CN110731263A - method for evaluating hybridization affinity of transgenic soybean and wild soybean - Google Patents
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Abstract
The invention belongs to the field of ecological safety evaluation of transgenic soybeans, and particularly relates to methods for evaluating hybridization affinity of transgenic soybeans and wild soybeans.
Description
Technical Field
The invention relates to methods for evaluating hybridization affinity of transgenic soybeans and wild soybeans, belonging 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 method is characterized in that wild soybeans (Glycine soja Sieb. et Zucc.) have the same number of chromosomes as soybean chromosomes 2n which is 40, belong to the same G chromosome group, are direct ancestors of the soybeans and are important components of genetic resources of the soybeans, and have important values in researches on the origin and evolution of the soybeans.A survey finds that the soybean planting areas except Hainan province in China have the distribution of the wild soybeans, and the overlapping area of the planting areas of the planted soybeans and the wild soybean distribution areas is gradually increased along with the increase of the planting area of the soybeans.soybean and the wild soybeans are not reproductive isolated, so that the soybeans are easy to cross, and the related researches at present show that the planted soybeans and the wild soybeans have gene mutual infiltration in the natural environment.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides methods for evaluating the hybridization affinity of transgenic soybeans and wild soybeans.
The technical scheme of the invention is as follows:
method for evaluating the hybridization affinity of transgenic soybean to wild soybean includes such steps as counting the number of saturated seeds in the case that transgenic soybean is used as male parent and wild soybean, crossing, and the number of saturated seeds in the case that wild soybean is self-crossed.
The method of evaluating the hybridization affinity of transgenic soybeans for wild soybeans according to claim 1, wherein said 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;
a complete emasculation technology, namely selecting a bud which is not subjected to selfing at the middle upper part of a wild soybean plant and has mature stigma when the wild soybean and the transgenic soybean meet in flowering phase, removing a calyx by using a 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 the anther , and then taking fresh leaves around to wrap the periphery of the 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;
the selfing bud setting technology is that the bud of wild soybean plant is selected and the growth state of the bud is when the wild soybean plant is hybridized, and the selected bud is marked 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 index and grade of 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.
, sowing the material in a plastic flowerpot with a hole at the bottom, uniformly mixing the planting matrix of farmland soil and organic cultivation soil in a ratio of 1: 1, planting 1 plant of each wild soybean pot, 8 plants of each wild soybean pot with repetitions, 4 repetitions in total, planting 1 batch of transgenic soybeans with 1 plant of each pot, 8 plants of each transgenic soybean pot with repetitions in total, planting 3 batches of the transgenic soybeans with 10-day intervals in each batch, selecting the transgenic soybeans capable of meeting the florescence of the wild soybeans, carrying out normal field management in the whole growth cycle of the material, including watering, weeding and killing insects, and inserting a bamboo rod with the length of 2 meters beside the plant when the wild soybeans grow to the 3 rd three-row compound leaf, so that the wild soybeans can grow on tendrils conveniently.
At step , the emasculation and pollination persons should be trained to perform the experiment when 50 emasculation pollinates at a 0% rate.
And , setting the corolla of the bud below the highest calyx by 0.5-1 mm, and making the stigma of the bud swell and transparent and disc-shaped without breaking anthers.
And , the operation time of the castration step is 17: 00-19: 00 in the afternoon.
And , the operation time of artificial pollination is 6: 00-8: 00 in the morning.
And , wrapping fresh leaves after artificial pollination is completed, keeping for 5-7 d, 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 allowing any young pod to naturally grow to be mature.
At step , at least 200 flower buds are selected from the soybean plants in the crossing and natural conditions.
At step , the pod bearing rate data from the hybridization experiments were subjected to group statistics, with at least 4 replicates per treatment, at least 20 pods per replicate, and less than 20 all statistics.
Further , the step of affinity assessment is performed using SPSS software for significance analysis.
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 methods for evaluating the hybridization affinity of the transgenic soybeans and the wild soybeans, solves the problem that no method for evaluating the hybridization affinity of the transgenic soybeans and the wild soybeans exists at present, not only can provide scientific reference standards for affinity experiments of the wild soybeans and the transgenic soybeans of different populations, but also can provide a theoretical basis for ecological risk research for evaluating the 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 shows the comparison of the pod bearing of wild soybean inbred with the pod bearing of transgenic soybean pollen, row for the pod bearing of wild soybean inbred, and row two for the pod bearing of transgenic soybean crossing with wild soybean.
FIG. 2 shows the comparison of the seeds obtained by selfing wild soybean with the seeds obtained by pollinating the pollen of the transgenic soybean, at line , the seeds obtained by selfing wild soybean are shown, and the seeds obtained by hybridizing the transgenic soybean with the wild soybean are shown in the second line.
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 is further described in detail in step by way of specific embodiments.
Example 1
As shown in FIG. 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, and 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 methods 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.
method for evaluating the hybridization affinity between transgenic soybean and wild soybean includes ensuring the meeting of florescence, thorough emasculation, successful pollination, choosing selfing bud, statistical data and evaluating affinity.
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;
a complete emasculation technology, namely selecting a bud which is not subjected to selfing at the middle upper part of a wild soybean plant and has mature stigma when the wild soybean and the transgenic soybean meet in flowering phase, removing a calyx by using a 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 the anther , and then taking fresh leaves around to wrap the periphery of the 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;
the selfing bud setting technology is that the bud of wild soybean plant is selected and the growth state of the bud is when the wild soybean plant is hybridized, and the selected bud is marked 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 flowering phase meeting planting technology is characterized in that on the basis of fully knowing the growth characteristics of transgenic soybeans and 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, planting matrix is farmland soil and organic cultivation soil is mixed in a ratio of 1: 1, each pot of the wild soybeans is 1, 8 plants are repeats for 4 times, 1 batch of the wild soybeans is planted, in order to ensure that the wild soybeans can meet the flowering phase of glyphosate-resistant transgenic soybeans, each pot of the transgenic soybeans is 1, 8 plants are repeats for 4 times, 3 batches of the transgenic soybeans are planted, each batch is separated by 10 days, normal field management is carried out in the whole growth cycle of the materials, the field management comprises watering, weeding and insect killing, and when the wild soybeans grow to the 3 rd three-end compound leaf, bamboo shoots with the length of 2 meters are inserted beside the plants, so that the wild soybeans can grow.
Hybridization of glyphosate-resistant transgenic soybeans with 18 wild soybean populations:
the complete emasculation technology comprises the steps of selecting 18 flower buds at the middle upper parts of wild soybean plants, enabling the corolla of the flower buds to be 0.5-1 mm lower than the highest calyx, enabling the stigma of the flower buds to be enlarged and transparent and to be in a disc shape at the moment, enabling anthers not to break and scatter and not to self-breed, selecting flower buds with fixed buds on the wild soybean population plants at 17: 00-19: 00 afternoon for emasculation, removing the calyx with a pair of forceps, pinching the middle parts of the whole corolla, slightly applying force in a direction of inclining a flag flap, removing the whole corolla and the anthers together, and wrapping fresh leaves around the periphery of the flower buds to protect the 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.
A self-copulating bud-setting technique for choosing 18 buds from wild soybean plant population features that the bud state is when the buds are hybridized, and the chosen buds are marked.
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 saturated seeds per pod and calculation standard, the pods grown in the experiment are grouped and counted, each treatment is divided into at least 4 times of repetition, at least 20 pods are counted each time, less than 20 pods are counted, significance analysis is performed by using SPSS software, according to pod-set rate (+) + standard error data, it can be known that the selfing rate of different wild soybean populations is high and is between 96.50% and 99.50%, wherein the lowest of gehin-2, gansu baiyin and cinnamoyl forest has a value of 96.5%, the highest of sheng yang has a value of 99.5%, according to significance analysis data, significant differences exist among the selfing rates of different wild soybean populations, while according to the statistics of the number of saturated seeds of transgenic soybean and wild soybean, the number of seeds under the situation of natural selfing is counted, and the highest of the number of hybrid seeds under the situation of the same wild soybean is found that the number of the transgenic soybean is 100% multiplied by the number of the selfing rate of the same wild soybean, and the highest of the selfing rate of the wild soybean is found that the southern and wild soybean are equal to the highest of the transgenic soybean inbred population of the same wild soybean, wherein the difference is found that the highest of the southern soybean is equal to the highest to the transgenic soybean hull-wild soybean, the highest difference of the transgenic soybean hull-wild soybean, the highest of the transgenic soybean, the transgenic soybean hull-wild soybean, the transgenic soybean hull-set difference is 3957, and the difference is equal to the difference of the southern soybean, and the southern soybean, wherein the highest difference is found that the difference of the southern-wild soybean, the difference is equal to the southern soybean, the difference is equal to be equal to the southern soybean, the.
2. Table 3 table 4 shows that when the average number of saturates per pod and the calculation criteria are met, the pods from the test are grouped and counted, each process is repeated at least 4 times, at least 20 pods are counted each time, and less than 20 pods are counted all statistically, and the significance analysis is performed by using SPSS software, according to the average number of saturates per pod ± criteria, the average number of saturates per pod of the same wild soybean population is significantly higher than the average number of saturates per pod of the wild soybean population when the wild soybean population is hybridized with the transgenic soybean, wherein the eastern east difference is at least 1.41, and the eastern dongtianhaerbin-1 difference is at most 2.39.
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
Table 5 shows that 3.2 percent of saturation ratio in chenzhou of hunnan is high in affinity, 0.41 percent of saturation ratio in north river is low in affinity, 2.16 to 2.88 percent of saturation ratio in north river is high in affinity, 352 percent of black dragon harbourne-1, black dragon harja-2, jehlin white city-2, shening yang, shandong jiang ying, hengsu, ansu, yinshun, ganjun, and tiengqing is low in affinity, and 0.96 percent of saturation ratio in south lake is low in affinity.
Claims (10)
- The method for evaluating the hybridization affinity of the transgenic soybean and the wild soybean is characterized by comprising the steps of counting the number of the saturated seeds which are grown by hybridizing the transgenic soybean serving as a male parent and the wild soybean under the same growth environment condition and the number of the saturated seeds which are grown by selfing the wild soybean, and evaluating the hybridization affinity of the transgenic soybean and the wild soybean by taking the ratio of the number of the saturated seeds which are grown by hybridizing and selfing as a saturation ratio.
- 2. The method of evaluating the hybridization affinity of transgenic soybeans for wild soybeans according to claim 1, wherein said method comprises: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 the anther , and then taking fresh leaves around to wrap the periphery of 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, leading the growth state to be 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: the saturation ratio (%) is calculated as (number of cross-saturation/number of self-saturation) × 100%, and the cross-compatibility of the transgenic soybean and the wild soybean is evaluated by the saturation ratio.
- 3. The method for evaluating the hybridization affinity of transgenic soybeans with wild soybeans according to claim 2, wherein the evaluation index and the classification of the 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.
- 4. The method for evaluating the hybridization affinity of transgenic soybeans and wild soybeans according to claim 2, wherein the material is sown in a plastic flowerpot with a hole at the bottom, the planting matrix is farmland soil and is mixed with organic cultivation soil in a ratio of 1: 1, the wild soybeans are planted in 1 batch with 1 pot and 8 pots and repetitions with 4 repetitions, the transgenic soybeans are planted in 1 batch with 1 pot and 8 repetitions with 8 pots and repetitions with 4 repetitions, 3 batches are planted with 10-day intervals in each batch to select the transgenic soybeans capable of meeting the flowering phase of the wild soybeans, normal field management including watering, weeding and insect killing is carried out in the whole growth cycle of the material, and when the wild soybeans grow to the 3 rd three-leaf complex, 2-meter bamboo is inserted beside the plants to facilitate the climbing of the wild soybeans to grow.
- 5. The method of varieties for evaluating the hybridization affinity of transgenic soybean to wild soybean according to claim 2, wherein the corolla of the bud is selected to be 0.5mm-1mm below the highest calyx, and the stigma of the bud is enlarged and transparent and is in the shape of a disc without breaking the anther.
- 6. The method for evaluating hybrid affinity of transgenic soybeans with wild soybeans as claimed in claim 2, wherein the emasculation step is performed at 17: 00-19: 00 pm.
- 7. The method for evaluating hybridization affinity of transgenic soybeans and wild soybeans according to claim 2, wherein the operation time of artificial pollination is 6: 00-8: 00 a.m.
- 8. The method for evaluating hybridization affinity of transgenic soybean and wild soybean according to claim 2, wherein the fresh leaves are covered after artificial pollination for 5-7 days, the flowers after artificial pollination are waited for to form young pods, the dried leaves originally covered around the hybridized flowers are removed, and the young pods are naturally grown to maturity.
- 9. The method of varieties for evaluating the hybridization affinity of transgenic soybeans for wild soybeans according to claim 2, wherein at least 200 flower buds are selected from soybean plants in hybridization and natural conditions.
- 10. The method of for evaluating the hybridization affinity of transgenic soybean to wild soybean according to claim 2, wherein the pod bearing rate data of hybridization test is grouped into statistics, each treatment is repeated at least 4 times, each time the repetition counts at least 20 pods, less than 20 all the statistics.
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