CN111887148A - Breeding method of double-main-stem semi-short-stalk cabbage type rape - Google Patents
Breeding method of double-main-stem semi-short-stalk cabbage type rape Download PDFInfo
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- CN111887148A CN111887148A CN202010752548.7A CN202010752548A CN111887148A CN 111887148 A CN111887148 A CN 111887148A CN 202010752548 A CN202010752548 A CN 202010752548A CN 111887148 A CN111887148 A CN 111887148A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/02—Methods or apparatus for hybridisation; Artificial pollination ; Fertility
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/04—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/06—Processes for producing mutations, e.g. treatment with chemicals or with radiation
Abstract
The invention belongs to the technical field of rape breeding, and discloses a method for breeding double-main-stem semi-short-stalk cabbage type rape, which comprises the following steps: carrying out mutagenesis treatment on seeds of the cabbage type rape inbred line by using ethyl methane sulfonate, selecting double-main-stem high-stalk plants for inbreeding, and selecting double-main-stem semi-short-stalk plants for inbreeding; culturing haploid plants, and performing doubling treatment to obtain double-main-stem semi-short-stalk cabbage type rape populations; and performing field identification to obtain the double-main-stem semi-short-stalk cabbage type rape. The double-main-stem semi-short-stalk cabbage type rape provided by the invention has the characters of double-main-stem semi-short-stalk, can solve the lodging problem, improve the yield and the quality of the rape, realize mechanical harvesting and improve the overall benefit of the rape; EMS mutagenesis is carried out on the cabbage type rape, the natural mutation rate of organisms can be improved, the method is simple and easy to implement, the specificity is strong, certain basic groups on DNA can be mutagenized and positioned, and progeny is easy to inherit stably.
Description
Technical Field
The invention belongs to the technical field of rape breeding, and particularly relates to a breeding method of double-main-stem semi-short-stalk cabbage type rape.
Background
At present, rape is an important edible oil crop in China, is mainly planted in Yangtze river basin and adjacent areas, is a main winter-free crop, is planted for hundreds of millions of acres all the year round, has 100 hundred million Kg of total yield every year, and is the first in the world regardless of area and total yield. In China, over 400 million tons of rapeseed oil can be produced from domestic rapeseeds every year, more than 40% of domestic vegetable oil accounts for nearly 20% of the total consumption of domestic vegetable oil, and the rapeseed oil accounts for 19% of the consumption of domestic edible oil. Besides being used for oil extraction, the rapeseed residues can also be used as high-protein feeding cakes in the feed industry after the oil extraction of commercial rape seeds, and can be 600 million tons of feed raw materials in the feed industry every year. However, in recent years, due to the impact of imported rapeseeds, and the factors of massive transfer of rural labor force, shortage of rural labor force, low comparative benefit of rapes and the like, the whole rape planting area in China shows a downward sliding trend. How to improve the actual yield of unit area, reduce the planting cost and turn back the downward sliding trend of rape planting area to ensure the edible oil safety in China becomes an important mission for rape breeding workers in China.
Therefore, increasing the actual harvest yield of oilseed rape is a constant theme. With the continuous maturation of the cross breeding technology and the continuous efforts of breeding workers, the yield per unit area of the rape in China is greatly improved, but also because of the application of the cross breeding technology, the plant height of the rape is continuously increased, so that the yield is reduced due to lodging of the rape, and the yield is reduced by more than 15% in severe cases, thereby causing the loss of the actual yield. The dwarfing breeding of crops is successfully applied to the crops such as rice, wheat and the like, but the research on the breeding of rape is less. The method is a good idea for breeding lodging-resistant rape varieties by reducing the plant height while ensuring the yield.
The yield and the quality of the rapeseed oil have important influence on the supply safety of edible vegetable oil in China, and with the continuous improvement of the requirement on the yield of the rapeseed oil, lodging becomes an important factor influencing the yield and the quality of the rapeseed oil. Lodging enables the stems and leaves of the rapes to be overlapped and concealed mutually to form a high-temperature and high-humidity space, and the resistance to stress and insect pest is reduced; the lodging rape transfers the nutrient supply center from the upper pod of the rape to the middle-lower part to regenerate branches, the small branches bloom but are not durable and cause the phenomenon of greedy and late maturity, the lodging can influence the normal photosynthesis of the rape plants, the quantity of blighted negative horns is increased, the effective horns are reduced, the yield and the quality are reduced, the yield of the lodging rape is reduced by 10 to 30 percent compared with the normal rape, and the serious yield can reach more than 50 percent. The lodging can increase the mechanical operation difficulty and even the mechanical harvesting cannot be realized, the loss is increased, the economic benefit of the rape is seriously influenced, and the enthusiasm of farmers for planting the rape is reduced.
The ideal rape plant type structure is medium-length siliques with semi-short stalk character, small branch angle and high density of upward growth. With the popularization of cabbage type rape hybrid varieties and the wide application of chemical fertilizers, the average height of the rape is increased by more than 20cm, the fertilizer resistance of the rape is reduced, the lodging risk is improved, and the lodging problem of the rape becomes an important factor for restricting the yield, the quality and the mechanized harvest of the rape. However, the breeding method of the half-dwarf cabbage type rape is not available at present, the quality of the rape is poor, and the economic benefit is low.
Meanwhile, because of the shortage of rural labor, the labor cost for rape planting is increased. Mechanized light and simplified cultivation is a trend of development of rape planting modes, and inevitably leads to increase of the seed consumption. At present, the traditional cabbage type rape is almost single main stem, namely the rape plant has no tillering, and one rape plant has only 1 main stem. If the main stem number of a single rape plant can be increased, the seed consumption can be reduced, and the seed cost is reduced.
In view of the above thought, if a breeding method is innovated, mature and stable materials are used to create the method, which can reduce the plant height of the cabbage type rape, realize lodging resistance to reduce yield loss and increase actual harvest yield, and increase the number of main stems of the cabbage type rape at the same time, reduce seeding amount to reduce seed cost, and has significant scientific research value and application prospect.
Through the above analysis, the problems and defects of the prior art are as follows: at present, a method for breeding half-short-stalk cabbage type rape is temporarily absent, the quality of the existing rape is poor, and the economic benefit is low; meanwhile, the existing researches in rape breeding are less.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a breeding method of double-main-stem semi-short-stalk cabbage type rape.
The invention is realized in such a way that the breeding method of the double-main-stem semi-short-stalk cabbage type rape comprises the following steps:
step one, castration is carried out by taking a recessive double-main-stem cabbage type rape material which is stable in maturity and good in comprehensive character as a female parent; dominant short-stalk cabbage type rape material pollen with stable maturity and better comprehensive characters is taken as a male parent to carry out hybridization.
Step two, in F1Planting into plant rows, bagging for selfing, threshing according to the harvest of single plant, and mutagenizing the seeds of the cabbage type rape selfing line by using ethyl methane sulfonate.
Step three, in F2Planting F separately1Single plant, investigating the character separation condition of the double main stem short stalk; selecting single plants with short stalk character and double main stem character, bagging, selfing, and threshing separately according to the single plant harvest.
Step four, in F3Respectively mix F2The selfing single plants are all planted, and the character separation condition of the double main stems is investigated; and (4) screening the whole plant rows with the double-main-stem short-stalk character, bagging, selfing, and harvesting and threshing respectively according to single plants.
Step five, in step F4Planting F separately3Selfing the individual plant of (1) to obtain F4:3Family members.
Step six, taking F4:3Culturing haploid plants by family plant pollen and repeatedly investigating the haploid plants in the field4:3The height character of the main stem of the family is doubled, the stable expression of the double-main-stem semi-short-stem character of the family haploid plant is doubled, and the double-main-stem semi-short-stem cabbage type rape population is obtained.
And seventhly, performing field identification on the inheritance and stability of the double-main-stem short-stalk character of the obtained double-main-stem semi-short-stalk brassica napus population, and reserving the population with the stable semi-short-stalk character in the field to obtain the double-main-stem semi-short-stalk brassica napus.
Further, in the second step, the method for mutagenizing the seeds of the brassica napus inbred line by using ethyl methanesulfonate comprises the following steps:
(1) selecting the cabbage type rape inbred line seeds, and selecting the seeds with full grains, no damage and obvious characters as the cabbage type rape inbred line seeds;
(2) placing the selected seeds in clear water, and soaking for 8-12 h;
(3) soaking the soaked seeds in ethyl methanesulfonate for 3-4 h;
(4) taking out the soaked seeds, washing the seeds by using clear water, and cleaning residual ethyl methanesulfonate on the surface;
(5) placing the cleaned seeds on dry filter paper, setting the culture temperature to be 20-25 ℃ and the humidity to be 40-60%, and performing seed culture to obtain the treated seeds of the brassica napus inbred line;
(6) cultivating the treated seeds of the cabbage type rape inbred line to obtain F1And (5) plant generation.
Further, in the step (3), the concentration of the ethyl methanesulfonate was 0.6%.
Further, in the step (3), the method for soaking the soaked seeds by using ethyl methanesulfonate comprises the following steps:
the oxygen atom position of guanine is alkylated; during DNA replication, alkylated guanines pair with thymines, resulting in base substitutions, i.e., G: C to A: T.
Further, in step five, F2:3The height of the plant of the family is 120-140 mm.
Further, in the sixth step, the method for culturing the haploid plant comprises the following steps: taking F in the early flowering phase2:3And (3) carrying out microspore culture on pollen of the young buds on the main inflorescence and the upper branch inflorescence of the family plant to obtain a haploid plant.
Further, the method for doubling the haploid plant of the family stably expressing the double main stem semi-short stalk character comprises the following steps:
(a) picking flower buds, separating free microspores by using a B5-13 culture medium, and then separately containing the free microspores by using an NLN-13 culture medium containing 200mg/L colchicine;
(b) placing the culture medium containing the free microspores in a dark room, setting the culture temperature to be 27-29 ℃, and culturing for 20-24 hours;
(c) replacing the culture medium with an NLN-13 culture medium without colchicine, placing the culture medium in a dark room, setting the culture temperature to be 26-30 ℃, reducing the temperature to 25 ℃ after culturing for 36-48 h, and continuing culturing for 6-9 days to obtain the induced embryoid.
Further, the method for doubling the haploid plant of the family stably expressing the double main stem semi-short stalk character further comprises the following steps:
1) culturing the induced embryoid until the embryoid can be seen by naked eyes;
2) transferring the embryoid to a shaking table, and culturing in a dark room at the temperature of 25-30 ℃ and the rotation speed of the shaking table of 55-65 r/min;
3) culturing on a shaking table until embryoid is in a cotyledon shape or torpedo shape, and treating the embryoid for 8-10 days at 2-4 ℃;
4) and transferring the processed embryoid into a B5 solid culture medium, culturing in a light environment at 23-25 ℃, and inducing regeneration seedlings.
Further, in step (a), the method for separating free microspores comprises:
(I) picking buds in the field during the flowering period of a haploid plant, selecting buds which are vigorous in growth, multiple in branches, main inflorescences and enough buds on primary branches of the plant during the bud picking, and picking buds in the 3 rd to 4 th rings from the outside to the inside of the selected buds;
(II) disinfecting the flower buds picked in the step (I) for 20-30 s by using 75% alcohol;
(III) continuously reversing, vibrating and disinfecting for 15-20 min by using 0.1% mercuric chloride, placing sterilized buds in a sterile centrifuge tube, and repeatedly cleaning by using sterile water;
(IV) adding a precooled sucrose solution with the concentration of 13%, crushing buds by using a sterilized glass rod, vibrating uniformly, filtering, and collecting filtrate;
(V) centrifuging the filtrate, wherein the centrifugal rotating speed is 1000-1200 r/min, and the centrifugal time is 3-5 min; precipitating microspores, and removing the supernatant to obtain the microspores.
The invention also aims to provide the double-main-stem semi-short-stem cabbage type rape bred by the breeding method of the double-main-stem semi-short-stem cabbage type rape.
By combining all the technical schemes, the invention has the advantages and positive effects that: the breeding method of the double-main-stem semi-short-stalk cabbage type rape provided by the invention utilizes a dominant short-stalk cabbage type rape material which is stable in maturity and good in comprehensive character and a recessive double-main-stem cabbage type rape material which is stable in maturity and good in comprehensive character, and screens the double-main-stem short-stalk cabbage type rape which is stable in maturity and good in comprehensive character in the later generation through hybridization and selfing. The invention can breed the double-main-stem short-stalk brassica napus material, improve the lodging resistance of the brassica napus, increase the actual harvest yield, reduce the seed cost, realize the purposes of cost saving and yield increase, increase the planting benefit of the brassica napus, improve the positivity of the brassica napus planting, help the development of the brassica napus industry and ensure the edible oil safety in China. The double-main-stem semi-short-stalk cabbage type rape bred by the breeding method has the characters of double-main-stem semi-short-stalk, can solve the lodging problem, improve the yield and the quality of the rape, realize mechanical harvesting and improve the overall benefit of the rape; EMS mutagenesis is carried out on the cabbage type rape, the natural mutation rate of organisms can be improved, the operability is strong, the method is simple and easy to implement, the specificity is strong, certain basic groups on DNA can be mutagenized and positioned, and offspring is easy to be inherited stably.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed to be used in the embodiments of the present application will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present application, and it is obvious for those skilled in the art that other drawings can be obtained from the drawings without creative efforts.
FIG. 1 is a flow chart of a method for breeding a double main stem semi-short stalk Brassica napus provided in an embodiment of the present invention.
FIG. 2 is a flow chart of the method for mutagenizing seeds of Brassica napus inbred line by using ethylmethane sulfonate provided by the embodiment of the present invention.
Fig. 3 is a flowchart of a method for soaking soaked seeds with ethyl methanesulfonate according to an embodiment of the present invention.
FIG. 4 is a flow chart of the method for doubling the haploid plants of the pedigree stably expressing the double-stem half-short-stalk trait provided by the embodiment of the invention.
FIG. 5 is a flow chart of a method for isolating free microspores in accordance with an embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Aiming at the problems in the prior art, the invention provides a method for breeding double-main-stem semi-short-stalk brassica napus, and the invention is described in detail with reference to the attached drawings.
As shown in fig. 1, the breeding method of the double-main-stem semi-dwarf brassica napus provided by the embodiment of the invention comprises the following steps:
s101, castration is carried out by using a recessive double-main-stem cabbage type rape material which is stable in maturity and good in comprehensive character as a female parent; dominant short-stalk cabbage type rape material pollen with stable maturity and better comprehensive characters is taken as a male parent to carry out hybridization.
S102, at F1Planting into plant rows, bagging for selfing, threshing according to the harvest of single plant, and mutagenizing the seeds of the cabbage type rape selfing line by using ethyl methane sulfonate.
S103, at F2Planting F separately1Single plant, investigating the character separation condition of the double main stem short stalk; selecting single plants with short stalk character and double main stem character, bagging, selfing, and threshing separately according to the single plant harvest.
S104, at F3Respectively mix F2All the inbred single plants ofPlanting, and investigating the separation condition of the short stalk character of the double main stems; and (4) screening the whole plant rows with the double-main-stem short-stalk character, bagging, selfing, and harvesting and threshing respectively according to single plants.
S105, at F4Planting F separately3Selfing the individual plant of (1) to obtain F4:3Family members.
S106, taking F4:3Culturing haploid plants by family plant pollen and repeatedly investigating the haploid plants in the field4:3The height character of the main stem of the family is doubled, the stable expression of the double-main-stem semi-short-stem character of the family haploid plant is doubled, and the double-main-stem semi-short-stem cabbage type rape population is obtained.
And S107, performing field identification on the inheritance and stability of the double-main-stem short-stalk character of the obtained double-main-stem semi-short-stalk brassica napus population, and reserving the population with the stable semi-short-stalk character in the field to obtain the double-main-stem semi-short-stalk brassica napus.
As shown in fig. 2, in step S102, the method for performing mutagenesis treatment on seeds of an inbred line of brassica napus by using ethyl methanesulfonate includes:
s201, selecting the seeds of the cabbage type rape inbred line, and selecting the seeds with full grains, no damage and obvious characters as the seeds of the cabbage type rape inbred line.
S202, placing the selected seeds in clear water, and soaking for 8-12 hours.
S203, soaking the soaked seeds in ethyl methanesulfonate for 3-4 h.
S204, taking out the soaked seeds, washing the seeds by using clean water, and cleaning the residual ethyl methanesulfonate on the surface.
S205, placing the cleaned seeds on dry filter paper, setting the culture temperature to be 20-25 ℃ and the humidity to be 40-60%, and performing seed culture to obtain the treated seeds of the brassica napus inbred line.
S206, cultivating the treated seeds of the cabbage type rape inbred line to obtain F1And (5) plant generation.
In step S203 provided in the embodiment of the present invention, the concentration of the ethyl methanesulfonate is 0.6%.
As shown in fig. 3, in step S203, the method for soaking the soaked seeds with ethyl methanesulfonate includes:
s301, the oxygen atom position of guanine is alkylated.
S302, during DNA replication, alkylated guanines pair with thymines, resulting in base substitutions, i.e., G: C to A: T.
In step S105 provided in the embodiment of the present invention, F is2:3The height of the plant of the family is 120-140 mm.
In step S106 provided in the embodiment of the present invention, the method for culturing a haploid plant includes: taking F in the early flowering phase2:3And (3) carrying out microspore culture on pollen of the young buds on the main inflorescence and the upper branch inflorescence of the family plant to obtain a haploid plant.
As shown in fig. 4, the method for doubling a haploid plant of a pedigree stably exhibiting the double-stem semi-short-stalk trait provided by the embodiment of the invention comprises:
s401, picking flower buds, separating free microspores by using a B5-13 culture medium, and then separately containing the free microspores by using an NLN-13 culture medium containing 200mg/L colchicine.
S402, placing the culture medium containing the free microspores in a dark room, setting the culture temperature to be 27-29 ℃, and culturing for 20-24 hours.
S403, replacing the culture medium with an NLN-13 culture medium without colchicine, placing the culture medium in a dark room, setting the culture temperature to be 26-30 ℃, reducing the temperature to 25 ℃ after culturing for 36-48 h, and continuing culturing for 6-9 days to obtain the induced embryoid.
The method for doubling the haploid plant of the family stably expressing the double-stem semi-short-stalk character provided by the embodiment of the invention further comprises the following steps:
1) and culturing the induced embryoid until the embryoid can be seen by naked eyes.
2) And transferring the embryoid to a shaking table, and culturing in a dark room at the temperature of 25-30 ℃ and the rotation speed of the shaking table of 55-65 r/min.
3) Culturing on a shaking table until the embryoid is in a cotyledon shape or torpedo shape, and treating the embryoid for 8-10 days at the temperature of 2-4 ℃.
4) And transferring the processed embryoid into a B5 solid culture medium, culturing in a light environment at 23-25 ℃, and inducing regeneration seedlings.
As shown in fig. 5, in step S401, the method for separating free microspores includes:
s501, picking buds in the field during the flowering phase of the haploid plant, selecting buds which are vigorous in growth, multiple in branches, main inflorescences and enough in buds on primary branches of the plant during the bud picking, and picking buds in the 3 rd to 4 th rings from the outside to the inside of the selected buds.
And S502, disinfecting the flower buds picked in the S501 for 20-30S by using 75% alcohol.
S503, continuously reversing and vibrating with 0.1% mercuric chloride for disinfection for 15-20 min, placing the sterilized flower buds into a sterile centrifuge tube, and repeatedly cleaning with sterile water.
S504, adding a precooled sucrose solution with the concentration of 13%, crushing buds by using a sterilized glass rod, shaking uniformly, filtering, and collecting filtrate.
S505, centrifuging the filtrate, wherein the centrifugal rotating speed is 1000-1200 r/min, and the centrifugal time is 3-5 min; precipitating microspores, and removing the supernatant to obtain the microspores.
The invention is further described with reference to specific examples.
Example (b): the dwarf cabbage type rape material D871 and the double-main-stem cabbage type rape material HSJ188 are used for breeding the double-main-stem dwarf material HSJ 1701.
(1) Respectively planting the short stalk material D871 and the double main stalk material HSJ188 into plant rows, emasculating by taking the double main stalk material HSJ188 as a female parent in a flowering period, and taking the pollen of the short stalk material D871 for sexual hybridization. Harvesting hybrid seed F1。
(2) At F1The plants are planted into plant rows, and 47 plants in the plant rows are found to have the short stalk character through investigation, but double main stem single plants are not found. Selecting good single plant and baggingSelfing, and threshing separately according to the harvest of single plant.
(3) At F2The population is expanded, 400 plants are planted according to the cell, and the segregation situation of the double-main-stem short-stalk character is investigated. . Investigation shows that 301 plants with short stalk property, 99 plants with high stalk property, 76 plants with double main stem short stalk property and 25 plants with double main stem high stalk property. Bagging and selfing 76 plants with the double main stem short stalk character, and threshing respectively according to single plant harvest to actually obtain 68 single plant seeds.
(4) At F368 single plants are respectively planted into plant rows, each single plant is planted into 2 rows, the total number of the plants is about 100, and the segregation situation of the double-main-stem short-stalk character is investigated. Investigation shows that 23 plant rows have the double-main-stem short-stalk character, and 45 plant rows have the double-main-stem separation of high stalks. And selecting 5-10 excellent plants from the 23 plant rows with the double main stem short stalk character for bagging and selfing, and respectively threshing according to the harvest of the single plants to obtain 172 single plants in total.
(5) At F4172 single plants are respectively planted into plant rows, each single plant is planted into 1 row, about 50 plants are planted in total, and the segregation situation of the double main stem short stalk character is investigated. The investigation shows that all plant rows have the character of double main stems and short stems.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention, and the scope of the present invention is not limited thereto, and any modification, equivalent replacement, and improvement made by those skilled in the art within the technical scope of the present invention disclosed herein, which is within the spirit and principle of the present invention, should be covered by the present invention.
Claims (10)
1. A breeding method of double-main-stem semi-short-stalk cabbage type rape is characterized by comprising the following steps:
step one, castration is carried out by taking a recessive double-main-stem cabbage type rape material which is stable in maturity and good in comprehensive character as a female parent; taking dominant short-stalk cabbage type rape material pollen which is mature and stable and has better comprehensive characters as a male parent to perform hybridization;
step two, in F1Planting into plant rows, bagging, selfing, and threshing according to the harvest of single plantCarrying out mutagenesis treatment on the seeds of the cabbage type rape inbred line by using ethyl methane sulfonate;
step three, in F2Planting F separately1Single plant, investigating the character separation condition of the double main stem short stalk; selecting single plants which have the characteristics of short stalk and double main stems, bagging, selfing, and respectively threshing according to the harvest of the single plants;
step four, in F3Respectively mix F2The selfing single plants are all planted, and the character separation condition of the double main stems is investigated; screening the whole plant rows with the double main stem short stalk character, bagging and selfing, and respectively threshing according to the harvest of single plant;
step five, in step F4Planting F separately3Selfing the individual plant of (1) to obtain F4:3Family tying;
step six, taking F4:3Culturing haploid plants by family plant pollen and repeatedly investigating the haploid plants in the field4:3The method comprises the following steps of performing doubling treatment on a family haploid plant stably expressing the double-main-stem and semi-short-stem character to obtain a double-main-stem and semi-short-stem cabbage type rape population;
and seventhly, performing field identification on the inheritance and stability of the double-main-stem short-stalk character of the obtained double-main-stem semi-short-stalk brassica napus population, and reserving the population with the stable semi-short-stalk character in the field to obtain the double-main-stem semi-short-stalk brassica napus.
2. The method for breeding brassica napus with double main stems and half short stalk as claimed in claim 1, wherein in the second step, the method for mutagenizing the seeds of the brassica napus inbred line by using ethyl methanesulfonate comprises:
(1) selecting the cabbage type rape inbred line seeds, and selecting the seeds with full grains, no damage and obvious characters as the cabbage type rape inbred line seeds;
(2) placing the selected seeds in clear water, and soaking for 8-12 h;
(3) soaking the soaked seeds in ethyl methanesulfonate for 3-4 h;
(4) taking out the soaked seeds, washing the seeds by using clear water, and cleaning residual ethyl methanesulfonate on the surface;
(5) placing the cleaned seeds on dry filter paper, setting the culture temperature to be 20-25 ℃ and the humidity to be 40-60%, and performing seed culture to obtain the treated seeds of the brassica napus inbred line;
(6) cultivating the treated seeds of the cabbage type rape inbred line to obtain F1And (5) plant generation.
3. The method for breeding the brassica napus with double main stems and half short stalk as claimed in claim 2, wherein the concentration of the ethyl methanesulfonate in the step (3) is 0.6%.
4. The method for breeding the brassica napus with double main stems and half short stalk as claimed in claim 2, wherein the step (3) of soaking the soaked seeds with ethyl methanesulfonate comprises:
the oxygen atom position of guanine is alkylated; during DNA replication, alkylated guanines pair with thymines, resulting in base substitutions, i.e., G: C to A: T.
5. The method for breeding a brassica napus with double main stems and half short stems as claimed in claim 1, wherein in step five, F2:3The height of the plant of the family is 120-140 mm.
6. The method for breeding the double main stem semi-dwarf brassica napus according to claim 1, wherein in the sixth step, the method for culturing the haploid plant comprises the following steps: taking F in the early flowering phase2:3And (3) carrying out microspore culture on pollen of the young buds on the main inflorescence and the upper branch inflorescence of the family plant to obtain a haploid plant.
7. The method for breeding the double main-stem semi-short-stalk brassica napus as claimed in claim 6, wherein the method for doubling the haploid plants of the family stably exhibiting the double main-stem semi-short-stalk trait comprises:
(a) picking flower buds, separating free microspores by using a B5-13 culture medium, and then separately containing the free microspores by using an NLN-13 culture medium containing 200mg/L colchicine;
(b) placing the culture medium containing the free microspores in a dark room, setting the culture temperature to be 27-29 ℃, and culturing for 20-24 hours;
(c) replacing the culture medium with an NLN-13 culture medium without colchicine, placing the culture medium in a dark room, setting the culture temperature to be 26-30 ℃, reducing the temperature to 25 ℃ after culturing for 36-48 h, and continuing culturing for 6-9 days to obtain the induced embryoid.
8. The method for breeding the double main-stem semi-dwarf brassica napus according to claim 7, wherein the method for doubling the haploid plants of the line stably expressing the double main-stem semi-dwarf trait further comprises:
1) culturing the induced embryoid until the embryoid can be seen by naked eyes;
2) transferring the embryoid to a shaking table, and culturing in a dark room at the temperature of 25-30 ℃ and the rotation speed of the shaking table of 55-65 r/min;
3) culturing on a shaking table until embryoid is in a cotyledon shape or torpedo shape, and treating the embryoid for 8-10 days at 2-4 ℃;
4) and transferring the processed embryoid into a B5 solid culture medium, culturing in a light environment at 23-25 ℃, and inducing regeneration seedlings.
9. The method for breeding brassica napus with double main stems as claimed in claim 7, wherein the method for separating free microspores in step (a) comprises:
(I) picking buds in the field during the flowering period of a haploid plant, selecting buds which are vigorous in growth, multiple in branches, main inflorescences and enough buds on primary branches of the plant during the bud picking, and picking buds in the 3 rd to 4 th rings from the outside to the inside of the selected buds;
(II) disinfecting the flower buds picked in the step (I) for 20-30 s by using 75% alcohol;
(III) continuously reversing, vibrating and disinfecting for 15-20 min by using 0.1% mercuric chloride, placing sterilized buds in a sterile centrifuge tube, and repeatedly cleaning by using sterile water;
(IV) adding a precooled sucrose solution with the concentration of 13%, crushing buds by using a sterilized glass rod, vibrating uniformly, filtering, and collecting filtrate;
(V) centrifuging the filtrate, wherein the centrifugal rotating speed is 1000-1200 r/min, and the centrifugal time is 3-5 min; precipitating microspores, and removing the supernatant to obtain the microspores.
10. The method for breeding the double-main-stem semi-dwarf cabbage type rape according to any one of claims 1 to 9.
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