CN114467743A - Method for creating new germplasm of soybean with phytophthora root rot resistance - Google Patents
Method for creating new germplasm of soybean with phytophthora root rot resistance Download PDFInfo
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Abstract
The invention discloses a method for creating a new germplasm of soybean with phytophthora root rot resistance, which comprises the steps of inoculating wild soybean leaves with phytophthora to soybean, screening out wild soybean with phytophthora root rot resistance, hybridizing the screened wild soybean with phytophthora root rot resistance with cultivated soybean to obtain filial generations, screening the phytophthora root rot resistance of an inbred line of the filial generations, and simultaneously screening by combining with agronomic characters to create the new germplasm with phytophthora root rot resistance. Therefore, the resistance resources and the progeny materials are accurately screened, and the screening reliability is greatly enhanced.
Description
Technical Field
The invention belongs to the field of wild plant resource utilization research, and particularly relates to a method for creating a new soybean germplasm capable of resisting phytophthora root rot.
Background
Phytophthora Root Rot (PRR) of soybean was first discovered in ohio and indiana in the united states, and then has occurred in soybean production areas in countries such as australia, argentina, hungary, brazil, china, japan, canada, and korea, and has now become a major worldwide soybean disease.
The most effective and direct method for soybean phytophthora root rot is the application of disease-resistant varieties, so that the screening of resistance sources is particularly important. China is the origin of soybeans, wild soybean germplasm resources are very rich, and the resistance to phytophthora root rot is common and has geographical differences. The resistance source screening of phytophthora root rot is respectively carried out on soybeans and wild soybean resources from northeast China, Huang-Huai-Hai and abroad, and the results show that the soybean varieties in different ecological regions in China have rich resistance diversity, and China has more germplasms for resisting the phytophthora root rot of soybean.
The existing method for identifying the disease resistance of phytophthora sojae to root rot disease is mature and general, and is a hypocotyl wound mycelium inoculation method, the strain is phytophthora sojae (phytophthora sojae), and the evaluation standard of the inoculation identification method for anti-infection materials is as follows: and adding a inoculation period, taking the plants which have wilting property of the whole plants and are broken off from the inoculated parts and die after inoculation as infected plants, taking the plants which have no change or have brown stain on the local inoculated parts after inoculation as disease-resistant plants after the plants continue to grow normally. The growth characteristics of wild soybeans and progeny thereof are that the stems of the wild soybeans are thin and weak, the stems are sprawling and low in lignification degree, when the hypocotyl wound mycelium inoculation method is used for identification, the stems are particularly easy to cut off when the cut wounds are cut on the stems due to the thin and weak stems, and the lignification degree of the stems is very low, so that the stems of the wild soybeans and the progeny thereof are bent and droop to present a disease-sensitive state when the cut wounds are inoculated and the inoculation is not developed, and the resistance response of a test material cannot be accurately described when the evaluation is carried out according to the resistance identification standard of the method, so that a new method is urgently needed to identify the disease resistance of the wild soybeans and the progeny thereof to the phytophthora root rot resistant new germplasm and the creation of the wild soybeans.
Disclosure of Invention
The invention discloses a method for creating a new soybean germplasm with phytophthora root rot resistance, which solves the problem that the anti-infection reaction of an experimental material cannot be accurately described easily due to thin and weak wild soybean stems and easy breakage.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for creating a new germplasm of soybean with phytophthora root rot resistance, which comprises the following steps:
s1, preparing a carrot culture medium: cleaning fresh carrot, cutting into small pieces, weighing 100g, mincing with a plant tissue mincing machine, adding an appropriate amount of distilled water, boiling for 30min, filtering with 3 layers of gauze, fixing the volume of the filtered carrot juice to 500mL, adding 10g of agar, sterilizing at 120 ℃ for 20min under high temperature and high pressure, pouring the sterilized culture medium into a sterilized flat dish, wherein the height of the culture medium is 0.5 cm;
s2, culture of test strains: the strain is phytophthora sojae, square blocks of the phytophthora sojae with the diameter of 10mm multiplied by 10mm are inoculated to the carrot culture medium S1, and dark culture is carried out for 7 days at the temperature of 25 ℃;
s3, potting: after the skin of the wild soybean seeds is broken, performing potted planting, planting 10 seeds in each pot, leaving 5 seedlings with consistent growth in each pot after emergence, and starting leaf inoculation identification after the first three compound leaves of the seedlings are completely unfolded;
s4, leaf inoculation identification: cutting off the first three-compound leaf after the first three-compound leaf is completely unfolded in S3, placing the first three-compound leaf in a stainless steel tray, placing sterile gauze at the bottom of the tray, spraying a proper amount of distilled water to keep the gauze moist, marking a scratch wound on the central position of the upper surface of the leaf by using a sterilized scalpel, cutting the phytophthora sojae cultured in S2 to obtain a circular fungus block, covering the circular fungus block on the scratch wound, enabling the hypha to face upwards, using the scratch leaf inoculated with a blank carrot culture medium as a blank control, covering the tray with a preservative film, and culturing the tray at the temperature of 24 ℃, the illumination time of 12h/d and the relative humidity of 100%;
s5, evaluation of the resistance level of wild soybean phytophthora root rot: performing disease phenotype observation on the inoculated leaves on the 5 th day after the S4 treatment, wherein the disease phenotype observation object is the leaf discoloration degree, calculating the disease susceptibility according to the observed disease phenotype to determine the resistance level of the wild soybeans to the phytophthora root rot, and counting the resistance level of the wild soybeans;
s6, determination of resistance resources: obtaining resistance grade data of wild soybeans according to S5, and determining wild soybean resources with obvious resistance difference to phytophthora root rot as resistant wild soybeans;
s7, obtaining interspecific filial generation: using a cultivated soybean material with excellent agronomic characters as a female parent, and using a wild soybean with resistance screened from S6 as a male parent to perform hybridization to obtain F0 generation hybrid particles; planting hybrid grains F0 in the next year and harvesting F1 seeds, continuing planting the seeds harvested in the previous year in each growing season from the F2 generation, and harvesting the seeds of the next generation in a 1-grain-passing manner until the F5 generation;
s8, identifying the resistance of phytophthora root rot of interspecific filial generation: carrying out phytophthora root rot resistance screening from the generation F2 to the generation F5 according to the method from the generation S1 to the generation S6, and reserving plants showing phytophthora root rot resistance;
s9, phenotype screening of interspecific filial generation: and F2-F5 generations, and further checking related characters by combining the plant resisting phytophthora root rot of S8 to screen out a new germplasm resisting phytophthora root rot.
Further, the length of the scratch wound is 1/3 and the diameter of the circular mushroom block is 1/3 of the length of the leaf at S4.
Further, the method for calculating the susceptibility in S5 includes: the disease rate is the number of the infected leaves/the total number of inoculated leaves multiplied by 100 percent, and the infected leaves comprise yellow leaves, brown leaves and green leaves.
Further, the resistance grade of S5 comprises disease resistance (R), intermediate type (I) and disease susceptibility (S), wherein the disease resistance (R) susceptibility rate is 0-30.0%, the intermediate type (I) susceptibility rate is 30.1-70.0%, and the disease susceptibility (S) susceptibility rate is 70.1-100%.
Furthermore, the wild soybean of S7 needs to be subjected to the peeling treatment before being sowed, and the female parent is sowed 7-10 days later than the male parent.
Further, after the female parent of S7 enters the full-bloom stage, selecting a flower bud with high development degree on the upper part of a plant stem, removing other flower buds at the same node, slightly pinching the lower part of the flower bud by hands, removing sepals and corolla of the flower bud by using forceps, then removing anther, and tying a blank mark plate.
Further, the pollination method of the cross of S7 is as follows: selecting a newly opened flower on the day, peeling off petals of the flower to expose anthers, slightly coating pollen on the stigmas of the female parent buds which are emasculated, wrapping the emasculated flower with fresh leaves after pollination, fixing the emasculated flower with a toothpick, and marking on the number plate.
Further, the method for checking whether the hybridization was successful in S7: and (3) after artificial pollination for 1 week, timely checking whether hybridization is successful, if the hybridization is successful, checking whether the ovary begins to expand, and simultaneously checking whether new buds germinate at the same node and are timely removed.
Further, the relevant traits of S9 include: growth period, lodging traits, quality traits, yield traits and comprehensive agronomic traits.
The invention has the following beneficial effects:
the invention utilizes the leaf inoculation method to screen the wild soybean resistance source, overcomes the problem that the wild soybean stem is thin and weak and easy to break, and can easily cause that the resistance reaction of experimental materials can not be accurately described. The invention determines a standard evaluation system of wild soybean resources for phytophthora root rot resistance, creates a method for creating a new phytophthora root rot resistance germplasm by interspecific hybridization from wild resource resistance evaluation screening to screened wild soybeans with resistance, and is simple, practical and efficient.
Drawings
FIG. 1: a flow schematic diagram of a method for creating a new soybean germplasm with phytophthora root rot resistance;
FIG. 2: a schematic diagram of phytophthora root rot inoculated leaves;
FIG. 3: a phenotype diagram of the reaction after phytophthora root rot inoculation of leaves;
FIG. 4: determining the experiment of the observation days after the leaves are inoculated with the phytophthora;
FIG. 5: a comparison test of inoculating a blank carrot culture medium to the scratch wound of the leaf;
the reference numbers in the figures are: three compound leaves-1, scratch wound-2 and round fungus block-3.
Detailed Description
A method for creating a new germplasm of soybean with phytophthora root rot resistance comprises the steps of inoculating wild soybean leaves with phytophthora to soybean, screening out wild soybean with phytophthora root rot resistance, hybridizing the screened wild soybean with phytophthora root rot resistance with cultivated soybean to obtain hybrid progeny, screening an inbred line of the hybrid progeny for phytophthora root rot resistance, and creating a new germplasm with phytophthora root rot resistance by combining agronomic character screening (shown in figure 1).
Example 1
A method for creating a new germplasm of soybean with phytophthora root rot resistance, which comprises the following steps:
s1, preparing a carrot culture medium: cleaning fresh carrot, cutting into small pieces, weighing 100g, mincing with plant tissue mincing machine, adding appropriate amount of distilled water, boiling for 30min, filtering with 3 layers of gauze, diluting filtered carrot juice to 500mL, adding 10g of agar, and sterilizing at 120 deg.C under high temperature and high pressure for 20 min. Pouring the sterilized culture medium into a dish sterilized in advance, wherein the height of the culture medium is 0.5 cm;
s2, culture of test strains: the strain is phytophthora sojae, square blocks of the phytophthora sojae with the diameter of 10mm multiplied by 10mm are inoculated to the carrot culture medium S1, and dark culture is carried out for 7 days at the temperature of 25 ℃;
s3, potting: after the skin of the wild soybean seeds is broken, performing potted planting, planting 10 seeds in each pot, leaving 5 seedlings with consistent growth in each pot after emergence, and starting leaf inoculation identification after the first three compound leaves 1 of the seedlings are completely unfolded;
s4, leaf inoculation identification: referring to fig. 2, after the first three compound leaves 1 in the S3 are completely unfolded, the first three compound leaves are cut off and placed in a stainless steel tray, sterile gauze is placed at the bottom of the tray and sprayed with a proper amount of distilled water to keep the gauze moist, a scratch wound 2 is marked on the center of the upper surface of each leaf by using a sterilized scalpel, the length of the scratch wound is 1/3, a circular bacterium block 3 is cut from the phytophthora sojae cultured in the S2, the diameter of the circular bacterium block is 1/3 of the length of each leaf, the circular bacterium block is covered on the scratch wound with hypha facing upwards, referring to fig. 2, meanwhile, the scratch leaf inoculated with a blank carrot culture medium is used as a blank control, the tray is covered by using a preservative film, and the tray is placed in an environment with the temperature of 24 ℃, the illumination time of 12h/d and the relative humidity of 100% for culture;
s5, evaluation of the resistance level of wild soybean phytophthora root rot: and (3) performing disease phenotype observation on the inoculated leaves on the 5 th day after the S4 treatment, wherein the disease phenotype observation is performed on the leaves, the discoloration degree of the leaves is the object of the disease phenotype observation, the disease susceptibility is calculated according to the observed disease phenotype to determine the resistance level of the wild soybeans to the phytophthora root rot, and the resistance level of the wild soybeans is counted, and the disease susceptibility calculation method comprises the following steps: the disease susceptibility rate is the number of the infected leaves/the total number of inoculated leaves multiplied by 100%, the infected leaves comprise yellow, brown and green leaves, the resistance level comprises disease resistance (R), intermediate type (I) and disease susceptibility (S), and the corresponding disease susceptibility rates are respectively 0-30.0%, 30.1-70.0% and 70.1-100% and are shown in table 1;
TABLE 1 resistance rating Scale
| Resistance grade | Disease resistance (R) | Intermediate type (I) | Susceptible disease (S) |
| Rate of infection | 0-30.0％ | 30.1-70.0％ | 70.1-100％ |
S6, determination of resistance resources: obtaining resistance grade data of wild soybeans according to S5, and determining wild soybean resources with obvious resistance difference to phytophthora root rot as resistant wild soybeans;
s7, obtaining interspecific filial generation: the method is characterized in that resistant wild soybeans are used as male parents, cultivated soybeans with excellent agronomic characters are used as female parents, the seeds of the wild soybeans are subjected to peeling treatment before sowing, and the female parents are sown 7-10 days later than the male parents in order to ensure that the flowering phases of the male and female parents meet. Planting the parent strain in a test field in the last ten days of 5 months, wherein the female parent strain and the male parent strain are adjacently planted in 1 row and in 10cm, the row spacing is 65cm, the hole spacing is 10cm, 1 seedling is planted in each hole after seedling emergence, and the parent strain is erected by using a bamboo pole to assist the stem of the parent strain to wind the bamboo pole for growth after seedling establishment. After the female parent enters the full-bloom stage, selecting a flower bud with higher development degree on the upper part of a plant stem, removing other flower buds at the same node position, slightly pinching the lower part of the flower bud by hands, removing sepals and corolla of the flower bud by tweezers, removing anther by a soybean pollination emasculation device (a device for removing the flower bud), observing whether a column head and a flower column are intact or not, tying a blank mark plate, waiting for pollination, and using a magnifying glass in the whole process of emasculation and pollination because the organs of the parent and the parent are small. When the pollen of the male parent is taken, flowers which bloom on the same day are selected, petals of the flowers are stripped, anthers are exposed, the pollen is lightly smeared on the stigmas of the female parent buds which are emasculated, the whole emasculated flowers are wrapped by fresh leaves after pollination, the flowers are fixed by toothpicks, and the marks are marked on the number plate. In order to confirm that the pollen of the male parent is in a pollen-scattering state, the anther of the male parent is lightly touched on the nail of the left hand before pollination, and if powdery substances exist on the nail, the anther can be confirmed to be in a pollen-scattering period. The female parent castration is carried out in the afternoon, after the castration is finished, a blank mark plate is tied on the stem of the female parent below the node adjacent to the castrated flower bud, and pollination is carried out in the afternoon on the next day. The pollination time is selected in the morning of fine day, the cloudy day or rainy day is not favorable for pollen scattering of the male parent, the pollen of the male parent is collected, the pollen is directly pollinated on the stigma of the castrated female parent in the afternoon of the previous day, and the pollen scattering of the male parent is finished in the morning, so that the hybridization work is finished to 12 o' clock in the noon. After pollination, the blank mark plate tied when the female parent is castrated is marked with the hybrid combination code, the material names of the two parents and the pollination date, and the record is made on the workbook. And (3) after pollination for 1 week, timely checking whether hybridization is successful, if the hybridization is successful, checking whether the ovary begins to expand, and simultaneously checking whether new buds germinate at the same node and are timely removed. After the plants are mature, timely harvesting the hybrid bean pods according to the combination, drying in the sun and threshing, putting the harvested hybrid grains (namely F0 generation seeds) and the number plate into a gauze bag, and warehousing for storage. The next year hybrid F0 was planted and F1 seeds were harvested, the seeds harvested in the previous year were planted further each growing season since F2 generation, and the next generation seeds were harvested in a 1-seed-pass manner until F5 generation.
S8, identifying the resistance of phytophthora root rot of interspecific filial generation: carrying out phytophthora root rot resistance screening from the generation F2 to the generation F5 according to the method from the generation S1 to the generation S6, and reserving plants showing phytophthora root rot resistance;
s9, phenotype screening of interspecific filial generation: and F2-F5 generations, combining the plant resisting phytophthora root rot of S8, and combining the growth period, lodging characters, quality characters, yield characters and comprehensive agronomic characters to determine excellent interspecific progeny, thereby further screening new germplasm resisting phytophthora root rot.
Example 2
Determination of the number of observation days after inoculation of the leaves with phytophthora sojae: after 2d of inoculation by using an in vitro leaf inoculation method, brown spots appear at the inoculation part of the disease-susceptible material ZYD 594; after 5 days of inoculation, the brown spots are obviously enlarged and almost spread on the whole leaf; after inoculation for 7d, the brown spot part is obviously green and rotten; the inoculation positions of the disease-resistant materials Longye 01-491 are not changed obviously all the time, so the disease condition is observed after the inoculation for 5 d. See fig. 4.
Example 3
Leaf mar wound inoculation blank medium contrast experiment, after drawing out the mar wound with disease-sensitive material ZYD594, divide into two sets ofly, the mar not inoculate as control 1 group, the blank carrot culture medium of mar scratch inoculation is control 2 groups, after the cultivation is handled 5d, the blade all has not taken place obvious change, see figure 5, show that mar wound itself and blank carrot culture medium all do not exert an influence to the blade, it is infected by the phytophthora sojae and causes also to have proved the morbidity condition after the blade inoculation.
Example 4
Feasibility verification of leaf inoculation: 4 resistant varieties, 4 intermediate varieties and 16 susceptible varieties are obtained by a hypocotyl inoculation method, wherein the resistant varieties, the intermediate varieties and the susceptible varieties are respectively 17%, 17% and 66% of the total number; leaf inoculation yielded 4 resistant, 6 intermediate and 14 susceptible varieties, 17%, 25% and 58% of the total (table 2), respectively.
Table 2 identification of disease resistance of 24 parts of cultivated soybeans by different inoculation methods
The consistency rate of the identification result reaches 91.67%. In comparison, 2 varieties appeared susceptible in hypocotyl inoculation and intermediate in ex vivo leaf inoculation.
The results of the analyses related to the in vitro leaf inoculation method and the hypocotyl inoculation method by DPS are shown in Table 3. The relevance of the in vitro leaf inoculation method and the hypocotyl inoculation method reaches an extremely remarkable level (P is less than 0.01). The data obtained by the independence test of r × c (r represents the number of strains of 3 different types; c represents 2 inoculations) were used to calculate X for 2 methods2＝0.5333＜X2 0.05,2Differences were not evident demonstrating the 2 methods (table 4). The results of the correlation and independence tests show that the identification result of the in vitro leaf inoculation method is as reliable as that of the hypocotyl inoculation method.
TABLE 3 correlation analysis between in vitro leaf inoculation and hypocotyl inoculation
Note: denotes P < 0.01; x1 leaf inoculation method; x2 hypocotyl inoculation method
TABLE 4 results of independence test of in vitro leaf inoculation and hypocotyl inoculation
The above examples further illustrate and explain the method for creating a new germplasm of soybean with phytophthora root rot resistance disclosed in the present invention, and the explanation of the above examples is only used to help understand the method and the core idea of the present application, and for those skilled in the art, according to the idea of the present application, there may be variations in the specific implementation manner and application scope, and the content of the present specification should not be understood as a limitation to the present application.
Claims (9)
1. A method for creating a new soybean germplasm capable of resisting phytophthora root rot is characterized by comprising the following steps of:
s1, preparing a carrot culture medium: cleaning fresh carrot, cutting into small pieces, weighing 100g, mincing with a plant tissue mincing machine, adding an appropriate amount of distilled water, boiling for 30min, filtering with 3 layers of gauze, fixing the volume of the filtered carrot juice to 500mL, adding 10g of agar, sterilizing at 120 ℃ for 20min under high temperature and high pressure, pouring the sterilized culture medium into a sterilized flat dish, wherein the height of the culture medium is 0.5 cm;
s2, culture of test strains: the strain is phytophthora sojae, a square bacterial block of 10mm multiplied by 10mm is inoculated to the carrot culture medium S1, and dark culture is carried out for 7d at 25 ℃;
s3, potting: after the skin of the wild soybean seeds is broken, performing potted planting, planting 10 seeds in each pot, leaving 5 seedlings with consistent growth in each pot after emergence, and starting leaf inoculation identification after the first three compound leaves of the seedlings are completely unfolded;
s4, leaf inoculation identification: cutting off the first three-compound leaf after the first three-compound leaf is completely unfolded in S3, placing the first three-compound leaf in a stainless steel tray, placing sterile gauze at the bottom of the tray, spraying a proper amount of distilled water to keep the gauze moist, marking a scratch wound on the central position of the upper surface of the leaf by using a sterilized scalpel, cutting the phytophthora sojae cultured in S2 to obtain a circular fungus block, covering the circular fungus block on the scratch wound, enabling the hypha to face upwards, using the scratch leaf inoculated with a blank carrot culture medium as a blank control, covering the tray with a preservative film, and culturing the tray at the temperature of 24 ℃, the illumination time of 12h/d and the relative humidity of 100%;
s5, evaluation of wild soybean phytophthora root rot resistance level: performing disease phenotype observation on the inoculated leaves on the 5 th day after the S4 treatment, wherein the disease phenotype observation object is the leaf discoloration degree, calculating the disease susceptibility according to the observed disease phenotype to determine the resistance level of the wild soybeans to the phytophthora root rot, and counting the resistance level of the wild soybeans;
s6, determination of resistance resources: acquiring resistance grade data of wild soybeans according to S5, and determining wild soybean resources with significant resistance difference to phytophthora root rot as resistant wild soybeans;
s7, obtaining interspecific filial generation: using a cultivated soybean material with excellent agronomic characters as a female parent, using a wild soybean with resistance screened from S6 as a male parent to perform hybridization to obtain F0 generation hybrid grains, planting hybrid grains F0 in the next year and harvesting F1 seeds, continuing planting the seeds harvested in the previous year in each growing season from F2 generation, and harvesting the next generation of seeds in a 1 grain-passing mode until the F5 generation;
s8, identifying the resistance of phytophthora root rot of interspecific filial generation: carrying out phytophthora root rot resistance screening from the generation F2 to the generation F5 according to the method from the generation S1 to the generation S6, and reserving plants showing phytophthora root rot resistance;
s9, phenotype screening of interspecific filial generation: and F2-F5 generations, and further checking related characters to screen out a new germplasm for resisting the phytophthora root rot by combining the phytophthora root rot resistant plant from S8.
2. The method for creating the new germplasm of soybean resisting phytophthora root rot according to claim 1, wherein the length of the scratch wound is 1/3, and the diameter of the circular fungus block is 1/3 of the length of the leaf at S4.
3. The method for creating the new germplasm of soybean resisting phytophthora root rot according to claim 1, wherein the infection rate calculation method in S5 is as follows: susceptible leaf rate = number of susceptible leaves/total number of inoculated leaves × 100%, said susceptible leaves including leaf yellowing, browning, and chlorosis.
4. The method for creating the new germplasm of soybean resisting phytophthora root rot according to claim 1, wherein the resistance grades of S5 comprise disease resistance (R), intermediate type (I) and disease susceptibility (S), the disease resistance (R) susceptibility is 0.1-30.0%, the intermediate type (I) susceptibility is 30.1-70.0%, and the disease susceptibility (S) susceptibility is 70.1-99.9%.
5. The method for creating the new germplasm of soybean with phytophthora root rot resistance according to claim 1, wherein the wild soybean at S7 needs to be subjected to a skin breaking treatment before being sown, and the female parent is sown 7-10 days later than the male parent.
6. The method for creating the new germplasm of soybean resisting phytophthora root rot according to claim 1, wherein after the female parent enters the full-bloom stage in S7, a flower bud with high development degree at the upper part of the stem of the plant is selected, other flower buds at the same node are removed, then the lower part of the flower bud is gently pinched by hands, the sepals and the corolla of the flower bud are removed by tweezers, then the anther is removed, and a blank mark board is fastened.
7. The method for creating the new germplasm of soybean resisting phytophthora root rot according to claim 1, wherein the pollination method for the cross in S7 is as follows: selecting a newly opened flower on the day, peeling off petals of the flower to expose anthers, slightly coating pollen on the stigmas of the female parent buds which are emasculated, wrapping the emasculated flower with fresh leaves after pollination, fixing the emasculated flower with a toothpick, and marking on the number plate.
8. The method for creating a new germplasm of soybean resistant to phytophthora root rot according to claim 1, wherein the method for checking whether hybridization is successful in S7 comprises: and (3) after artificial pollination for 1 week, timely checking whether hybridization is successful, if the hybridization is successful, checking whether the ovary begins to expand, and simultaneously checking whether new buds germinate at the same node and are timely removed.
9. The method for creating the new germplasm of soybean resisting phytophthora root rot according to claim 1, wherein the relevant traits of S9 include: growth period, lodging traits, quality traits, yield traits and comprehensive agronomic traits.
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