CN110731263B - Method for evaluating hybridization affinity of transgenic soybean and wild soybean - Google Patents
Method for evaluating hybridization affinity of transgenic soybean and wild soybean Download PDFInfo
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Abstract
本发明属于转基因大豆生态安全评估领域，具体涉及一种评价转基因大豆与野生大豆杂交亲和性的方法，统计相同生长环境条件下转基因大豆为父本与野生大豆杂交所结荚的饱粒种子数和野生大豆自交所结荚的饱粒种子数的饱粒比，评价转基因大豆与野生大豆杂交亲和性。具体试验操作步骤包括花期相遇的种植技术、彻底去雄技术、成功授粉技术、自交花蕾定蕾技术、数据统计方法、亲和性评价技术。本发明提出了转基因大豆与野生大豆杂交亲和性评价试验实施的技术和评价标准，对转基因大豆安全地投入生产时的环境风险管理及预防具有重要作用。本发明具有试验操作性强、数据处理简单、结果客观、准确性高等优点。
The invention belongs to the field of ecological safety assessment of transgenic soybeans, and in particular relates to a method for evaluating the hybridization compatibility of transgenic soybeans and wild soybeans. The cross-compatibility between transgenic soybean and wild soybean was evaluated by the ratio of saturated seeds to the number of saturated seeds podded by wild soybean self-crossing. The specific experimental operation steps include planting technology of meeting in flowering period, thorough emasculation technology, successful pollination technology, self-fertilization budding technology, data statistics method, and affinity evaluation technology. The invention proposes the technology and evaluation standard for the implementation of the hybridization compatibility evaluation test of the transgenic soybean and the wild soybean, which plays an important role in the environmental risk management and prevention when the transgenic soybean is safely put into production. The invention has the advantages of strong test operability, simple data processing, objective results and high accuracy.
Description
Technical Field
The invention relates to a method for evaluating hybridization affinity of transgenic soybeans and wild soybeans, and belongs to the field of ecological safety evaluation of transgenic soybeans.
Background
Transgenic crops began to grow commercially in 1996. By 2018, the planting area of transgenic crops is increased to 1.917 hundred million hectares. The transgenic soybean (Glycine max) in all transgenic crops has the largest planting area, reaches 0.959 hundred million hectares in 2018 and accounts for 50 percent of the global transgenic crop planting area, and all the planted transgenic soybeans contain herbicide-resistant genes. In recent years, the import of Chinese soybeans has been continuously increased, and 9553 million tons has been reached by 2017. China is a main distribution area of wild soybean resources, and in order to prevent foreign genes from polluting the wild soybean resources, the risk of gene drift possibly brought by transgenic soybeans is evaluated systematically and reluctantly before commercial production.
The number of chromosomes of wild soybeans (Glycine soja Sieb. et Zucc.) is the same as that of soybean chromosomes 2 n-40, the wild soybeans belong to the G chromosome group, are direct ancestors of the soybeans, are important components of genetic resources of the soybeans, and have important values in the research of soybean origin and evolution. According to investigation, the distribution of wild soybeans is found in other soybean planting areas except Hainan province in China, and the overlapping area of the soybean planting areas and the wild soybean distribution areas is gradually increased along with the increase of the soybean planting area. The soybean and the wild soybean have no reproductive isolation and are easy to hybridize, and related researches show that the cultivated soybean and the wild soybean are subjected to gene cross-infiltration in the natural environment at present. Pollen-mediated gene drift is the major pathway of gene drift, and the possibility of gene drift is influenced by the affinity of transgenic crops and kindred species, the degree of flowering overlap, the degree of distribution overlap, environmental (e.g., wind direction) conditions, etc., and the fitness of progeny carrying resistance genes. Affinity determined by genetic background is the determining factor. Wild soybean resources in different areas of China have certain genetic differences, and abundant genetic differentiation occurs in the process of adapting to different growth environments for a long time. Due to the difference of the affinities of the transgenic soybeans and wild soybeans with different genetic backgrounds, the risks of planting the transgenic soybeans in different production areas are different under the condition of overlapping flowering periods; meanwhile, due to the difference of the genetic background, the pod setting rate and the seed setting rate of different wild soybean populations under natural conditions have significant difference, and the evaluation of the affinity by using the pod setting rate and the seed setting rate alone is not scientific. Therefore, the scientific and accurate evaluation method for the hybridization affinity of the transgenic soybeans and the wild soybeans is provided, not only can provide scientific reference standards for affinity experiments of the wild soybeans and the transgenic soybeans in different populations, but also can provide theoretical basis for ecological risk research for evaluating the gene drift of the transgenic soybeans to the wild soybeans.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a method for evaluating the hybridization affinity of transgenic soybeans and wild soybeans.
The technical scheme of the invention is as follows:
a method of evaluating the hybridization affinity of a transgenic soybean to a wild soybean, the method comprising: counting the number of the saturated seeds which are subjected to pod formation by taking the transgenic soybean as a male parent and a wild soybean under the same growth environment condition and the number of the saturated seeds which are subjected to pod formation by selfing of the wild soybean, and taking the ratio of the number of the saturated seeds subjected to pod formation by hybridization and selfing as a grain saturation ratio to evaluate the hybridization affinity of the transgenic soybean and the wild soybean.
The method of claim 1, wherein the method comprises:
the planting technology of flowering synchronization: on the basis of fully understanding the growth characteristics of the transgenic soybean and the wild soybean, the transgenic soybean and the wild soybean are planted in batches, so that the flowering phases of the transgenic soybean and the wild soybean meet each other;
complete castration technology: when the flowering phases of the wild soybean and the transgenic soybean meet, selecting a flower bud which is not subjected to selfing at the middle upper part of a wild soybean plant and has a mature stigma, removing a calyx by using a forceps, pinching the middle part of the whole flower crown, slightly applying force in a direction of inclining a flag flap, removing the whole flower crown together with anther, and then wrapping fresh leaves around the flower bud to protect the naked stigma;
the successful pollination technology comprises the following steps: firstly, taking leaves wrapped on the castrated buds of the wild soybeans, selecting the buds of the transgenic soybeans of the male parent, the corolla of which is not completely opened and the flag petals of which are higher than the sepals, taking out anthers of the buds and rubbing the anthers on the castrated stigmas of the wild soybeans to ensure that pollen of the male parent falls on the stigmas of the female parent buds, and wrapping fresh leaves after pollination is finished;
selfing bud setting technology: selecting flower buds on wild soybean plants, wherein the growth state of the flower buds is consistent with that of the flower buds during hybridization, and marking the selected flower buds without other treatment;
and (3) data statistics: when the seeds are mature, harvesting the pods which grow mature by self-crossing under natural conditions and the pods which grow mature successfully by crossing, and respectively counting the number of the mature full-grain seeds under the two methods;
affinity evaluation technique: according to the formula: the saturation ratio (%) was calculated as (number of saturated hybrid/number of self-saturated hybrid) x 100%, the hybridization affinity of the transgenic soybean and the wild soybean was evaluated by the saturation ratio, and the hybridization affinity of the population was evaluated by five grades of 1, 2, 3, 4, and 5 according to the saturation ratio.
Furthermore, the evaluation indexes and grades of the hybridization affinity in the affinity evaluation step are named as:
level 1: the saturated grain ratio is more than 3.0 percent, the affinity is extremely high,
and 2, stage: 3.0 percent or more than 2.0 percent of saturated grain ratio, high affinity,
and 3, level: 2.0% or more of the saturated particle ratio of more than 1.0%, and in affinity,
4, level: 1.0% or more of saturated grain ratio of more than 0.5%, low affinity,
and 5, stage: the saturated particle ratio is more than or equal to 0.5 percent, and the affinity is extremely low.
Further, the material is sowed in the porose plastic flowerpot in bottom, and the planting matrix is farmland soil and organic cultivation soil 1: 1 and mixing uniformly. Wild soybeans were planted in 1 pot with 8 replicates for each pot, for a total of 4 replicates, and 1 batch was planted. 1 plant is planted in each pot of transgenic soybeans, 8 plants are repeated for 4 times, 3 batches are planted, and the interval between every two batches is 10 days; to select transgenic soybeans that meet the wild soybean in flowering phase; normal field management including watering, weeding and killing insects is carried out in the whole growth period of the material. When the wild soybeans grow to the 3 rd three-leaf complex, bamboo poles with the length of 2 meters are inserted beside the plants, so that the wild soybeans can grow on tendrils conveniently.
Further, the experimenter in emasculation and pollination should be trained to perform the experiment when 50 emasculation and pollination are carried out with a pod bearing rate of 0%.
Furthermore, the corolla of the bud in the bud setting step is 0.5mm-1mm lower than the highest calyx, and the bud stigma expands, is transparent and disc-shaped, but anthers are not broken and scattered.
Furthermore, the operation time of the castration step is 17: 00-19: 00 in the afternoon.
Further, the operation time of artificial pollination is 6: 00-8: 00 in the morning.
Further, wrapping fresh leaves for 5-7 days after artificial pollination is completed, waiting for the transgenic soybean pollen and the wild soybean stigma to combine to form a young pod, removing the dried leaves wrapped around the hybridized flower originally, and then allowing any young pod to naturally grow to be mature.
Further, at least 200 flower buds are selected from the soybean plants in the crossing and natural conditions.
Further, the pod bearing rate data of the hybridization test is subjected to grouping statistics, each treatment is repeated at least 4 times, at least 20 pods are counted in each repetition, and all statistics are counted when less than 20 pods are counted.
Further, the step of affinity evaluation was performed by significance analysis using SPSS software.
Has the advantages that:
the method provided by the invention counts the number of saturated seeds of the hybrid seeds of the transgenic soybeans and the wild soybeans and also counts the number of saturated seeds of the seeds under the natural selfing condition, and takes the ratio of the saturated seeds (%) (the number of saturated seeds of the hybrid/the number of saturated seeds of the selfing) multiplied by 100% as a reference standard for evaluating the affinity between the transgenic soybeans and the wild soybeans, so that errors caused by different pod setting rates and seed setting rates of wild soybean populations in various places are eliminated.
The invention can provide a method for evaluating the hybridization affinity of transgenic soybeans and wild soybeans, and solves the problem that no method for evaluating the hybridization affinity of the transgenic soybeans and the wild soybeans exists at present. The method can provide scientific reference standards for affinity experiments of wild soybeans and transgenic soybeans of different populations, and can also provide theoretical basis for ecological risk research for evaluating gene drift of the transgenic soybeans to the wild soybeans.
The invention provides a method for evaluating hybridization affinity of transgenic soybeans and wild soybeans, which can be used for evaluating the hybridization affinity of wild soybeans and transgenic soybeans all over the country, has an important role in scientifically evaluating the hybridization affinity of the transgenic soybeans and the wild soybeans, and is a premise for safely releasing the transgenic soybeans, so that prevention work can be done on the release of the transgenic soybeans in advance according to an affinity index.
Drawings
FIG. 1 is a comparison of the pod bearing of wild soybean selfing and the pod bearing of transgenic soybean pollen; the first row is the pod bearing of wild soybean selfing; the second row is the pod bearing when the transgenic soybean is crossed with the wild soybean.
FIG. 2 is a comparison of seeds obtained by selfing wild soybean and transgenic soybean pollen; the first row is seeds obtained by wild soybean selfing; the second row is seeds obtained by crossing the transgenic soybeans and wild soybeans.
FIG. 3 shows a flower selected from budding.
Detailed Description
The present invention will be described in detail with reference to specific examples.
The present invention will be described in further detail with reference to the following embodiments.
Example 1
As shown in figure 1, the pod formed by selfing wild soybean is obviously different from the pod formed by pollinating transgenic soybean pollen in appearance, the pods formed by selfing are all in a full state, while the pods formed by hybridizing the transgenic soybean and the wild soybean are all in an atrophied state, the number of the full pods is very small, which is a phenomenon caused by incompatibility of the two, so that the method is very important for establishing a method for evaluating the hybridization affinity of the transgenic soybean and the wild soybean.
As shown in fig. 2, the seeds produced by crossing the transgenic soybean with the wild soybean are significantly different from the seeds produced by selfing the wild soybean in morphology, the seeds produced by crossing the transgenic soybean with the wild soybean are mostly shrunken, and the seeds produced by selfing the wild soybean are mostly full. This results in a very significant difference between the two in the index of the number of saturated grains, so that the ratio of saturated grains is very critical for our evaluation of hybridization affinity.
As shown in figure 3, the top end of the calyx wrapped outside the bud is slightly opened, so that the corolla is light purple, the stigma of the bud is enlarged and transparent and is in a disc shape, but anthers are not broken and scattered, and the flower is not selfed.
A method for evaluating the hybridization affinity of transgenic soybean and wild soybean includes ensuring the meeting of flowering phases, ensuring thorough emasculation, ensuring successful pollination, selecting selfing buds, carrying out data statistics and carrying out affinity evaluation.
The method specifically comprises the following steps:
the planting technology of flowering synchronization: on the basis of fully understanding the growth characteristics of the transgenic soybean and the wild soybean, the transgenic soybean and the wild soybean are planted in batches, so that the flowering phases of the transgenic soybean and the wild soybean meet each other;
complete castration technology: when the flowering phases of the wild soybean and the transgenic soybean meet, selecting a flower bud which is not subjected to selfing at the middle upper part of a wild soybean plant and has a mature stigma, removing a calyx by using a forceps, pinching the middle part of the whole flower crown, slightly applying force in a direction of inclining a flag flap, removing the whole flower crown together with anther, and then wrapping fresh leaves around the flower bud to protect the naked stigma;
the successful pollination technology comprises the following steps: firstly, taking leaves wrapped on the castrated buds of the wild soybeans, selecting the buds of the transgenic soybeans of the male parent, the corolla of which is not completely opened and the flag petals of which are higher than the sepals, taking out anthers of the buds and rubbing the anthers on the castrated stigmas of the wild soybeans to ensure that pollen of the male parent falls on the stigmas of the female parent buds, and wrapping fresh leaves after pollination is finished;
selfing bud setting technology: selecting flower buds on wild soybean plants, wherein the growth state of the flower buds is consistent with that of the flower buds during hybridization, and marking the selected flower buds without other treatment;
and (3) data statistics: when the seeds are mature, harvesting the pods which grow mature by self-crossing under natural conditions and the pods which grow mature successfully by crossing, and respectively counting the number of the mature full-grain seeds under the two methods;
affinity evaluation technique: according to the formula: the saturation ratio (%) was calculated as (number of saturated hybrid/number of self-saturated hybrid) x 100%, the hybridization affinity of the transgenic soybean and the wild soybean was evaluated by the saturation ratio, and the hybridization affinity of the population was evaluated by five grades of 1, 2, 3, 4, and 5 according to the saturation ratio.
The evaluation indexes and grades of the hybridization affinity in the affinity evaluation step are named as:
level 1: the saturated grain ratio is more than 3.0 percent, the affinity is extremely high,
and 2, stage: 3.0 percent or more than 2.0 percent of saturated grain ratio, high affinity,
and 3, level: 2.0% or more of the saturated particle ratio of more than 1.0%, and in affinity,
4, level: 1.0% or more of saturated grain ratio of more than 0.5%, low affinity,
and 5, stage: the saturated particle ratio is more than or equal to 0.5 percent, and the affinity is extremely low.
Example 2
Evaluation of hybridization affinity of glyphosate-resistant transgenic soybeans and 18 wild soybean populations
The implementation method comprises the following steps:
collection of experimental materials: the glyphosate-resistant transgenic soybean is provided by the national soybean improvement center of Nanjing agriculture university. Wild soybean populations were collected in 18 different geographical locations throughout the country, see table 1.
TABLE 1 basic information on the population of wild soybeans tested
The planting technology of flowering synchronization: on the basis of fully understanding the growth characteristics of the transgenic soybeans and the wild soybeans, the transgenic soybeans and the wild soybeans are planted in batches, a seeding container is a plastic flowerpot with a hole at the bottom, and planting substrates are farmland soil and organic cultivation soil 1: 1 and mixing uniformly. Wild soybeans were planted in 1 pot with 8 replicates for each pot, for a total of 4 replicates, and 1 batch was planted. In order to ensure that the wild soybeans can meet the flowering phase of the glyphosate-resistant transgenic soybeans, 1 plant of each pot of the transgenic soybeans is planted, 8 plants are repeated for 4 times, and 3 batches of the transgenic soybeans are planted at an interval of 10 days. Normal field management including watering, weeding and killing insects is carried out in the whole growth period of the material. When the wild soybeans grow to the 3 rd three-leaf complex, bamboo poles with the length of 2 meters are inserted beside the plants, so that the wild soybeans can grow on tendrils conveniently.
Hybridization of glyphosate-resistant transgenic soybeans with 18 wild soybean populations:
complete castration technology: selecting 18 flower buds at the middle upper parts of wild soybean plants, wherein the corolla of the flower bud is 0.5-1 mm lower than the highest calyx, and the stigma of the flower bud is enlarged, transparent and disc-shaped, but anthers are not broken and scattered, and selfing does not occur. And (3) selecting flowers with fixed buds on each wild soybean population plant at 17: 00-19: 00 afternoon for emasculation, removing calyxes by using forceps, pinching the middle part of the whole corolla, slightly applying force in a direction of inclining a flag flap, removing the whole corolla together with anthers, and then wrapping fresh leaves around the periphery of the flower buds to protect naked stigma.
The successful pollination technology comprises the following steps: at 6: 00-8: 00 in the morning, firstly taking leaves wrapped on emasculated buds of wild soybeans, selecting buds of male parent glyphosate-resistant transgenic soybeans, wherein crowns are not completely opened and petals of the flowers are higher than sepals, taking out anthers of the buds, slightly rubbing the buds on emasculated stigmas of the wild soybeans to ensure that pollen of the male parents falls on the stigmas of the female parent buds, hanging a hang tag after pollination is completed, clearly writing hybridization combinations and dates, wrapping fresh leaves for 5-7 days, waiting for flowers subjected to artificial hybridization pollination to form young pods, removing the dried leaves originally wrapped around the hybridized flowers, and naturally growing the young pods to be mature.
Selfing bud setting technology: and selecting the flower buds of 18 wild soybean population plants, wherein the growth state of the flower buds is consistent with that of the flower buds during hybridization, and marking the selected flower buds without other treatment.
And (3) data statistics: and harvesting the pods grown to be mature by selfing under natural conditions and the pods grown to be mature successfully by hybridization, and respectively counting the pod bearing rate and the number of mature saturated seeds under the two methods.
Affinity evaluation technique: according to the formula: the saturation ratio (%) was calculated as (number of saturated hybrid/number of self-saturated hybrid) x 100%, the hybridization affinity between the transgenic soybean and the wild soybean was evaluated by the saturation ratio, and the hybridization affinity of the population was evaluated by five grades of 1, 2, 3, 4, and 5 according to the saturation ratio, and the classification criteria are shown in table 2.
TABLE 2 evaluation of affinity grading criteria
Test results and analysis:
TABLE 3 transgenic Soybean and wild Soybean hybridization data
TABLE 4 wild Soybean inbred data
TABLE 5 castration and non-pollination pod-set rate data for wild soybeans
1. Table 3 table 4 statistics of average number of satiations per pod and calculation of standard errors, pod held in the experiment was subjected to group statistics, each treatment was divided into at least 4 replicates, at least 20 pods were counted per replicate, less than 20 pods were counted all, and significance analysis was performed using SPSS software. From the pod bearing rate (%) ± standard error data, it can be known that: the selfing and pod-bearing rate of different wild soybean populations is high and is between 96.50% and 99.50%, wherein Jilin Baicheng-2, Gansu Baiyin and Guangxi Guilin are the lowest in value and 96.5%, and Liaoning Shenyang is the highest in value and 99.5%. According to significance analysis data, the self-pod-bearing rates of different wild soybean populations have significance differences, the method also counts the seed saturation number under the natural self-breeding condition while counting the seed saturation number of the hybrid seeds of the transgenic soybeans and the wild soybeans, and takes the saturation number ratio (%) (the hybrid saturation number/the self-selfing saturation number) multiplied by 100% as a reference standard for evaluating the high and low affinity of the transgenic soybeans and the wild soybeans, so that errors caused by the fact that the pod-bearing rates and the seed-bearing rates of different wild soybean populations are different are eliminated. The pod bearing rate of the transgenic soybean and the wild soybean in hybridization is between 4.92% and 18.03%, wherein Hebei baoding is the lowest, the value is 4.92%, and Chenzhou, Hunan is the highest, the value is 18.03%. It can be easily seen that the hybrid pod bearing rate of the transgenic soybean and the wild soybean is greatly different from the selfing pod bearing rate of the same wild soybean population, the hybrid pod bearing rate of the transgenic soybean and the wild soybean is obviously lower than the selfing pod bearing rate of the same wild soybean population by 80.47% -94.08%, wherein the minimum difference of Chenzhou in Hunan province is 80.47%, and the maximum difference of Baoding in Hebei province is 94.08%.
2. Table 3 table 4 statistics of mean number of satiations per pod and calculation of standard errors, pod from the trial was grouped and counted for at least 4 replicates per treatment, at least 20 pods were counted per replicate, less than 20 were counted for all, and significance analysis was performed using SPSS software. From the mean number of particles per pod ± standard error, it can be known that: the average number of grains per pod of the same wild soybean population is obviously higher than that of grains per pod of the wild soybean population when the wild soybean population is hybridized with transgenic soybeans, wherein the minimum difference between east and east of Shandong is 1.41, and the maximum difference between Haerbin-1 of Heilongjiang is 2.39. The average number of saturated grains per pod under the selfing condition of different populations is between 1.7 and 2.69, and significant differences exist, wherein the highest value of Jiangsu Hezhou is 2.69, and the lowest value of Shandong Dongying is 1.7. The average number of grains per pod of filial generations of different wild soybean populations and transgenic soybeans is between 0.2 and 0.48, and significant differences exist, wherein Helongjiang Harbin-1 is 0.2 at the lowest, and Hebei handan is 0.48 at the highest.
3. Table 5 statistics of emasculation and non-pollination pod-bearing rates, which can be derived from the emasculation and non-pollination data: the pod bearing rate of emasculation and non-pollination in 4 repetitions is 0%, which shows that the bud fixing and emasculation steps in the test process are accurate and scientific, the phenomenon that buds have self-pollinated does not occur, and emasculation is thorough.
Table 618 wild soybean population and glyphosate-resistant transgenic soybean hybridization affinity expression
The number of saturated seeds in the cross in table 6 is the cross podding rate × 200 × average number of saturated seeds per pod, and the number of filled seeds obtained by crossing 200 flowers was obtained. The self-crossing plump seed number is the self-crossing pod-bearing rate multiplied by 200 multiplied by the average plump seed number per pod, and 200 flowers are obtained and are subjected to self crossing to obtain the plump seed number. As can be seen from table 5: chenzhou, Hunan, has a full grain ratio of 3.2% and extremely high affinity; hebei was confirmed to have a saturated grain ratio of 0.41%, and the affinity was the lowest. The satiety ratio of Jilin white city-1, Hebei Handan, Zhejiang Hainin, inner Mongolia Baotou, Henan Shangqiu and Jiangxi Yingtan is 2.16% -2.88%, and the affinity is high; the saturation particle ratio of Heilongjiang Harbin-1, Heilongjiang Harbin-2, Jilin Baicheng-2, Shenyang Liaoning, Dongying in Shandong, Changzhou in Jiangsu, Anhui Anqing, Gansu silver and Guangxi Guilin is 1.17% -1.84%, and the affinity is shown in middle; the saturation ratio of Feinging TieLing is 0.96%, and the affinity is low.
Claims (6)
1. A method for evaluating the hybridization affinity of a transgenic soybean to a wild soybean, said method comprising:
planting transgenic soybean and wild soybean in batches to ensure that the flowering phases of the transgenic soybean and the wild soybean meet each other;
when the flowering phases of the wild soybean and the transgenic soybean meet, selecting a flower bud which is not subjected to selfing at the middle upper part of a wild soybean plant and has a mature stigma, removing a calyx by using a forceps, pinching the middle part of the whole flower crown, slightly applying force in a direction of inclining a flag petal, removing the whole flower crown together with anther, and then wrapping fresh leaves around the flower bud to protect the naked stigma;
firstly, taking leaves wrapped on the castrated buds of the wild soybeans, selecting the buds of the transgenic soybeans of the male parent, the corolla of which is not completely opened and the flag petals of which are higher than the sepals, taking out anthers of the buds and rubbing the anthers on the castrated stigmas of the wild soybeans to ensure that pollen of the male parent falls on the stigmas of the female parent buds, and wrapping fresh leaves after pollination is finished;
selecting flower buds on wild soybean plants, wherein the growth state of the flower buds is consistent with that of the flower buds during hybridization, and marking the selected flower buds without other treatment;
when the seeds are mature, harvesting the pods which grow mature by self-crossing under natural conditions and the pods which grow mature successfully by crossing, and respectively counting the number of the mature full-grain seeds under the two methods;
according to the formula: calculating a saturated grain ratio by using a saturated grain ratio (%) = (number of cross saturated grains/number of self-cross saturated grains) × 100%, and evaluating the cross affinity of the transgenic soybean and the wild soybean according to the saturated grain ratio; evaluation indexes and grades of hybridization affinity are named as:
level 1: the saturated grain ratio is more than 3.0 percent, the affinity is extremely high,
and 2, stage: 3.0 percent or more than 2.0 percent of saturated grain ratio, high affinity,
and 3, level: 2.0% or more of the saturated particle ratio of more than 1.0%, and in affinity,
4, level: 1.0% or more of saturated grain ratio of more than 0.5%, low affinity,
and 5, stage: the saturated particle ratio is more than or equal to 0.5 percent, and the affinity is extremely low;
selecting at least 200 flower buds on soybean plants in hybridization and natural conditions;
and (4) carrying out grouping statistics on pod bearing rate data of the hybridization test, wherein each treatment is repeated at least 4 times, at least 20 pods are counted in each repetition, and all the pods are counted if the number of the pods is less than 20.
2. The method for evaluating the hybridization affinity of the transgenic soybeans and the wild soybeans as claimed in claim 1, wherein the materials are sown in plastic flowerpots with holes at the bottom, and planting substrates are farmland soil and organic cultivation soil 1: 1, uniformly mixing; 1 strain of wild soybean is planted in each pot, 8 strains are repeated for 4 times, and 1 batch of wild soybean is planted; 1 plant of transgenic soybean is planted in each pot, 8 plants are repeated for 4 times, 3 batches are planted, and the interval between each batch is 10 days, so that the transgenic soybean which can meet the florescence of the wild soybean is selected; normal field management including watering, weeding and killing insects is carried out in the whole growth period of the material; when the wild soybeans grow to the 3 rd three-leaf complex, bamboo poles with the length of 2 meters are inserted beside the plants, so that the wild soybeans can grow on tendrils conveniently.
3. The method of claim 1, wherein the corolla of the flower bud is 0.5mm to 1mm below the highest calyx, and the stigma of the flower bud is enlarged and transparent and is in the shape of a disc without anthers breaking.
4. The method for evaluating the hybridization affinity of the transgenic soybean and the wild soybean according to claim 1, wherein the emasculation step is performed at 17:00 to 19:00 in the afternoon.
5. The method for evaluating the hybridization affinity of the transgenic soybean and the wild soybean according to claim 1, wherein the operation time of artificial pollination is 6: 00-8: 00 in the morning.
6. The method for evaluating the hybridization affinity of transgenic soybeans and wild soybeans as claimed in claim 1, wherein the fresh leaves are covered and kept for 5-7 days after the artificial pollination is completed, the flowers subjected to the artificial hybridization pollination are waited for forming young pods, the dried leaves originally covered around the hybridized flowers are removed, and the young pods are naturally grown to be mature.
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