CN113950977B - Method for improving Chinese cabbage germplasm creation efficiency - Google Patents
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Abstract
The invention discloses a method for improving Chinese cabbage germplasm creation efficiency, which specifically comprises the following steps: s1, preparation of rootstocks and scions: selecting a hybrid variety with a large root as a stock material, selecting a bolting resistant spring Chinese cabbage hybrid variety as a scion material, sowing the stock and the scion seed, and vernalizing; s2, planting; s3, management of grafting environment: the plant is grown by controlling the environmental temperature at 15-20 ℃, and when bolting for 20cm, the root is irrigated by using chlormequat chloride diluent; s4, selecting grafting conditions: making a grafting part at the second primary branch of the stock; the scion is selected as a first-level branch with the length of 6-10 cm; s5, grafting: grafting the scion bud on a stock plant; s6, management after grafting: managing the grafted plants by controlling the temperature of the growing environment after grafting is finished; the method can effectively improve the germ-shedding rate of the isolated microspores of the materials difficult to germ-shed, accelerate the purification speed of the Chinese cabbage materials and improve the germ plasm creation efficiency.
Description
Technical Field
The invention belongs to the technical field of plant breeding science, and particularly relates to a method for improving the creation efficiency of Chinese cabbage germplasm.
Background
China is the origin of Chinese cabbage (Raphanus sativus L.), and the cultivation history is long; chinese cabbage resources are relatively rich; such as Qingdao 'jiao zhou Chinese cabbage', weifang 'yellow bud Chinese cabbage' and the like.
In recent years, with the improvement of living standard of people and the development of situations at home and abroad, consumption habits of people on Chinese cabbages are changed, spring Chinese cabbages are gradually increased, and higher requirements on the quality of the Chinese cabbages are provided; however, due to the difference of breeding concepts and the lagging technical level, the breeding work of the spring Chinese cabbage variety is slow, and the Korean spring Chinese cabbage variety accounts for a large proportion in the Chinese market, so that the Korean spring Chinese cabbage variety is favored by consumers.
The domestic Chinese cabbage has few excellent varieties meeting the market demand, and the development and improvement of the quality improvement of the Chinese cabbage are needed to ensure the healthy and sustainable development of the Chinese cabbage industry; the breeding of good varieties requires good self-bred line materials; the creation of excellent germplasm is mainly realized by technical means such as filial generation separation, mutant induction, free microspore culture, transgenosis and the like; wherein, the culture of the free microspore is an efficient way for obtaining the homozygous inbred line.
Chinese cabbage is one of the species in cruciferous vegetable crops where free microspore culture is relatively easy to perform, but the genotypes of many donors still limit the application of the free microspore culture technology to many Chinese cabbage materials.
In the prior art, the method for improving the induction rate and the direct seedling rate of the microspore embryos of the hybrids of Chinese cabbages and Chinese cabbages CN201010248752.1 improves the induction rate of embryoids by 6.2 times by using an NLN culture medium added with auxin 2- (4-chlorophenoxy) isobutyric acid with the concentration of 40 mu M and 130 g.L-1 sucrose.
Research on efficient system of Chinese cabbage isolated microspore culture technology (Chinese vegetable 2014, (08)) researches the influence of mechanical microspore extraction by using a cell disruption instrument and microspore extraction by manual extrusion on the activity and extraction amount of Chinese cabbage isolated microspores.
Research on Chinese cabbage microspore culture influencing factors (Chinese vegetable 2006, (07)) confirms that the comprehensive influence of auxin and cytokinin is the superposition of the influences of the auxin and the cytokinin, an NLN culture medium is a basic culture medium which is most suitable for culturing Chinese cabbage free microspores, and active carbon has an inhibiting effect on the generation of Chinese cabbage microspore embryos.
There is no report on the improvement of the germ production rate by grafting Chinese cabbage after bolting.
Disclosure of Invention
The invention aims to solve the main technical problem of providing the method for improving the germ plasm creation efficiency of the Chinese cabbage, which can effectively improve the germ production rate of the free microspores of the materials difficult to produce germs, accelerate the purification speed of the Chinese cabbage materials and improve the germ plasm creation efficiency.
In order to solve the technical problems, the invention provides the following technical scheme:
a method for improving the creation efficiency of Chinese cabbage germplasm specifically comprises the following steps:
s1, preparation of rootstocks and scions: selecting a hybrid variety which has strong resistance, is easy to produce embryos and has large root fertilizer as a stock material, selecting a spring Chinese cabbage hybrid variety which has beautiful appearance, excellent quality, yellow heart and bolting resistance as a scion material, sowing the stock and the scion seeds and smoothly performing vernalization;
s2, planting: in the middle-upper 3 months, the stock and the scion seed plants are fixedly planted in an arched shed just before bolting;
s3, management of grafting environment: the environmental temperature is controlled at 15-20 ℃ to lead the plants to grow, and when the shoots are bolting for 20cm, the chlormequat chloride diluent is used for irrigating roots;
s4, selecting grafting conditions: taking a grafting part at the second primary branch of the stock; the scion is selected as a first-level branch with the length of 6-10 cm;
s5, grafting: grafting the scion bud on a stock plant;
s6, management after grafting: and after grafting is finished, managing the grafted plants by controlling the temperature of the growing environment.
The following is a further optimization of the above technical solution of the present invention:
the creation method further comprises the following steps: s7, culturing free microspores, wherein the free microspores are cultured according to the following steps:
(1) Controlling the growth environment temperature of the grafting body to be 15-25 ℃, removing main stems, selecting 2.5-3mm buds in the mononuclear border period or the binuclear early period to be connected with flower branches when 5 flowers are grown on the first-stage lateral branches, preserving moisture of the buds, placing the buds in a refrigerator at 4 ℃, carrying out cold treatment for 24 hours under the dark condition, and then sterilizing;
(2) Adding a B5-13 culture medium into the sterilized flower buds to dissociate microspores;
(3) Before inoculation, soaking the flower buds in 70% alcohol for 30 s, pouring off the alcohol, adding 7% sodium hypochlorite solution, soaking for 12min, and washing with sterile water for 3 times;
(4) Extruding microspores from the cleaned flower buds in a B5 culture medium; filtering the suspension with sterile microporous gauze of 50 μm, and centrifuging the filtrate with a centrifuge to obtain precipitate;
(5) Adding 10mL of B5 culture medium into the precipitate, shaking up, and centrifuging the precipitate by adopting a centrifuge to obtain a precipitate, namely microspores;
(6) Transferring the free microspore into NLN culture medium, regulating concentration to 1-2X 10 5 Is/ml and then transferred intoThe incubator is heat-shocked at 33 ℃ for 24h, and then transferred to dark culture at 25 ℃ until embryogenic callus is formed.
And (4) further optimizing: in the step S2, during field planting, the row spacing is 1m, and the plant spacing is 1m; the Chinese cabbage is buried in soil.
Further optimization: in step S3, the concentration of the chlormequat chloride diluent is 500 mg/l, and the root irrigation amount of the chlormequat chloride diluent is as follows: each plant is 100-200 ml.
Further optimization: in step S5, the grafting process is performed as follows:
1) Sterilizing the stock and the blade before grafting; the terminal bud and two lateral branches of the rootstock are remained, and the redundant lateral branches are cut off from the position of 1 cm;
2) Selecting a lateral branch of 6-10cm of the rootstock as a grafting incision, cutting off the lateral branch, and failing to cut the central medullary cavity part of the rootstock;
3) The scion cut is the same as the stock cut, and the xylem of the upper-level branch is reduced as much as possible;
4) Before grafting, ensuring the humidity of the cuts of the stock and the scion and having exudates; aligning the scion notch with the stock notch and then immediately fastening the scion notch and the stock notch with a preservative film;
5) After the cut is bound, removing terminal buds of the scion, leaving 1 leaf, pinching side buds of the scion, and leaving 1 leaf.
Further optimization: in the step 2), the lateral branches respectively generate 1.5-2.5cm long cuts up and down, the widest part of the cut is about 0.6-1cm, and the thickest part is 0.2-0.4mm.
Further optimization: the management after grafting is carried out according to the following steps:
a. watering the grafted body in time after grafting is finished, shading the sun, and controlling the growth environment temperature of the grafted body to be 15-25 ℃ so as to heal the wound;
b. after the wound is healed and the scion recovers the growth vigor, removing the terminal bud and the redundant lateral branches of the rootstock, and reserving a main branch with the distance of 10-15cm from the terminal bud to the scion;
c. grafting for about 25 days, removing the preservative film at the wound, and binding the scion and the stock;
d. controlling the growth environment temperature of the grafting body at 15-25 ℃, fixing the first-level lateral branch on the bracket after the first-level branch of the main branch of the scion grows, removing the top of the main branch of the scion, and collecting 2.5-3mm buds for free microspore culture when 5-10 flowers bloom in the second-level branch.
According to the invention, by adopting the technical scheme, the Chinese cabbage hybrid which is strong in resistance and easy to produce embryos is used as the stock, branches of the Chinese cabbage which is difficult to produce embryos and is subjected to bolting are used as the scions, the environment temperature is controlled to be 15-20 ℃, the illumination time is controlled to be 14-15 hours, and the isolated microspore culture is combined, so that the embryo production rate of the material which is difficult to produce embryos is improved.
According to the invention, the Chinese cabbage with vigorous root activity, strong resistance and high germ production rate can be used as the stock to supply the upper material, so that the resistance signal conduction and resistance level of the upper material can be effectively improved, and the germ production rate of the material difficult to produce germ is improved.
The method for improving the creation efficiency of the Chinese cabbage germplasm disclosed by the invention has the advantages of high grafting success rate (more than 90%), obvious effect (more than 2 times of embryo yield improvement), suitability for creation of Chinese cabbage germplasm materials and wide application value.
The present invention will be further described with reference to the following examples.
Detailed Description
Example 1:
a method for improving the creation efficiency of Chinese cabbage germplasm specifically comprises the following steps:
s1, preparation of rootstocks and scions: hybrid varieties which are strong in resistance, easy to produce embryos and fertile in roots are selected as stock materials, spring cabbage hybrid varieties which are attractive in appearance, good in quality, yellow in core and resistant to bolting are selected as scion materials, then the stocks and scion seeds are sowed in 50-hole seedling trays in the middle ten days of 12 months, and the stocks are placed in a sunlight greenhouse, so that the stocks can smoothly pass vernalization.
In the step S1, the rootstock breed is provided by the Weichai Ministry of agriculture academic institute of Weichai Ministry of 2, and the Weichai Ministry of 2 Chinese cabbage is normally sold in the market.
The scion variety is the Weichai county Chinese cabbage hybrid variety ' Weichai Chun Bai No. 3 ', provided by the Weichai Fang City agricultural academy of sciences, and the Weichai Chun Bai No. 3 ' Chinese cabbage variety is normally sold in the city.
In the step S1, the sunlight greenhouse is built by building a small arched shed and covering two films, the temperature in the sunlight greenhouse is controlled to be more than 0 ℃ and less than 10 ℃, and the rootstocks can pass through vernalization smoothly in the environment of the sunlight greenhouse.
S2, planting: in the middle-upper ten days of 3 months, the stock and the scion seed plants are fixedly planted in an arched shed when bolting is just needed, the row spacing is 1m, and the plant spacing is 1m; during field planting, the immature Chinese cabbages are completely buried in the soil.
S3, management of grafting environment: the growth speed of the lateral branches and the main shoots after the Chinese cabbage is bolting is high, the internal tissues are loose, the growth speed of plants is slowed down by controlling the environmental temperature at 15 ℃, the tissues are compact, and when the shoots are bolting for 20cm, 100 ml of chlormequat chloride diluent with the concentration of 500 mg/L can be used for root irrigation of each plant.
In the step S3, the growth speed of the Chinese cabbage can be slowed down by controlling the growth environment of the Chinese cabbage seed plant, so that the tissue of the plant is compact, and the root is irrigated by using the chlormequat chloride diluent, so that the internode of the plant can be shortened, the tissue is compact, and the later-stage grafting is facilitated and the survival rate of the grafting is improved.
In the step S3, the chlormequat chloride diluent is prepared by diluting chlormequat chloride with water.
S4, selecting grafting conditions: taking a grafting part at the second primary branch of the stock; the selection of the scions is wide, and the scions are preferably light green in color, strong in growth and 6-10cm long in primary branch.
S5, grafting: grafting scion buds on a stock plant, wherein the grafting process is carried out according to the following steps:
1) Before grafting, spraying sterilization and disinfection medicines such as chlorothalonil and the like to disinfect the stock, and disinfecting appliances such as a blade and the like; and (4) reserving the top bud and the two lateral branches of the rootstock, and cutting off the redundant lateral branches from the position of 1 cm.
2) Selecting a lateral branch of 6-10cm of the rootstock as a grafting incision, cutting the lateral branch to form incisions with the length of 1.5cm on the upper part and the lower part of the lateral branch respectively, wherein the widest part of the incision is about 0.6cm, the thickest part of the incision is about 0.2mm, and the central medullary cavity part of the rootstock cannot be cut.
3) The incision of the scion is the same as the incision of the stock, the upper and lower sides of the lateral branch of the scion are respectively 1cm, the widest position of the incision is about 0.6cm, the thickest position is about 0.2mm, the xylem of the upper-level branch is reduced as much as possible, and the incision is neat and smooth.
Design like this, can make and produce great grafting incision on scion bud and the stock plant, can guarantee through this grafting incision that scion and stock cambium have bigger area of contact, survive easily, improve the survival rate.
4) Before grafting, ensuring the humidity of the cuts of the stock and the scion and having exudates; and then, aligning the scion cut with the stock cut, and immediately tightening and binding the scion cut with a preservative film to prevent moisture from evaporating from the wound and influencing wound healing.
5) After the cut is bound, removing terminal buds on the scion, and leaving 1 leaf, if the leaf is larger, reducing the length to 1cm, pinching side buds of the scion, and leaving 1 little leaf.
S6, management after grafting: after grafting, managing the grafted plants by controlling the temperature of the growing environment, wherein the management after grafting is carried out according to the following steps:
a. and watering the grafted body in time after grafting is finished, shading the grafted body by using a shading net, and controlling the growth environment temperature of the grafted body to be 15 ℃ so that the wound can be healed slowly.
b. After the wound is healed and the scion recovers to grow, the terminal bud and the redundant lateral branches of the rootstock are removed, a main branch with the distance of 10-15cm is reserved between the terminal bud and the scion, the scion is prevented from being influenced after the main branch is gradually shrunk, and meanwhile, the scion is ensured to have sufficient nutrition to grow.
c. And (3) after grafting for about 25 days, removing the preservative film at the wound, and binding the scion and the stock one by one to avoid failure caused by wound tearing due to vigorous growth and more branches.
d. Controlling the growth environment temperature of the grafting body at 15 ℃, fixing the first-level lateral branch on the bracket after the first-level branch of the main branch of the scion grows, removing the top of the main branch of the scion, and collecting 2.5-3mm flower buds for free microspore culture when 5-10 flowers bloom in the second-level branch.
S7, culturing the free microspores according to the following steps:
(1) Controlling the growth environment temperature of the grafting body to be 15 ℃, promoting the development consistency of microspores, removing main stems, selecting 2.5 mm flower buds located in the side period of a single nucleus or the early period of a double nucleus to be connected with flower branches when 5 flowers are planted on the first-level lateral branches, moisturizing the flower buds, placing the flower buds in a refrigerator at 4 ℃, carrying out cold treatment for 24 hours under the dark condition, and then sterilizing.
(2) Adding B5-13 culture medium into sterilized bud, and extruding to release microspore.
(3) Before inoculation, the flower buds are transferred into a sterile beaker on a super clean workbench, then 70% alcohol is poured into the sterile beaker for surface disinfection, the alcohol is poured out after soaking for 30 seconds, then 7% sodium hypochlorite solution is added for soaking disinfection for 12min, and then the sterile beaker is washed by sterile water for 3 times.
The B5-13 medium is purchased from Beijing Virgo commercial GmbH, and can be purchased directly from the market.
(4) Putting the cleaned flower bud into a mortar, extruding the flower bud in a B5 culture medium, and extruding microspores in the flower bud; filtering the suspension by using sterile microporous gauze with the diameter of 50 mu m, collecting filtrate, filling the filtrate into a 15ml centrifugal tube, then centrifuging the filtrate in the centrifugal tube by using a centrifuge, wherein the highest rotation speed of the centrifuge is 850-900rpm, the centrifuging time is 4min, and obtaining a precipitate after the centrifuging is finished.
(5) Adding 10mL of B5 culture medium into the precipitate, shaking up, centrifuging the precipitate by using a centrifuge, wherein the highest rotation speed of the centrifuge is 850-900rpm, the centrifuging time is 3-5min, and repeating twice to obtain the precipitate, namely microspores.
The B5 medium in the step (4) and the step (5) is produced by Beijing Western Mejie science and technology Co., ltd and can be directly purchased from the market.
(6) Transferring the free microspores into an NLN culture medium, adjusting the concentration to be 1-2 multiplied by 105/ml, then transferring the free microspores into an incubator to carry out heat shock treatment at 33 ℃, wherein the heat shock treatment time is 24 hours, and after the heat shock treatment is finished, transferring the culture medium into a dark environment at 25 ℃ until embryogenic callus is formed.
Example 2:
a method for improving the creation efficiency of Chinese cabbage germplasm specifically comprises the following steps:
s1, preparation of rootstocks and scions: the method comprises the steps of selecting a hybrid variety which is strong in resistance, easy to produce embryos and large in root fertility as a stock material, selecting a spring Chinese cabbage hybrid variety which is attractive in appearance, good in quality, yellow in core and bolting resistant as a scion material, then sowing seeds in a 50-hole seedling tray in the middle ten days of 12 months, and placing the seedling tray in a sunlight greenhouse, so that the stock can pass vernalization smoothly.
In the step S1, the stock cultivar "Weichai Bai No. 2" was provided by Weichai Fang City agricultural academy, and the "Weichai Bai No. 2" Chinese cabbage cultivar was normally marketed in the market.
The scion variety is the Weichai county Chinese cabbage hybrid variety ' Weichai Chun Bai No. 3 ', provided by the Weichai Fang City agricultural academy of sciences, and the Weichai Chun Bai No. 3 ' Chinese cabbage variety is normally sold in the city.
In the step S1, the sunlight greenhouse is built by building a small arched shed and covering two films, the temperature in the sunlight greenhouse is controlled to be more than 0 ℃ and less than 10 ℃, and the rootstocks can smoothly pass through vernalization in the environment of the sunlight greenhouse.
S2, field planting: in the middle-upper ten days of 3 months, the stock seed plants are fixedly planted in the arched shed when bolting is carried out, the row spacing is 1m, and the plant spacing is 1m; during field planting, the immature Chinese cabbages are completely buried in the soil.
S3, management of grafting environment: the growth speed of the lateral branches and the main bolt after bolting of the Chinese cabbage is high, the internal tissues are loose, the growth speed of plants is slowed down by controlling the environmental temperature at 17 ℃, the tissues are compact, and when the bolting is carried out for 20cm, 150 ml of chlormequat chloride diluent per plant can be used for root irrigation at the same time.
In the step S3, the growth speed of the Chinese cabbage can be slowed down by controlling the growth environment of the Chinese cabbage seed plant, so that the tissue of the plant is compact, and the root is irrigated by using the chlormequat chloride diluent, so that the internode of the plant can be shortened, the tissue is compact, and the later-stage grafting is facilitated and the survival rate of the grafting is improved.
S4, selecting grafting conditions: the second primary branch of the stock is used as a grafting part, the selection range of the scion is wide, and the primary branch with light green color, strong growth and 8cm length is preferred.
S5, grafting: grafting scion buds on a stock plant, wherein the grafting process is carried out according to the following steps:
1) Before grafting, spraying sterilization and disinfection medicines such as chlorothalonil and the like to disinfect the stock, and disinfecting the instruments such as blades and the like; the terminal bud and two lateral branches are remained, and the redundant lateral branches are cut off from the position of 1 cm.
2) And selecting a lateral branch of 6-10cm of the rootstock as a grafting incision, wherein the lateral branch can not be cut at the same root by cutting, 2 cm-long incisions are respectively formed on the upper part and the lower part of the lateral branch, the widest part of the incision is about 0.8cm, the thickest part of the incision is about 0.3mm, and the central medullary cavity part of the rootstock can not be cut.
3) The incision of the scion is the same as the incision of the stock, the upper side and the lower side of the lateral branch of the scion are respectively 1.2cm, the widest position of the incision is about 0.8cm, the thickest position is about 0.3mm, xylem of the upper-level branch is reduced as much as possible, and the incision is neat and smooth.
Design like this, can make and produce great grafting incision on scion bud and the stock plant, can guarantee through this grafting incision that scion and stock cambium have bigger area of contact, survive easily, improve the survival rate.
4) Before grafting, ensuring the humidity of the cuts of the stock and the scion and having exudates; and then, aligning the scion cut with the stock cut, and immediately tightening and binding the scion cut with a preservative film to prevent moisture from evaporating from the wound and influencing wound healing.
5) After the cut is bound, removing terminal buds on the scion, and leaving 1 leaf, if the leaf is larger, reducing the length to 1cm, pinching side buds of the scion, and leaving 1 leaflet.
S6, management after grafting: after grafting, managing the grafted plants by controlling the temperature of the growing environment, wherein the management after grafting is carried out according to the following steps:
a. and watering the grafted body in time after grafting is finished, shading the grafted body by using a shading net, and controlling the growth environment temperature of the grafted body to be 20 ℃ so that the wound can be healed slowly.
b. After the wound is healed and the scion recovers the growth vigor, the terminal bud and the redundant lateral branches of the rootstock are removed, a main branch with the distance of 12cm is reserved between the terminal bud and the scion, the scion is prevented from being influenced after the main branch is gradually shrunk, and meanwhile, the scion is ensured to have sufficient nutrition to grow.
c. And (3) grafting for about 25 days, removing the preservative film at the wound, and binding the scion and the stock one by one to avoid failure caused by the fact that the scion and the stock grow vigorously and branch more and the wound is torn.
d. Controlling the growth environment temperature of the grafting body at 20 ℃, fixing the first-level lateral branch on the bracket after the first-level branch of the main branch of the scion grows, removing the top of the main branch of the scion, and collecting 2.7mm buds for free microspore culture when 7 flowers bloom in the second-level branch.
S7, culturing the free microspores according to the following steps:
(1) Controlling the growth environment temperature of the grafting body to be 20 ℃, promoting the development consistency of microspores, removing main stems, selecting 2.7mm flower buds located in the side period of a single nucleus or the early period of a double nucleus to be connected with flower branches when 5 flowers are planted on the first-level lateral branches, moisturizing the flower buds, placing the flower buds in a refrigerator at 4 ℃, carrying out cold treatment for 24 hours under the dark condition, and then sterilizing.
(2) Adding B5-13 culture medium into sterilized bud, and extruding to release microspore.
(3) Before inoculation, the flower buds are transferred into a sterile beaker on a super clean workbench, then 70% alcohol is poured into the sterile beaker for surface disinfection, the alcohol is poured out after the flower buds are soaked for 30 seconds, then 7% sodium hypochlorite solution is added for soaking disinfection for 12min, and then the flower buds are washed by sterile water for 3 times.
(4) Putting the cleaned flower buds into a mortar, extruding the flower buds in a B5 culture medium, and extruding microspores; filtering the suspension by using sterile microporous gauze with the diameter of 50 mu m, collecting filtrate, filling the filtrate into a 15ml centrifugal tube, and then centrifuging the filtrate in the centrifugal tube by using a centrifuge, wherein the highest rotation speed of the centrifuge is 870rpm, the centrifuging time is 4min, and precipitates are obtained after the centrifuging is finished.
(5) Adding 10mL of B5 culture medium into the precipitate, shaking up, and then centrifuging the precipitate by using a centrifuge, wherein the highest rotation speed of the centrifuge is 870rpm, the centrifuging time is 4min, and repeating the centrifuging twice to obtain the precipitate, namely the microspore.
(6) Transferring the free microspore into NLN culture medium, adjusting the concentration to 1.5X 10 5 And/ml, then performing heat shock treatment at 33 ℃ for 24 hours, and transferring to dark culture at 25 ℃ after the heat shock treatment is completed until embryogenic callus is formed.
Example 3:
a method for improving the creation efficiency of Chinese cabbage germplasm specifically comprises the following steps:
s1, preparation of rootstocks and scions: the method comprises the steps of selecting a hybrid variety which is strong in resistance, easy to produce embryos and large in root fertility as a stock material, selecting a spring Chinese cabbage hybrid variety which is attractive in appearance, good in quality, yellow in core and bolting resistant as a scion material, then sowing seeds in a 50-hole seedling tray in the middle ten days of 12 months, and placing the seedling tray in a sunlight greenhouse, so that the stock can pass vernalization smoothly.
In the step S1, the stock cultivar "Weichai Bai No. 2" was provided by Weichai Fang City agricultural academy, and the "Weichai Bai No. 2" Chinese cabbage cultivar was normally marketed in the market.
The scion variety is the Weichai county Chinese cabbage hybrid variety ' Weichai Chun Bai No. 3 ', provided by the Weichai Fang City agricultural academy of sciences, and the Weichai Chun Bai No. 3 ' Chinese cabbage variety is normally sold in the city.
In the step S1, the sunlight greenhouse is built by building a small arched shed and covering two films, the temperature in the sunlight greenhouse is controlled to be more than 0 ℃ and less than 10 ℃, and the rootstocks can smoothly pass through vernalization in the environment of the sunlight greenhouse.
S2, planting: in the middle-upper ten days of 3 months, the stock seed plants are fixedly planted in the arched shed when bolting is carried out, the row spacing is 1m, and the plant spacing is 1m; during field planting, the immature Chinese cabbages are completely buried in the soil.
S3, management of grafting environment: the growth speed of the lateral branches and the main bolt after bolting of the Chinese cabbage is high, the internal tissues are loose, the growth speed of plants is slowed down by controlling the environmental temperature at 20 ℃, the tissues are compact, and when the bolting is carried out for 20cm, 200 ml of chlormequat chloride diluent per plant can be used for root irrigation at the same time.
In the step S3, the growth speed of the Chinese cabbage can be slowed down by controlling the growth environment of the Chinese cabbage seed plant, so that the tissue of the plant is compact, and the root irrigation is carried out through the chlormequat chloride diluent, so that the internode of the plant can be shortened, the tissue is compact, and the later-stage grafting is facilitated and the survival rate of the grafting is improved.
S4, selecting grafting conditions: the second primary branch of the stock is used as a grafting part, the scion is selected widely, and the primary branch which is light green in color, strong in growth and 10cm in length is preferred.
S5, grafting: grafting the scion bud on a stock plant, wherein the grafting process is carried out according to the following steps:
1) Before grafting, spraying sterilization and disinfection medicines such as chlorothalonil and the like to disinfect the stock, and disinfecting the instruments such as blades and the like; the terminal bud and two lateral branches are remained, and the redundant lateral branches are cut off from the position of 1 cm.
2) Selecting 10cm lateral branches of the rootstock as grafting incisions, wherein the lateral branches can not be cut at the same root, 2.5cm long incisions are respectively formed on the upper part and the lower part of each lateral branch, the widest part of the incision is about 1cm, the thickest part of the incision is about 0.4mm, and the central medullary cavity part of the rootstock can not be cut.
3) The scion cut is the same as the stock cut, the upper side and the lower side of each lateral branch of the scion are 1.5cm, the widest position of the cut is about 1cm, the thickest position of the cut is about 0.4mm, the xylem of the upper-level branch is reduced as much as possible, and the cut is neat and smooth.
Design like this, can make and produce great grafting incision on scion bud and the stock plant, can guarantee through this grafting incision that scion and stock cambium have bigger area of contact, survive easily, improve the survival rate.
4) Before grafting, ensuring the humidity of the cuts of the rootstock and the scion and having exudates; and then, aligning the scion cut with the stock cut, and immediately tightening and binding the scion cut with a preservative film to prevent moisture from evaporating from the wound and influencing wound healing.
5) After the cut is bound, removing terminal buds on the scion, and leaving 1 leaf, if the leaf is larger, reducing the length to 1cm, pinching side buds of the scion, and leaving 1 little leaf.
S6, management after grafting: after grafting, managing the grafted plants by controlling the temperature of the growing environment, wherein the management after grafting is carried out according to the following steps:
a. and watering the grafting body in time after grafting is finished, shading the sun by using a shading net, and controlling the growth environment temperature of the grafting body to be 25 ℃ so that the wound can be healed slowly.
b. After the wound is healed and the scion recovers to grow, the terminal bud and the redundant lateral branches of the rootstock are removed, the main branch with the distance of 15cm between the terminal bud and the scion is reserved, the scion is prevented from being influenced after the main branch gradually shrinks, and meanwhile, the scion is ensured to have sufficient nutrition to grow.
c. And (3) grafting for about 25 days, removing the preservative film at the wound, and binding the scion and the stock one by one to avoid failure caused by the fact that the scion and the stock grow vigorously and branch more and the wound is torn.
d. Controlling the growth environment temperature of the grafting body at 25 ℃, fixing the first-level lateral branch on the bracket after the first-level branch of the main branch of the scion grows, removing the top of the main branch of the scion, and collecting 3mm buds for free microspore culture when 10 flowers bloom in the second-level branch.
S7, culturing the free microspore according to the following steps:
(1) Controlling the growth environment temperature of the grafting body to be 25 ℃, promoting the development consistency of microspores, removing main stems, selecting 3mm flower buds located in the side period of a single nucleus or the early period of a double nucleus to be connected with flower branches when 5 flowers are planted on the first-level lateral branches, preserving moisture of the flower buds, placing the flower buds in a refrigerator at 4 ℃, carrying out cold treatment for 24 hours under the dark condition, and then sterilizing.
(2) Adding B5-13 culture medium into sterilized bud, and separating microspore by squeezing method.
(3) Before inoculation, the flower buds are transferred into a sterile beaker on a super clean workbench, then 70% alcohol is poured into the sterile beaker for surface disinfection, the alcohol is poured out after the flower buds are soaked for 30 seconds, then 7% sodium hypochlorite solution is added for soaking disinfection for 12min, and then the flower buds are washed by sterile water for 3 times.
(4) Putting the cleaned flower buds into a mortar, extruding the flower buds in a B5 culture medium, and extruding microspores; filtering the suspension by using sterile microporous gauze with the diameter of 50 mu m, collecting filtrate, filling the filtrate into a 15ml centrifugal tube, and then centrifuging the filtrate in the centrifugal tube by using a centrifuge, wherein the highest rotation speed of the centrifuge is 900rpm, the centrifuging time is 4min, and precipitates are obtained after the centrifuging is finished.
(5) Adding 10mL of B5 culture medium into the precipitate, shaking up, and then centrifuging the precipitate by using a centrifuge, wherein the highest rotation speed of the centrifuge is 900rpm, the centrifuging time is 5min, and repeating twice to obtain the precipitate, namely the microspore.
(6) Transferring the free microspore into NLN culture medium, regulating concentration to 1-2X 10 5 Per ml, 10 dishes per treatment; then heat shock treatment is carried out at 33 ℃, the heat shock treatment time is 24 hours, and after the heat shock treatment is finished, dark culture is carried out at 25 ℃ until embryogenic callus is formed.
The method for improving the germplasm creation efficiency of the Chinese cabbage provided by the embodiment of the invention can effectively improve the germ production rate of free microspores of materials difficult to produce germs, accelerate the purification speed of the Chinese cabbage materials and improve the germplasm creation efficiency.
The Chinese cabbage germplasm provided by the invention is sown to detect the survival rate, the flowering phase, the plant robustness and the germ production rate of the Chinese cabbage germplasm.
And the Wei white No. 2 Chinese cabbage and the Wei spring white No. 3 Chinese cabbage and the graft are adopted: the germplasm of the scion (Weichai Bai No. 2) + the stock (Weichai Chun Bai No. 3) is synchronously sown, the management processes of all groups are the same, and the germplasm detection results are shown in the following table:
as can be seen from the table above, the Chinese cabbage has the germplasm survival rate of 95 percent, slightly delayed flowering phase and short and thick lateral branch dark green internodes; the embryo emergence rate is 3.07 +/-0.29 embryos and buds, is improved by 1.47 times compared with the embryo emergence rate of the Wei white No. 2 seedlings, and the effect is obvious.
It will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in the embodiments described above without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims.
Claims (7)
1. A method for improving the creation efficiency of Chinese cabbage germplasm is characterized by comprising the following steps: the method specifically comprises the following steps:
s1, preparation of rootstocks and scions: selecting a hybrid variety which is strong in resistance, easy to produce embryos and large in root fertilizer as a stock material, selecting a bolting-resistant spring cabbage hybrid variety as a scion material, and sowing the stock and scion seeds and smoothly performing vernalization; the germ output rate of the rootstock is superior to that of the scion;
s2, field planting: in the middle-upper 3 months, the stock and the scion seed plants are fixedly planted in an arched shed just before bolting;
s3, management of grafting environment: the environmental temperature is controlled at 15-20 ℃ to lead the plants to grow, and when the shoots are bolting for 20cm, the chlormequat chloride diluent is used for irrigating roots;
s4, selecting grafting conditions: making a grafting part at the second primary branch of the stock; the scion is selected as a first-level branch with the length of 6-10 cm;
s5, grafting: grafting scion buds on a stock plant;
s6, management after grafting: and after grafting is finished, managing the grafted plants by controlling the temperature of the growing environment.
2. The method for improving the germplasm creation efficiency of Chinese cabbages according to claim 1, which is characterized by comprising the following steps of: the creation method further comprises the following steps: s7, culturing free microspores, wherein the free microspores are cultured according to the following steps:
(1) Controlling the growth environment temperature of the grafting body to be 15-25 ℃, removing the main flower shoots, selecting 2.5-3mm flower buds in the first-level lateral period or the double-core early period to be connected with flower branches when 5 flowers are planted on the first-level lateral branches, preserving moisture of the flower buds, placing the flower buds in a refrigerator at 4 ℃, performing cold treatment for 24 hours under the dark condition, and then sterilizing;
(2) Adding a B5-13 culture medium into the sterilized flower buds to dissociate microspores;
(3) Before inoculation, soaking the flower bud in 70% alcohol for 30 s, pouring off the alcohol, adding 7% sodium hypochlorite solution, soaking for 12min, and washing with sterile water for 3 times;
(4) Extruding microspores from the cleaned flower buds in a B5 culture medium; filtering the suspension with sterile microporous gauze of 50 μm, and centrifuging the filtrate with a centrifuge to obtain precipitate;
(5) Adding 10mL of B5 culture medium into the precipitate, shaking up, and centrifuging the precipitate by adopting a centrifuge to obtain the precipitate, namely microspores;
(6) Transferring the free microspore into NLN culture medium, regulating the concentration to 1-2X 10 5 And/ml, then transferred to an incubator for heat shock treatment at 33 ℃ for 24h, and then transferred to dark culture at 25 ℃ until embryogenic callus is formed.
3. The method for improving the germplasm creation efficiency of Chinese cabbages according to claim 2, which is characterized in that: in the step S2, during field planting, the row spacing is 1m, and the plant spacing is 1m; the Chinese cabbage is buried in soil.
4. The method for improving Chinese cabbage germplasm creation efficiency according to claim 3, wherein the method comprises the following steps: in the step S3, the concentration of the chlormequat chloride diluent is 500 mg/L, and the root irrigation amount of the chlormequat chloride diluent is as follows: each plant is 100-200 ml.
5. The method for improving the germplasm creation efficiency of Chinese cabbages according to claim 4, which is characterized in that: in step S5, the grafting process is performed as follows:
1) Sterilizing the stock and the blade before grafting; reserving the terminal bud and two lateral branches of the rootstock, and cutting off the redundant lateral branches from a position of 1 cm;
2) Selecting a lateral branch of 6-10cm of the rootstock as a grafting incision, cutting off the lateral branch, and failing to cut the central medullary cavity part of the rootstock;
3) The scion cut is the same as the stock cut, and the xylem of the upper-level branch is reduced as much as possible;
4) Before grafting, ensuring the humidity of the cuts of the stock and the scion and having exudates; aligning the scion notch with the stock notch and then immediately fastening the scion notch and the stock notch with a preservative film;
5) After the cut is bound, removing terminal buds of the scion, leaving 1 leaf, pinching side buds of the scion, and leaving 1 leaf.
6. The method for improving the germplasm creation efficiency of Chinese cabbages according to claim 5, which is characterized in that: in the step 2), the upper and lower parts of the lateral branches respectively generate 1.5-2.5cm long cuts, the widest part of the cut is 0.6-1cm, and the thickest part is 0.2-0.4mm.
7. The method for improving the germplasm creation efficiency of Chinese cabbages according to claim 6, which is characterized in that: the management after grafting is carried out according to the following steps:
watering the grafted body in time after grafting, shading the sun, and controlling the growth environment temperature of the grafted body at 15-25 ℃ to enable the wound to heal;
b. after the wound is healed and the scion recovers the growth vigor, removing the terminal bud and the redundant lateral branches of the rootstock, and reserving a main branch with the distance of 10-15cm from the terminal bud to the scion;
c. grafting for about 25 days, removing the preservative film at the wound, and binding the scion and the stock;
d. controlling the growth environment temperature of the grafting body at 15-25 ℃, fixing the first-level lateral branch on the bracket after the first-level branch of the main branch of the scion grows, removing the top of the main branch of the scion, and collecting 2.5-3mm buds for free microspore culture when 5-10 flowers bloom in the second-level branch.
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