the seed germinated in the dark by the glyphosate solution exists, and whether the embryo radicle of the embryo is smooth and has no root hair, wherein the embryo radicle is a non-resistant rape variety, and the embryo radicle with root hair is a non-resistant variety.
Example 2
S1, placing the rape seeds to be detected on filter paper soaked in 0.1g/L glyphosate solution, and preserving moisture for 10 hours at room temperature;
s2, placing the soaked seeds of S1 at 25 ℃ for germination dark culture for 48 h;
and S3, observing whether the root hair of the embryo radicle of the seed germinated in the dark by the glyphosate solution exists, and whether the embryo radicle of the embryo is smooth and has no root hair, wherein the embryo radicle is a non-resistant rape variety, and the embryo radicle with root hair is a non-resistant variety.
Example 3
S1, placing the rape seeds to be detected on filter paper soaked in 1.5g/L glyphosate solution, and keeping the moisture for 8 hours at room temperature;
s2, placing the soaked seeds of S1 at 28 ℃ for germination dark culture for 60 h;
and S3, observing whether the root hair of the embryo radicle of the seed germinated in the dark by the glyphosate solution exists, and whether the embryo radicle of the embryo is smooth and has no root hair, wherein the embryo radicle is a non-resistant rape variety, and the embryo radicle with root hair is a non-resistant variety.
Example 4
S1, placing the rape seeds to be detected on filter paper soaked in 1.5g/L glyphosate solution, and keeping the moisture for 10 hours at room temperature;
s2, placing the soaked seeds of S1 at 25 ℃ for germination dark culture for 48 h;
and S3, observing whether the root hair of the embryo radicle of the seed germinated in the dark by the glyphosate solution exists, and whether the embryo radicle of the embryo is smooth and has no root hair, wherein the embryo radicle is a non-resistant rape variety, and the embryo radicle with root hair is a non-resistant variety.
Example 5
S1, placing the rape seeds to be detected on filter paper soaked in 0.5g/L glyphosate solution, and keeping moisture for 8 hours at room temperature;
s2, placing the soaked seeds of S1 at 28 ℃ for germination dark culture for 60 h;
and S3, observing whether the root hair of the embryo radicle of the seed germinated in the dark by the glyphosate solution exists, and whether the embryo radicle of the embryo is smooth and has no root hair, wherein the embryo radicle is a non-resistant rape variety, and the embryo radicle with root hair is a non-resistant variety.
Example 6
S1, placing the rape seeds to be detected on filter paper soaked in 0.5g/L glyphosate solution, and preserving moisture for 10 hours at room temperature;
s2, placing the soaked seeds of S1 at 25 ℃ for germination dark culture for 48 h;
and S3, observing whether the root hair of the embryo radicle of the seed germinated in the dark by the glyphosate solution exists, and whether the embryo radicle of the embryo is smooth and has no root hair, wherein the embryo radicle is a non-resistant rape variety, and the embryo radicle with root hair is a non-resistant variety.
Example 7
S1, placing the rape seeds to be detected on filter paper soaked in 1.0g/L glyphosate solution, and keeping moisture for 8 hours at room temperature;
s2, placing the soaked seeds of S1 at 28 ℃ for germination dark culture for 60 h;
and S3, observing whether the root hair of the embryo radicle of the seed germinated in the dark by the glyphosate solution exists, and whether the embryo radicle of the embryo is smooth and has no root hair, wherein the embryo radicle is a non-resistant rape variety, and the embryo radicle with root hair is a non-resistant variety.
Example 8
S1, placing the rape seeds to be detected on filter paper soaked in 1.0g/L glyphosate solution, and preserving moisture for 10 hours at room temperature;
s2, placing the soaked seeds of S1 at 25 ℃ for germination dark culture for 48 h;
and S3, observing whether the root hair of the embryo radicle of the seed germinated in the dark by the glyphosate solution exists, and whether the embryo radicle of the embryo is smooth and has no root hair, wherein the embryo radicle is a non-resistant rape variety, and the embryo radicle with root hair is a non-resistant variety.
Control group
S1, respectively placing the rape seeds to be detected in ddH2O (contrast) soaking filter paper, and keeping the moisture at room temperature for 8-10 h;
s2, placing the seeds of S1 at 25-28 ℃ for germination dark culture for 48-60 h;
s3, Observation of the use of ddH in S22O dark culture of the existence and elongation of the root hair of the embryonic root of the young seed embryo.
When the young embryos of the seeds cultured in examples 1 to 8 were compared with those of the control group, it was found that
The young embryo root hairs of the seeds of the embodiments 1 to 8 grow normally and the length of the radicle is obviously longer than that of the contrast, namely the resistant rape variety; the embryonic roots of the young embryos of the control group are smooth and have no root hairs, and the length of the embryonic roots is obviously shorter than that of the control group, namely the non-resistant rape variety.
Experimental example:
1. test materials and reagents
1.1 test materials, as in Table 1
TABLE 1 rape materials for identification
Wherein, BC1 (Zhongshuang) (note: Glyphosate-resistant transgenic rape transferred by taking Zhongshuang No. 11 as a receptor), F2 (103469-4) (note: Glyphosate-resistant transgenic rape transferred by taking SC4 as a receptor), SC4 (note: SC4 as a male parent of the oil Fense 682 of the hybrid rape variety), 103421-2 and 103426-6 (note: the homozygous resistant strain of the glyphosate-resistant transgenic rape transferred by taking SC4 as a receptor) and 20A as a female parent of the oil Feng 827 of the hybrid rape variety;
1.2 reagents
"Nondada" (41% aqueous glyphosate isopropylamine salt), manufactured by Monsanto, USA; domestic agar (agar) was purchased from Beijing Soilebao technologies, Inc.
2. Construction of rapid identification method for glyphosate-resistant rape seeds
2.1 Glyphosate solution treatment concentration screening
3 layers of filter paper cut into proper size are placed in a culture dish, and glyphosate solutions with different mass concentrations (5.0 g/L, 1.0g/L, 0.5g/L, 0.25g/L and 0.1 g/L) are poured until the filter paper is fully moistened. The culture dish is divided into two parts, 25 seeds of non-resistant materials and homozygous resistant offspring are respectively sown on a culture medium, and dark culture is carried out for 48-60h after moisture preservation is carried out for 8-10h at room temperature. And observing the elongation condition of the immature embryo and the existence of root hair of the radicle of each material in the glyphosate solutions with different concentrations, and screening the glyphosate solution concentration capable of effectively distinguishing the resistant rape from the non-resistant rape. The results were recorded for the number of young embryos with or without root hairs as shown in Table 2. The result shows that the glyphosate concentration within the dosage range of 0.5-1.0 g/L is the optimal concentration for rapidly identifying the resistant rape strain.
TABLE 2 identification of Glyphosate resistance phenotype
FIGS. 1 and 2 are the development status of the radicle of the rape after the germination culture of 1.0g/L glyphosate solution in the identification method of glyphosate-resistant rape seeds of the embodiment of the invention;
wherein, A in figure 1 is SC4 rape line negative control, B in figure 1 is 103421-2 rape line positive control, C in figure 2 is 103421-2 rape line positive control, D in figure 2 is SC4 rape line negative control.
As shown in fig. 1 and 2: negative controls all germinated, but radicles were short and none had root hair growth; the positive controls all germinate, the radicles are longer and all have root hairs to grow, so that the resistant rape strains can be quickly identified within the dosage range of the glyphosate concentration of 0.5-1.0 g/L, and the rape strains can be identified as resistant or non-resistant rape by comparing the root hairs of the radicles with the elongation length of the negative control bacon according to the existence or nonexistence of the combination of the radicles and the root hairs of the radicles.
The radicle reaches the preset length or has root hair, and the rape variety is judged to be resistant rape; and judging the rape variety to be non-resistant rape when the radicle can not reach the preset length or the rootless hair.
2.2 identification of resistant Individual plants in progeny segregating population
2.2.1 selection of immature embryo phenotypes in segregating populations
Respectively placing seeds BC1 (middle double resistance) and F2 (103469-4) of the rape resistance segregation population to be detected in ddH2O (control) and 0.5-1.0 g/L glyphosate solution are soaked on filter paper, moisture is kept for 8-10h at room temperature, and then germination dark culture is carried out for 48-60h at 25-28 ℃; observation with ddH2And O and glyphosate solution germinates and secretly cultures the existence and the elongation of the root hair of the embryo radicle of the seed, the embryo radicle is smooth and has no root hair, the length of the embryo radicle is obviously shorter than that of the contrast, the embryo radicle is a non-resistant single plant, and the normal growth of the root hair and the embryo radicle of the embryo are the non-resistant single plant. Phenotypic identification results (i.e., total number of resistant and non-resistant particles) results are reported in table 3, and the chi-square test indicates that BC1 isolates population-resistant individuals: non-resistant individuals met the 1:1 segregation ratio, F2 isolate population-resistant individuals: the non-resistant individual plants meet the 3:1 segregation ratio, which indicates that the resistant and non-resistant rapes can be effectively identified within the dosage range of 0.5-1.0 g/L of glyphosate concentration.
TABLE 3F 2, BC1 TABLE RESULT Kafang test X2
2.2.2 Co-segregation verification of isolation phenotype identification and PCR amplification identification results
And (3) transplanting the resistant and non-resistant immature embryos obtained in the step 2.2.1 into matrix soil for culture, selecting young stems and leaves to extract DNA when four to five true leaves are obtained, carrying out PCR by using FMV35S, GOX (modified) and CP4-EPSPS as primers, carrying out agarose gel electrophoresis on PCR products, photographing by using a gel imaging system, and recording results. And spraying glyphosate solution with field weeding concentration on the seedlings, and observing the resistance performance of the rapes in the seedling stage after five to seven days.
Extracting the genomic DNA of the immature embryo in the step 2.2.1 by using a CTAB method, and performing PCR amplification by using FMV35S, GOX (modified) and CP4-EPSPS as primers. The PCR reaction system is as follows:
dNTPs(2.5 mM) 0.8ul
10×Taq polymerase buffer (MgCl2) 2.0 ul
Primer F(5 uM) 1.0ul
Primer R(5 uM) 1.0ul
Taq polymerase(5 U/uL) 0.4ul
DNA template 1.0ul
ddH2O made up to 20 ul.
3.3 PCR reaction procedure: 94 ℃ for 5 min; 94 ℃, 20 s; at 59 ℃ for 40 s; 72 ℃ for 40 s; 40 cycles; 72 ℃ for 3 min; 15 ℃ for 2 min.
PCR reaction was performed with ddH2O as a blank, all PCR products were electrophoresed through agarose gel, photographed by a gel imaging system, and the results were recorded. The primer sequences are shown in Table 4, the amplification length of FMV35S and CP4-EPSPS gene is 200bp, and the amplification length of GOX gene is 450 bp.
TABLE 4 primer sequences
The experimental results are as follows:
the FMV35S is used as a primer to respectively amplify single plants of the double-resistant BC1 segregation population and the 103469-4F 2 segregation population, PCR positive and non-positive performances and a phenotype identification result are co-separated, and the results show that when the glyphosate concentration is 0.5-1.0 g/L, the rape seeds are germinated and cultured, and whether the seeds to be detected contain glyphosate resistant genes can be effectively identified.
The single plants which are positive are shown through agarose gel electrophoresis, no death phenomenon is found after glyphosate is sprayed, and all the single plants which are negative are dead in 7-12 days, and the results show that when the concentration of the glyphosate is 0.5-1.0 g/L, rape seeds with roots and hairs on young embryos contain herbicide-resistant genes, and the seedlings show resistance to the glyphosate.
FIG. 5 shows the results of the coseparation analysis of the positive expression and the presence of immature embryo root hair for different rape varieties by PCR amplification with different primers in the method for identifying glyphosate-resistant rape seeds of the embodiment of the present invention;
in the figure, a, c, e and g are positive controls, and b, d, f and h are negative controls.
As shown in fig. 5:
FMV35S is used as a primer to amplify the Zhejiang rape strain on the upper left, and GOX is used as a primer to amplify the Zhejiang rape strain on the lower left;
the top right uses FMV35S as primer to amplify 103426-6 rape strain, and the bottom right uses GOX as primer to amplify 103426-6 rape strain.
FIG. 3 shows the results of the group co-segregation verification of different rape varieties when the glyphosate concentration of the method for identifying glyphosate-resistant rape seeds of the embodiment of the present invention is 1 g/L; FIG. 4 shows the results of the group co-segregation verification of different rape varieties when the glyphosate concentration of the method for identifying glyphosate-resistant rape seeds of example 1 of the present invention is 0.5 g/L;
wherein E, H indicates that rape line is 103421-2, F, I indicates that rape line is 103469-4, and G, J indicates that rape line is SC 4.
As shown in fig. 4 and 5: the number of strains showing resistance has no significant difference from the segregation ratio of BC1 (medium double resistance) and F2 (103469-4) populations, and the method proves that the resistant and non-resistant rapes can be effectively identified within the dosage range of 0.5-1.0 g/L glyphosate concentration.
FIG. 6 shows the results of the co-segregation analysis of the seedling resistance expression and the presence of immature embryo root hairs in the method for identifying glyphosate-resistant oilseed rape seeds according to the embodiment of the invention;
wherein, 1-6 uses FMV35S as primer, 7-12 uses GOX as primer, and 13-18 uses CP4-EPSPS as primer.
As shown in fig. 6: the results show that when the concentration of the glyphosate is 0.5-1.0 g/L, rape seeds with young embryos and roots contain herbicide-resistant genes, and the rape seeds show resistance to the glyphosate in the seedling stage.
The invention has the beneficial effects that:
(1) has great significance in plant transgenic research, has good application prospect in rape germplasm purity identification, can greatly reduce investment of fund and time, and has certain practical value in large-scale detection.
(2) The invention can be obtained that the resistance and non-resistance rape plants can be completely and effectively distinguished by adding additional screening pressure in the germination stage of glyphosate-resistant progeny seeds.
(3) The detection method is rapid, simple, convenient, cost-saving, labor-saving and reliable in result, and has certain practical value in large-scale rape germplasm purity identification and glyphosate-resistant rape seed screening.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (5)
1. The identification method of the glyphosate-resistant rape seeds is characterized by comprising the following steps:
s1, placing the rape seeds to be detected in a solution with glyphosate resistance concentration of 0.1-1.5 (g/L) for soaking for 8-10 h;
s2, dark culturing the soaked seeds in the S1;
s3, judging the rape variety to be glyphosate-resistant rape when the embryo root of the young embryo of the seed has root hair; the embryonic roots of the young embryos have no root hairs, and the young embryos are the non-glyphosate-resistant rape.
2. The method for identifying glyphosate-resistant oilseed rape seeds of claim 1, wherein the glyphosate-resistant concentration is 0.5-1.0 (g/L).
3. The method for identifying glyphosate-resistant oilseed rape seeds of claim 1 or 2, wherein the dark culture time is 48-60 h.
4. The method of claim 1, further comprising determining whether said rapeseed cultivar is resistant or non-resistant rapeseed based on whether said young embryonic radicles of said seeds have reached a predetermined length.
5. The method for identifying glyphosate resistant rape seeds of claim 1, further comprising transplanting the tested rape immature embryos into a substrate for culture, and performing resistance identification verification according to seedlings.
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| CN101939435A (en) * | 2007-09-21 | 2011-01-05 | 巴斯夫植物科学有限公司 | Plants with increased yield |
| CN105340743A (en) * | 2015-11-11 | 2016-02-24 | 山西省农业科学院棉花研究所 | Method for rapidly detecting transgenic glyphosate-resistant cotton |
| CN105637091A (en) * | 2013-08-12 | 2016-06-01 | 巴斯夫农业公司 | Plants having increased tolerance to herbicides |
| CN105941145A (en) * | 2016-05-04 | 2016-09-21 | 中国农业科学院特产研究所 | Germination method of Oreorchis patens seeds |
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| CN101939435A (en) * | 2007-09-21 | 2011-01-05 | 巴斯夫植物科学有限公司 | Plants with increased yield |
| CN105637091A (en) * | 2013-08-12 | 2016-06-01 | 巴斯夫农业公司 | Plants having increased tolerance to herbicides |
| CN105340743A (en) * | 2015-11-11 | 2016-02-24 | 山西省农业科学院棉花研究所 | Method for rapidly detecting transgenic glyphosate-resistant cotton |
| CN105941145A (en) * | 2016-05-04 | 2016-09-21 | 中国农业科学院特产研究所 | Germination method of Oreorchis patens seeds |
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