CN115462312A - Method for obtaining distant hybridization progeny of oriental cherry through embryo rescue technology - Google Patents
Method for obtaining distant hybridization progeny of oriental cherry through embryo rescue technology Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
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Abstract
The invention provides a method for obtaining cherry blossom distant hybridization progeny through an embryo rescue technology, which belongs to the technical field of planting and comprises the steps of pollen obtaining, emasculation, hybridization, hybrid embryo obtaining, hybrid embryo culture to obtain hybrid seedlings, hardening seedlings, transplanting and domesticating. The invention has the advantages that: the pollen activity is high: the pollen detection activity reaches 92 percent; obtaining multiple filial generations: compared with the method for collecting seeds and raising seedlings after hybridization, the method can obtain more filial generations, improve the success rate of hybridization, ensure that the success rate of the hybridization of the seed raising seedlings is less than 25 percent, and ensure that the success rate of the method reaches 90 percent; shortening the seedling culture period: the method can avoid the defect of long seedling period of seed sowing, shortens the seedling period to 90 days, can obtain a large amount of filial generation seedlings in the current year of hybridization, and accelerates the time of distant hybridization breeding; strong seedlings can be obtained, and the survival rate of the seedlings is improved: in the embryo rescue process, strong seedling treatment can be carried out on the seedlings, the survival rate of the seedlings is increased, and the defect that weak seedlings are easy to survive when the hybrid seeds are sown is overcome.
Description
Technical Field
The application relates to the technical field of plant planting, in particular to a method for obtaining cherry blossom distant hybridization progeny through an embryo rescue technology.
Background
The Cerasus plant belongs to Rosaceae Prunuseidea, about 150 species are distributed in northern hemisphere temperate zone from European continental to northern America, most of the cherry blossom has the gorgeous of peach and the fragrance of plum, and is a famous spring woody flower plant and autumn leaf tree species. The wild oriental cherry resources in China are rich, the existing wild oriental cherry resources exceed 52 varieties and occupy more than 1/3 of the oriental cherry plant resources in the world, a plurality of oriental cherry varieties with very high ornamental value exist, the oriental cherry breeding work in China is still in a relatively early stage, most of the oriental cherry germplasm resources are still in a state of waiting for utilization and development, and therefore, the hybridization breeding of the oriental cherry resources to breed new varieties is a very necessary work.
The cherry blossom distant hybridization breeding has important significance for cherry blossom breeding work, and particularly has profound significance for the development work of Chinese native cherry blossom. The problem that the oriental cherry distant hybridization is low in affinity and easy to abortion also exists, and the embryo rescue technology is an important technology for solving the problem and is an important technical means for overcoming the problems of incompatibility and abortion of distant hybridization.
The patent with patent publication number of CN110050690A and patent name of southern area sweet cherry and Chinese cherry interspecific hybrid embryo rescue seedling method comprises the following steps:
1) Pollen collection and pollination: collecting natural flowers of the bell in a florescence by taking Chinese cherries (Prunus L.) as male parents; stripping off anther indoors, and drying in the shade at 20-30 deg.C; collecting the anther in a small glass bottle after the anther cracks and looses powder, sealing and storing in a refrigerator at 1-6 ℃ for later use; using European sweet cherry (Prunus L.) as female parent, self-incompatibility of female parent, and artificial pollination without emasculation of flowers in bell period; immediately covering a sulfuric acid paper bag after pollination, and replacing the mesh bag after 5-10 days;
2) Fruit picking and low-temperature lamination treatment: respectively picking hybrid young fruits of which the female parent is in the hard kernel stage and the embryo length/seed length PF is more than 0.5, putting the fresh fruits into a transparent plastic preservation box, and storing the fresh fruits in a low-temperature refrigerator at the temperature of 1-6 ℃ for 30-40 days;
3) Inoculating young embryos and carrying out germination culture: after the low-temperature treatment is finished, washing the fruits, removing the pulp and taking embryos; sterilizing in a clean bench; peeling off the inner seed coat, inoculating on a 1/2MS solid culture medium containing a first exogenous growth promoting additive, and culturing in a culture chamber;
4) Subculture propagation culture: taking the sterile young sprout, cutting stem section with length of 1.5-2.5cm, inoculating on subculture medium MS, and adding second exogenous growth promoting additive into the subculture medium MS; culturing in culture room for 35-45d;
5) Rooting culture: after the hybrid seedlings are subjected to subculture, the shoots of the strong cluster buds with the plant height of more than 2cm are inoculated into a rooting culture medium 1/2MS added with a third exogenous growth promoting additive; culturing in culture room for 35-45 days to obtain hybrid seedling;
6) Hardening and transplanting seedlings: hardening the hybrid seedlings for 10-20 days, and transplanting the hybrid seedlings into a nutrition pot; the transplanting time is 2 middle of the month.
The above patents suffer from the following disadvantages: the method has the advantages of insufficient universality with other cherry blossom varieties, large species difference between the sweet cherry and the cherry blossom, large difference between the method and the material adopted for embryo rescue, complex operation and low survival rate.
Disclosure of Invention
In order to achieve the purpose, the invention provides a method for obtaining the distant hybridization progeny of oriental cherry through an embryo rescue technology. The invention aims to overcome the obstacle of incompatibility of cherry blossom distant hybridization and provide an obtained cherry blossom distant hybridization progeny propagation method. Solves the problems of incompatible distant hybridization of oriental cherry and low survival rate of hybrid embryo.
The invention adopts the following technical scheme: a method for obtaining a cherry blossom distant hybridization progeny by an embryo rescue technology comprises the following steps:
1) Pollen acquisition: selecting oriental cherries without anther pollen scattering from a male parent tree, taking off the anthers by using forceps, placing the anthers in a culture dish filled with a sulfuric acid paper bag, drying to crack stamens and scatter pollen, then putting the scattered pollen and the anthers into a 5ml centrifugal tube, and refrigerating at 4 ℃ for later use or freezing and storing at-80 ℃;
2) The castration method comprises the following steps: selecting unopened large-bud-stage oriental cherries 1 day before pollination for artificial emasculation, and bagging the flowers with a sulfuric acid paper bag after emasculation;
3) The hybridization method comprises the following steps: restoring the refrigerated or frozen pollen to normal temperature, dipping the pollen by using a small brush, pollinating pistils which are castrated in advance, and pollinating 3-4 flowers once by dipping, and then carrying out pollination post-treatment;
4) Obtaining a hybrid embryo: after the flowers of the female parent are pollinated for 25-30 days, collecting young fruits, cleaning and disinfecting the young fruits, and transferring the young fruits to a tray for later use; obtaining clean hybrid embryos by a hybrid embryo stripping method;
5) Hybrid embryo culture to obtain hybrid seedlings: inoculating the hybrid embryo into a culture bottle of an embryo rescue culture medium in a way that the embryo tip is downward and is shallowly inserted into the culture medium, culturing for 30-35 days after inoculation, transferring the induced seedling into a strong seedling culture medium for strong seedling culture, transferring the strong seedling into a rooting culture medium for rooting culture after 30 days, and obtaining the hybrid seedling after 25-30 days;
6) Hardening, transplanting and domesticating: after rooting culture for 25-30 days, selecting 4-5 leaves and 3-4 root seedlings, opening a tissue culture bottle for hardening seedlings to ensure that the temperature and humidity of the growing environment of the hybrid seedlings are consistent with the external environment, cleaning the residual culture medium at the roots of the hybrid seedlings after 7-14 days, transplanting for planting, watering after planting, transferring to a small arched shed for moisturizing, and opening the small arched shed for normal management after 7-15 days.
Preferably, in step 1), the pollen drying method comprises: naturally dispersing and drying for 12-24 hours, and then putting into a drying dish for drying for 8 hours.
Preferably, in step 3), post-pollination treatment: bagging with sulphuric acid paper bag after pollination, removing paper bag after 5-7 days, and bagging with nylon mesh bag.
Preferably, in the step 4), the young fruit is cleaned and disinfected by the following steps: cleaning with tap water, transferring to a clean bench, sterilizing with 75% alcohol for 30s-1min, cleaning with sterile water for 2-3 times, soaking in 10% sodium hypochlorite solution for 8 min or 0.1% HgCl solution for 5 min, and cleaning with sterile water for 4-5 times.
Preferably, in step 4), the hybrid embryo stripping method comprises: slightly clamping young fruits at an angle vertical to the abdominal suture line of the fruits by using operating forceps, opening seed shells along the clamping seams by using forceps, taking out seeds, and peeling off the seed shells by using the forceps to obtain clean hybrid embryos, wherein the embryo tip part cannot be injured in the peeling process.
Preferably, in step 5), the embryo rescue medium: MS +6-BA1.0mg/L + KT0.5mg/L + NAA0.2mg/L.
Preferably, in the step 5), the culture mode in the embryo rescue culture medium is as follows: culturing for 5-7 days under the condition of no light with the temperature of 25-28 ℃ and the humidity of 70-80% after inoculation, then transferring to the condition of light for continuous culture, and performing strong seedling culture after culturing for 30-35 days.
Preferably, in step 5), the strong seedling culture medium: MS +6-BA0.5mg/L + GA30.5mg/L + NAA0.2mg/L.
Preferably, in step 5), the rooting medium: WPM + IBA1.0mg/L + NAA1.0mg/L.
Preferably, in step 6), the transplanting plants are selected to be filled with peat: perlite =1 substrate planted in 50 hole forest high foot plug trays.
The method for rescuing the cherry blossom distant hybrid embryos has the advantages that:
1. the pollen activity is high: the pollen obtained by the pollen obtaining method in the method has the pollen detection activity up to 92%;
2. obtaining multiple filial generations: compared with the method for harvesting and raising the seedlings after hybridization, the embryo rescue method can obtain more filial generations, improve the success rate of hybridization, the success rate of hybridization of the seedling raising of the seeds is less than 25 percent, the success rate of the method reaches 90 percent, and greatly improve the survival rate of distant filial generations;
3. shortening the seedling culture period: the method can avoid the defect of long seedling period of seed sowing and seedling raising, the seedling raising period is shortened to 90 days, a large number of filial generation seedlings can be obtained in the current year of hybridization, and the time of distant hybridization breeding is shortened;
4. strong seedlings can be obtained, and the survival rate of the seedlings is improved: in the embryo rescue process, strong seedling treatment can be carried out on the seedlings, the survival rate of the seedlings is increased, and the defect that weak seedlings are easy to survive when the hybrid seeds are sown is overcome.
5. The time for rescuing the cherry blossom embryo is determined to be 25-30 days after pollination.
6. The operation is simple: the fruit does not require low temperature stratification.
7. The embryo rescue culture medium is adjusted to be MS culture medium, and the rooting culture medium is adjusted to be WPM culture medium.
8. The peat and perlite (1) mixed matrix is adopted, the seedling hardening survival rate is higher, the survival rate is more than 95%, and the survival rate is improved by 10% compared with the survival rate of the prior art.
Detailed Description
The features and advantages of the present application will become more apparent and appreciated from the following detailed description of the application.
In the description of the present application, it should be noted that the terms "upper", "lower", "inner", "outer", "front", "rear", "left" and "right" and the like indicate orientations or positional relationships based on operational states of the present application, and are only used for convenience of description and simplification of description, but do not indicate or imply that the referred device or element must have a specific orientation, be constructed in a specific orientation and be operated, and thus, should not be construed as limiting the present application. Furthermore, the terms "first," "second," "third," and "fourth" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance.
The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the embodiments of the present application, and it is obvious that the described embodiments are only a part of the embodiments of the present application, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.
Example 1
A method for obtaining cherry blossom distant hybridization offspring by an embryo rescue technology comprises the following steps:
1) Pollen acquisition: selecting oriental cherries without anther pollen scattering from a male parent tree, taking off the anthers by using forceps, placing the anthers in a culture dish filled with a sulfuric acid paper bag, naturally dispersing light and drying for 12 hours, then placing the anthers in a drying dish and drying for 8 hours by using silica gel, so that stamens crack and pollen is scattered, then putting the scattered pollen and the anthers into a 5ml centrifugal tube, and refrigerating at 4 ℃ for later use;
2) The castration method comprises the following steps: selecting unopened large-bud-stage oriental cherries 1 day before pollination for artificial emasculation, and bagging the flowers with a sulfuric acid paper bag after emasculation;
3) The hybridization method comprises the following steps: restoring cold-stored or frozen pollen to normal temperature, dipping pollen with a small brush, pollinating pistil which has been castrated in advance, dipping 3 flowers which can be pollinated once, bagging with a sulfuric acid paper bag after pollination, removing the paper bag after 5 days, and bagging with a nylon mesh bag.
4) Obtaining a hybrid embryo: after female parent flowers pollinate for 25 days, picking young fruits, cleaning the young fruits by using tap water, transferring the young fruits to a super clean workbench, sterilizing for 1min by using 75% alcohol, cleaning for 2-3 times by using sterile water, soaking and sterilizing for 8 min by using a 10% sodium hypochlorite solution, cleaning for 4 times by using the sterile water, and transferring the young fruits to a tray for later use; lightly clamping young fruits at an angle vertical to the abdominal suture line of the fruits by using an operating forceps, opening seed shells along the clamping seams by using the forceps, taking out seeds, and peeling off the seed shells by using the forceps to obtain clean hybrid embryos, wherein the embryo tip parts cannot be damaged in the peeling process.
5) And (3) culturing the hybrid embryo to obtain a hybrid seedling: inoculating the hybrid embryo into a culture bottle filled with an MS +6-BA1.0mg/L + KT0.5mg/L + NAA0.2mg/L culture medium in a way that the embryo tip faces downwards, inserting the hybrid embryo in a shallow manner on the culture medium, culturing for 5 days under a lightless condition with the temperature of 25 ℃ and the humidity of 70% after inoculation, then transferring to a light condition for continuous culture, transferring the induced seedling into the MS +6-BA0.5mg/L + GA30.5mg/L + NAA0.2mg/L culture medium for strong seedling culture after 30 days, transferring the strong seedling into the WPM + IBA1.0mg/L + NAA1.0mg/L culture medium for rooting culture after 30 days, and obtaining the hybrid seedling after 25 days.
6) Hardening, transplanting and domesticating: after rooting culture for 25 days, selecting 4 leaves and 3 seedlings, opening a tissue culture bottle for hardening seedlings to ensure that the temperature and the humidity of the growth environment of the hybrid seedlings are consistent with those of the external environment, cleaning the residual culture medium at the roots of the hybrid seedlings after 7 days, and transplanting the hybrid seedlings to a container filled with peat: and (2) planting perlite (1) in a 50-hole forest high-foot plug tray of a substrate, watering thoroughly after planting, transferring the plug tray to a small arched shed for moisturizing, and opening the small arched shed for normal management after 7 days.
Example 2
A method for obtaining cherry blossom distant hybridization offspring by an embryo rescue technology comprises the following steps:
1) Pollen acquisition: selecting oriental cherries without anther pollen scattering from a male parent tree, taking off the anthers by using forceps, placing the anthers in a culture dish filled with a sulfuric acid paper bag, naturally dispersing light and drying for 24 hours, then placing the anthers in a drying dish and drying for 8 hours by using silica gel, so that stamens crack and pollen is scattered, and then putting the scattered pollen and the anthers into a 5ml centrifugal tube for freezing and storing at minus 80 ℃;
2) The castration method comprises the following steps: selecting unopened large-bud-stage oriental cherries for artificial emasculation 1 day before pollination, and bagging with sulfuric acid paper bags after emasculation;
3) The hybridization method comprises the following steps: restoring cold-stored or frozen pollen to normal temperature, dipping pollen with a small brush, pollinating pistil which has been castrated in advance, dipping 4 flowers which can be pollinated once, bagging with a sulfuric acid paper bag after pollination, removing the paper bag after 7 days, and bagging with a nylon mesh bag.
4) Obtaining a hybrid embryo: after the female parent flowers pollinate for 30 days, picking young fruits, cleaning the young fruits by using tap water, transferring the young fruits to a super clean workbench, sterilizing for 1min by using 75% alcohol, cleaning for 3 times by using sterile water, soaking and sterilizing for 5 min by using 0.1% HgCl solution, cleaning for 5 times by using the sterile water, and transferring the young fruits to a tray for later use; lightly clamping young fruits at an angle vertical to the abdominal suture line of the fruits by using an operating forceps, opening seed shells along the clamping seams by using the forceps, taking out seeds, and peeling off the seed shells by using the forceps to obtain clean hybrid embryos, wherein the embryo tip parts cannot be damaged in the peeling process.
5) And (3) culturing the hybrid embryo to obtain a hybrid seedling: inoculating the hybrid embryo into a culture bottle filled with MS +6-BA1.0mg/L + KT0.5mg/L + NAA0.2mg/L culture medium in a way that the embryo tip is downward, inserting the hybrid embryo in a shallow manner on the culture medium, culturing for 7 days under a lightless condition with the temperature of 28 ℃ and the humidity of 80% after inoculation, then transferring to a light condition for continuous culture, transferring the induced seedling into MS +6-BA0.5mg/L + GA30.5mg/L + NAA0.2mg/L culture medium for strong seedling culture after 35 days, transferring the strong seedling into WPM + IBA1.0mg/L + NAA1.0mg/L culture medium for rooting culture after 30 days, and obtaining the hybrid seedling after 30 days.
6) Hardening, transplanting and domesticating: after rooting culture is carried out for 30 days, 5 leaves and 4 seedlings are selected, a tissue culture bottle is opened for hardening seedlings, the temperature and the humidity of the growing environment of the hybrid seedlings are consistent with the external environment, after 14 days, the residual culture medium at the roots of the hybrid seedlings is cleaned, and the hybrid seedlings are transplanted to a container filled with peat: and (2) planting the perlite (1).
Example 3
A method for obtaining cherry blossom distant hybridization offspring by an embryo rescue technology comprises the following steps:
1) Pollen acquisition: selecting oriental cherries without anther pollen scattering from a male parent tree, taking off the anthers by using forceps, placing the anthers in a culture dish filled with a sulfuric acid paper bag, naturally dispersing light and drying for 20 hours, then placing the anthers in a drying dish and drying for 8 hours by using silica gel, so that stamens crack and pollen is scattered, then putting the scattered pollen and the anthers into a 5ml centrifugal tube, and refrigerating at 4 ℃ for later use;
2) The castration method comprises the following steps: selecting unopened large-bud-stage oriental cherries for artificial emasculation 1 day before pollination, and bagging with sulfuric acid paper bags after emasculation;
3) The hybridization method comprises the following steps: restoring cold-stored or frozen pollen to normal temperature, dipping pollen with a small brush, pollinating pistil which has been castrated in advance, dipping 4 flowers which can be pollinated once, bagging with a sulfuric acid paper bag after pollination, removing the paper bag after 7 days, and bagging with a nylon mesh bag.
4) Obtaining a hybrid embryo: after female parent flowers pollinate for 28 days, picking young fruits, cleaning the young fruits by using tap water, transferring the young fruits to a super clean workbench, sterilizing for 1min by using 75% alcohol, cleaning for 3 times by using sterile water, soaking and sterilizing for 8 min by using a 10% sodium hypochlorite solution, cleaning for 5 times by using the sterile water, and transferring the young fruits to a tray for later use; lightly clamping young fruits at an angle vertical to the abdominal suture line of the fruits by using an operating forceps, opening seed shells along the clamping seams by using the forceps, taking out seeds, and peeling off the seed shells by using the forceps to obtain clean hybrid embryos, wherein the embryo tip parts cannot be damaged in the peeling process.
5) Hybrid embryo culture to obtain hybrid seedlings: inoculating the hybrid embryo into a culture bottle filled with an MS +6-BA1.0mg/L + KT0.5mg/L + NAA0.2mg/L culture medium in a way that the embryo tip faces downwards and is shallowly inserted into the culture medium, culturing for 6 days under the dark condition that the temperature is 26 ℃ and the humidity is 75% after inoculation, then transferring to the light condition for continuous culture, transferring the induced seedling into the MS +6-BA0.5mg/L + GA30.5mg/L + NAA0.2mg/L culture medium for strong seedling culture after 33 days, transferring the strong seedling into a WPM + IBA1.0mg/L + NAA1.0mg/L culture medium for rooting culture after 30 days, and obtaining the hybrid seedling after 28 days.
6) Hardening, transplanting and domesticating: after rooting culture is carried out for 28 days, 5 leaves and 4 seedlings are selected, a tissue culture bottle is opened for hardening seedlings, the temperature and the humidity of the growing environment of the hybrid seedlings are consistent with the external environment, after 10 days, the residual culture medium at the roots of the hybrid seedlings is cleaned, and the hybrid seedlings are transplanted to a container filled with peat: and (2) planting the perlite (1).
The key innovation point of the method for rescuing the oriental cherry distant hybridization embryos is as follows:
1. and (3) drying the pollen: naturally dispersing light, drying for 12-24 hours, and then putting into a drying dish for drying for 8 hours;
2. the castration method comprises the following steps: selecting unopened large-bud-stage oriental cherries 1 day before pollination for artificial emasculation, and bagging the flowers with a sulfuric acid paper bag after emasculation;
3. and (3) pollination post-treatment: bagging with sulfuric acid paper bag after pollination, removing the paper bag after 5-7 days, and bagging with nylon mesh bag;
4. hybrid embryo extraction time: pollinating female parent flowers for 25-30 days;
5. a young fruit disinfection method: cleaning tap water, transferring to a superclean workbench, disinfecting for 30s with 75% alcohol, cleaning for 2-3 times with sterile water, soaking and disinfecting for 8 minutes with 10% sodium hypochlorite solution, and cleaning for 4-5 times with sterile water;
6. the hybrid embryo stripping method comprises the following steps: lightly clamping young fruits at an angle vertical to the abdominal suture line of the fruits by using an operating forceps, opening seed shells along the clamping seams by using the forceps, taking out seeds, and peeling off the seed shells by using the forceps to obtain clean hybrid embryos;
7. embryo rescue culture medium: MS +6-BA1.0mg/L + KT0.5mg/L + NAA0.2mg/L;
8. strong seedling culture medium: MS +6-BA0.5mg/L + GA30.5mg/L + NAA0.2mg/L;
9. rooting culture medium: WPM + IBA1.0mg/L + NAA1.0mg/L.
Having thus described the basic principles and principal features of the invention, and advantages thereof, it will be apparent to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, but is capable of other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Furthermore, it should be understood that although the present specification describes embodiments, not every embodiment includes only a single embodiment, and such description is for clarity purposes only, and it is to be understood that all embodiments may be combined as appropriate by one of ordinary skill in the art to form other embodiments as will be apparent to those of skill in the art from the description herein.
Claims (10)
1. A method for obtaining cherry blossom distant hybridization offspring by an embryo rescue technology is characterized in that: the method comprises the following steps:
1) Pollen acquisition: selecting oriental cherries without anther pollen scattering from a male parent tree, taking off the anthers by using forceps, placing the anthers in a culture dish filled with a sulfuric acid paper bag, drying to crack stamens and scatter pollen, then putting the scattered pollen and the anthers into a 5ml centrifugal tube, and refrigerating at 4 ℃ for later use or freezing and storing at-80 ℃;
2) The castration method comprises the following steps: selecting unopened large-bud-stage oriental cherries 1 day before pollination for artificial emasculation, and bagging the flowers with a sulfuric acid paper bag after emasculation;
3) The hybridization method comprises the following steps: restoring the cold-stored or frozen pollen to normal temperature, dipping the pollen by using a small brush, pollinating pistils which are emasculated in advance, dipping 3-4 flowers which can be pollinated once, and then carrying out pollination post-treatment;
4) Obtaining a hybrid embryo: after the flowers of the female parent are pollinated for 25-30 days, collecting young fruits, cleaning and disinfecting the young fruits, and transferring the young fruits to a tray for later use; obtaining clean hybrid embryos by a hybrid embryo stripping method;
5) Hybrid embryo culture to obtain hybrid seedlings: inoculating the hybrid embryo into a culture bottle of an embryo rescue culture medium in a way that the embryo tip is downward and is shallowly inserted into the culture medium, culturing for 30-35 days after inoculation, transferring the induced seedling into a strong seedling culture medium for strong seedling culture, transferring the strong seedling into a rooting culture medium for rooting culture after 30 days, and obtaining the hybrid seedling after 25-30 days;
6) Hardening, transplanting and domesticating: after rooting culture for 25-30 days, selecting 4-5 leaves and 3-4 root seedlings, opening a tissue culture bottle for hardening seedlings to ensure that the temperature and humidity of the growing environment of the hybrid seedlings are consistent with the external environment, cleaning the residual culture medium at the roots of the hybrid seedlings after 7-14 days, transplanting for planting, watering after planting, transferring to a small arched shed for moisturizing, and opening the small arched shed for normal management after 7-15 days.
2. The method for obtaining the progeny of the oriental cherry distant hybridization by the embryo rescue technology as claimed in claim 1, wherein the method comprises the following steps: in the step 1), a pollen drying mode is as follows: naturally dispersing and drying for 12-24 hours, and then putting into a drying dish for drying for 8 hours.
3. The method for obtaining the progeny of the oriental cherry distant hybridization by the embryo rescue technology as claimed in claim 1, wherein the method comprises the following steps: in the step 3), pollination post-treatment: bagging with sulphuric acid paper bag after pollination, removing paper bag after 5-7 days, and bagging with nylon mesh bag.
4. The method for obtaining the progeny of the oriental cherry distant hybridization by the embryo rescue technology as claimed in claim 1, wherein the method comprises the following steps: in the step 4), the method for cleaning and disinfecting the young fruits comprises the following steps: cleaning with tap water, transferring to an ultra-clean bench, sterilizing with 75% alcohol for 30s-1min, cleaning with sterile water for 2-3 times, soaking in 10% sodium hypochlorite solution for 8 min or soaking in 0.1% HgCl solution for 5 min, and cleaning with sterile water for 4-5 times.
5. The method for obtaining the distant hybrid progeny of oriental cherry through embryo rescue technology as claimed in claim 1, wherein the method comprises the following steps: in the step 4), the hybrid embryo stripping method comprises the following steps: slightly clamping young fruits at an angle vertical to the abdominal suture line of the fruits by using operating forceps, opening seed shells along the clamping seams by using forceps, taking out seeds, and peeling off the seed shells by using the forceps to obtain clean hybrid embryos, wherein the embryo tip part cannot be injured in the peeling process.
6. The method for obtaining the progeny of the oriental cherry distant hybridization by the embryo rescue technology as claimed in claim 1, wherein the method comprises the following steps: in step 5), the embryo rescue medium: MS +6-BA1.0mg/L + KT0.5mg/L + NAA0.2mg/L.
7. The method for obtaining the progeny of the oriental cherry distant hybridization by the embryo rescue technology as claimed in claim 1, wherein the method comprises the following steps: in the step 5), the culture mode in the embryo rescue culture medium is as follows: culturing for 5-7 days under the condition of no light with the temperature of 25-28 ℃ and the humidity of 70-80% after inoculation, then transferring to the condition of light for continuous culture, and performing strong seedling culture after culturing for 30-35 days.
8. The method for obtaining the progeny of the oriental cherry distant hybridization by the embryo rescue technology as claimed in claim 1, wherein the method comprises the following steps:
in the step 5), the strong seedling culture medium: MS +6-BA0.5mg/L + GA30.5mg/L + NAA0.2mg/L.
9. The method for obtaining the progeny of the oriental cherry distant hybridization by the embryo rescue technology as claimed in claim 1, wherein the method comprises the following steps:
in step 5), the rooting medium: WPM + IBA1.0mg/L + NAA1.0mg/L.
10. The method for obtaining the progeny of the oriental cherry distant hybridization by the embryo rescue technology as claimed in claim 1, wherein the method comprises the following steps:
in step 6), selecting peat to be loaded in the transplanting and planting step: perlite =1 substrate planted in 50 hole forest high foot plug trays.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103283606A (en) * | 2013-06-24 | 2013-09-11 | 四川农业大学 | Novel isolated culture method for cherry intraspecies and intraspecific hybrid embryo |
| CN110050690A (en) * | 2019-05-10 | 2019-07-26 | 浙江省农业科学院 | Southern area sweet cherry and cherry interspecific hybridization rescue culture seedling establishment method |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103283606A (en) * | 2013-06-24 | 2013-09-11 | 四川农业大学 | Novel isolated culture method for cherry intraspecies and intraspecific hybrid embryo |
| CN110050690A (en) * | 2019-05-10 | 2019-07-26 | 浙江省农业科学院 | Southern area sweet cherry and cherry interspecific hybridization rescue culture seedling establishment method |
Non-Patent Citations (1)
| Title |
|---|
| ELISABETH GARIN等: ""Somatic embryogenesis in wild cherry (Prunus avium)"", 《PLANT CELL，TISSUE AND ORGAN CULTURE》 * |
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