CN113854150A - Efficient plant regeneration method through induced germination of Chinese rose somatic embryos - Google Patents
Efficient plant regeneration method through induced germination of Chinese rose somatic embryos Download PDFInfo
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Abstract
The invention discloses a high-efficiency plant regeneration method by inducing germination of Chinese rose somatic embryos, which specifically comprises the following steps: (1) obtaining a sterile explant; (2) obtaining a sterile mother plant; (3) multiplication of aseptic seedlings; (4) inducing young callus; (5) induction of embryonic callus; (6) multiplication of somatic embryos; (7) and (4) germinating the somatic embryo. According to the invention, the plant regeneration process of inducing the high-frequency germination of the Chinese rose somatic embryos is adopted, so that the method is simple and stable; can provide sufficient intermediate material for the supply of subsequent transgenic receptors; also can lay a high-frequency plant regeneration system for obtaining transgenic regeneration plants subsequently.
Description
Technical Field
The invention relates to the technical field of plant regeneration, in particular to a high-efficiency plant regeneration method by inducing germination of Chinese rose somatic embryos.
Background
The Chinese rose (Rosa hybrid) is a Rosa (Rosa) plant of Rosaceae (Rosaceae), is native to China, has a long cultivation history, and has a record of Chinese rose planting in the period of Hanwudi.
As one of ten traditional flowers in China, China rose has unique flower fragrance, rich flower colors and various flower types, can bloom in four seasons, has long flowering phase and extremely high ornamental value and commercial value, and is widely applied to landscape architecture and environmental greening. However, powdery mildew and black spot are common diseases of roses; in addition, although China roses are rich in flower color, blue China roses are still scarce. The cultivated Chinese rose variety has disease resistance, the blue parent resources are lack, and the genetic background is complex, so that the conventional breeding improvement process is slow, and the efficiency is low.
The molecular breeding has the advantages of strong purposiveness, short breeding period and the like. Therefore, whether the resistance and flower color of Chinese rose can be improved by this means is an essential research basis for establishing a high-frequency plant regeneration system.
Disclosure of Invention
In view of the above, the present invention aims to provide a method for regenerating plants efficiently by inducing germination through a Chinese rose embryo, so as to solve the defects in the prior art.
In order to achieve the purpose, the invention adopts the following technical scheme:
a high-efficiency plant regeneration method for inducing germination through Chinese rose somatic embryos specifically comprises the following steps:
(1) obtaining of sterile explants
Selecting a robust branch with axillary buds from a Chinese rose plant as an explant, and then washing, shearing and sterilizing the explant to obtain a sterile explant;
(2) obtaining of sterile stock Strain
Inoculating the sterile explant to a sterile stock screening culture medium for illumination culture to obtain a sterile stock;
(3) multiplication of sterile seedlings
Cutting aseptic buds of an aseptic stock plant to form aseptic seedlings with wedge-shaped bases, inoculating the aseptic seedlings to an all-round culture medium for illumination culture, selecting the aseptic seedlings which grow rapidly, are strong and obviously proliferate after 4 weeks, and performing subculture with the same formula once every 4 weeks to obtain single-bud clone aseptic seedlings;
(4) induction of young calli
Cutting off the leaf with the petiole of the single-bud clone aseptic seedling, cutting 2-3 knives along the vertical direction of the main vein of the leaf, then inoculating the leaf back downwards to a high-concentration 2,4-D callus induction culture medium for dark culture, and cutting off the young callus after 1 month;
(5) induction of embryogenic callus
Inoculating the tender callus onto a low-concentration 2,4-D embryogenic induction culture medium for dark culture, carrying out subculture with the same formula once every 2 weeks, observing the formation of granular somatic embryos after twice subculture, and then inoculating the granular somatic embryos into an all-round culture medium for light culture to obtain somatic embryos;
(6) multiplication of somatic embryos
Inoculating the somatic embryos into an all-purpose culture medium for subculture in the same formula, appropriately cutting, and aggregating into clusters to obtain clustered somatic embryos;
(7) germination of somatic embryos
Separating the cluster somatic embryos to obtain single cotyledon somatic embryos, inoculating the single cotyledon somatic embryos into a totipotent culture medium for illumination culture, germinating the somatic embryos after 3 weeks, cutting the somatic embryos, inoculating the somatic embryos into the totipotent culture medium for illumination culture, and obtaining monthly rose regeneration plants germinated by the somatic embryos after 3 weeks.
Further, in the step (1), the Chinese rose plants are 4-5 years old, have no plant diseases and insect pests, are robust and have variety typicality; the branches grow for 1 year and have the diameter of 3-5 mm; the washing time is 1-2 h; the length of the shearing is 1-2 cm; the disinfection method specifically comprises the following steps: firstly, putting the explant into 75% alcohol by mass concentration to be soaked for 30s, taking out the explant, putting the explant into 0.1% mercury bichloride by mass concentration to be soaked for 7-9min, and finally, washing the explant with sterile water for 3 times.
The further technical scheme has the advantages that by washing the explant, the pollutants such as fungi and bacteria attached to the surface of the plant can be removed, and the yield of the sterile material is improved; by shearing the explants with proper sizes, the simple disinfection treatment of the explants can be ensured, the pollution rate is reduced, and the survival rate of materials is ensured; by the sterilization process, it can be ensured that viable sterile material is obtained to the maximum possible extent.
Further, in the step (2), the formulation of the sterile mother strain screening medium is as follows: MS + BA0-3.0mg/L + 3% sucrose + 0.8% agar, pH 6.0; the photoperiod of the light culture is 16h light/8 h dark, the light intensity is 2000Lx, and the culture time is 10 days.
The method has the advantages that the sterile mother strain screening culture medium selected by the method can obtain the germinated sterile seedlings as much as possible.
Further, in the step (3), the formula of the totipotent medium is as follows: MS + BA 0.1-3.0mg/L + IBA0.1-2.0mg/L + 3% sucrose + 0.8% agar, and pH is 6.0.
The method has the advantages that the selected full-energy culture medium is the most suitable formula, only one culture medium formula is needed for sterile seedling multiplication, somatic embryo multiplication and somatic embryo germination, and the whole regeneration induction process of Chinese rose plants is almost covered.
Further, in the step (4), the size of the blade is 5mm × 10 mm; the length of the petiole is 1 mm; the formula of the high-concentration 2,4-D callus induction culture medium is as follows: MS +2, 4-D1.0-10.0 mg/L.
The further technical scheme has the beneficial effect that the callus formation of the Chinese rose leaf tissue can be ensured by the high-concentration 2,4-D callus induction culture medium selected by the invention.
Further, in the step (5), the formula of the low-concentration 2,4-D embryogenesis induction medium is as follows: MS +2, 4-D0.1-5.0 mg/L.
The further technical scheme has the beneficial effects that the low-concentration 2,4-D callus induction culture medium selected by the invention can ensure the embryogenesis of the Chinese rose callus, and finally, somatic embryos can be formed.
Further, in the step (6), the cluster-shaped somatic embryos comprise 3-5 somatic embryos, and the size of the somatic embryos is 3-5 mm.
Further, in the step (7), the size of the single cotyledonary somatic embryo is 4-7 mm; and if the rooting is needed, cutting off the somatic embryos, inoculating the somatic embryos into an MS solid culture medium for illumination culture, and obtaining the Chinese rose rooting seedlings after 3 weeks.
According to the technical scheme, compared with the prior art, the invention has the following beneficial effects:
1. according to the invention, the plant regeneration process of inducing the high-frequency germination of the Chinese rose somatic embryos is adopted, so that the method is simple and stable;
2. the invention particularly establishes the high-concentration 2,4-D induced callus, the low-concentration 2,4-D induced callus is embryogenic, and the generation process of the Chinese rose somatic embryos obtained by removing the 2,4-D is stable and reliable;
3. the somatic embryo proliferation is induced by the clustered culture of the somatic embryos, and the strategy for singly culturing and inducing the somatic embryo germination is simple and easy to implement;
4. the invention can provide sufficient intermediate material for the supply of subsequent transgenic receptors;
5. the invention can also lay a high-frequency plant regeneration system for obtaining transgenic regeneration plants subsequently.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all 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 invention.
It is worth to be noted that the regeneration method of the present invention can be used not only for the regeneration of Chinese rose, but also for the regeneration of other similar species or similar plants with high efficiency, and all fall into the protection scope of the present invention.
Example 1
The efficient plant regeneration method for inducing germination through the Chinese rose somatic embryos specifically comprises the following steps:
(1) obtaining of sterile explants
Selecting a Chinese rose 'tannike' plant which is 5 years old, free of diseases and insect pests, robust in individual and typical in variety as a donor stock plant, selecting an annual robust branch with the diameter of 4mm as an explant from the donor stock plant at 10 am in a sunny spring, washing the explant for 2 hours in running water, cutting the branch into a stem section with the length of 1.8cm and leaf marks by using a dissecting scissors, firstly soaking the stem section in 75% alcohol for 30 seconds, taking out, soaking in 0.1% mercury bichloride for 8 minutes, continuously shaking a small beaker during the period to ensure that each explant is thoroughly sterilized, and finally washing with sterile water for 3 times to obtain an aseptic explant;
(2) obtaining of sterile stock Strain
Inoculating the sterile explants onto a sterile stock screening medium (the formula is MS + BA0-3.0mg/L + 3% sucrose + 0.8% agar, and the pH is 6.0) according to the morphological upper and lower ends, inoculating one sterile explant in each bottle, setting the photoperiod to 16h illumination/8 h dark, the illumination intensity to 2000Lx, and performing illumination culture for 10 days, wherein the growth and pollution conditions of the sterile explants are observed during the period, and the polluted and ungerminated materials are discarded, so as to obtain the sterile stock;
(3) multiplication of sterile seedlings
Cutting aseptic buds of an aseptic mother plant to form aseptic seedlings with wedge-shaped bases, then inoculating the aseptic seedlings to a full-energy culture medium (the formula is MS + BA 1.5mg/L + IBA 1.0mg/L + 3% sucrose + 0.8% agar, and the pH value is 6.0) for illumination culture, observing the proliferation condition of the aseptic seedlings, selecting an aseptic seedling single plant which grows rapidly, robustly and obviously and proliferating after 4 weeks to determine the aseptic seedling single plant as a single bud clone, and then carrying out subculture with the same formula once every 4 weeks to obtain single bud clone aseptic seedlings;
(4) induction of young calli
Cutting 1mm petiole of the single bud clone aseptic seedling with the size of 5mm multiplied by 10mm, cutting 2 knives along the vertical direction of each main vein of the leaf, then inoculating the leaf back downwards to a high-concentration 2,4-D callus induction culture medium (the formula is MS +2, 4-D10.0 mg/L) for dark culture, observing the formation of callus after 1 month, and cutting off young and tender callus in time;
(5) induction of embryogenic callus
Inoculating the tender callus to a low-concentration 2,4-D embryogenic induction culture medium (the formula is MS +2, 4-D0.1 mg/L) for dark culture, carrying out subculture once in the same formula every 2 weeks, observing the formation of granular somatic embryos after twice subculture, and immediately inoculating the tender callus to an all-purpose culture medium for light culture (for somatic embryo proliferation induction) to obtain somatic embryos;
(6) multiplication of somatic embryos
Inoculating the somatic embryos into a totipotent culture medium for subculture with the same formula, moderately cutting 4mm somatic embryos, and aggregating 4 individual embryos into clusters to obtain clustered somatic embryos;
(7) germination of somatic embryos
Separating the cluster somatic embryos to obtain single cotyledon somatic embryos with the size of 4mm, then inoculating the single cotyledon somatic embryos into an all-purpose culture medium for illumination culture, germinating the somatic embryos after 3 weeks, cutting the somatic embryos, inoculating the cut somatic embryos into the all-purpose culture medium for illumination culture, and obtaining monthly rose regeneration plants germinated by the somatic embryos after 3 weeks.
Example 2
The efficient plant regeneration method for inducing germination through the Chinese rose somatic embryos specifically comprises the following steps:
(1) obtaining of sterile explants
Selecting a Chinese rose 'Zhumei' plant which is 4 years old, free of diseases and insect pests, robust in individual and typical in variety as a donor stock plant, selecting an annual robust branch with the diameter of 3mm and an axillary bud from the donor stock plant at 9 am in a sunny spring, washing the explant for 1h in running water, cutting the branch into a stem section with the length of 1.5cm and leaf marks by using a dissecting scissors, soaking the stem section in 75% alcohol for 30s, taking out, soaking in 0.1% mercury bichloride for 7min, continuously shaking a small beaker during the period to ensure that each explant is thoroughly sterilized, and finally washing with sterile water for 3 times to obtain an aseptic explant;
(2) obtaining of sterile stock Strain
Inoculating the sterile explants onto a sterile stock screening medium (the formula is MS + BA0-3.0mg/L + 3% sucrose + 0.8% agar, and the pH is 6.0) according to the morphological upper and lower ends, inoculating one sterile explant in each bottle, setting the photoperiod to 16h illumination/8 h dark, the illumination intensity to 2000Lx, and performing illumination culture for 10 days, wherein the growth and pollution conditions of the sterile explants are observed during the period, and the polluted and ungerminated materials are discarded, so as to obtain the sterile stock;
(3) multiplication of sterile seedlings
Cutting aseptic buds of an aseptic mother plant to form aseptic seedlings with wedge-shaped bases, then inoculating the aseptic seedlings to a full-energy culture medium (the formula is MS + BA0.1 mg/L + IBA0.1 mg/L + 3% sucrose + 0.8% agar, and the pH value is 6.0) for illumination culture, observing the proliferation condition of the aseptic seedlings, selecting aseptic seedling single plants which grow rapidly, strongly and obviously to be determined as single bud clones after 4 weeks, and then carrying out subculture with the same formula once every 4 weeks to obtain single bud clone aseptic seedlings;
(4) induction of young calli
Cutting 1mm petiole of the single bud clone aseptic seedling, 5mm multiplied by 10mm in size, cutting 2 knives along the vertical direction of each main vein of the leaf, then inoculating the leaf back downwards to a high-concentration 2,4-D callus induction culture medium (the formula is MS +2, 4-D1.0 mg/L) for dark culture, observing the formation of callus after 1 month, and cutting off young and tender callus in time;
(5) induction of embryogenic callus
Inoculating the tender callus to a low-concentration 2,4-D embryogenic induction culture medium (the formula is MS +2, 4-D0.1 mg/L) for dark culture, carrying out subculture once in the same formula every 2 weeks, observing the formation of granular somatic embryos after twice subculture, and immediately inoculating the tender callus to an all-purpose culture medium for light culture (for somatic embryo proliferation induction) to obtain somatic embryos;
(6) multiplication of somatic embryos
Inoculating the somatic embryos into a totipotent culture medium for subculture with the same formula, moderately cutting 3mm somatic embryos, and aggregating 5 individual embryos into clusters to obtain clustered somatic embryos;
(7) germination of somatic embryos
Separating the cluster somatic embryos to obtain single cotyledon somatic embryos with the size of 4mm, then inoculating the single cotyledon somatic embryos into an all-purpose culture medium for illumination culture, germinating the somatic embryos after 3 weeks, cutting the somatic embryos, inoculating the cut somatic embryos into the all-purpose culture medium for illumination culture, and obtaining monthly rose regeneration plants germinated by the somatic embryos after 3 weeks.
Example 3
The efficient plant regeneration method for inducing germination through the Chinese rose somatic embryos specifically comprises the following steps:
(1) obtaining of sterile explants
Selecting a Chinese rose 'kennedy' plant which is 5 years old, free of diseases and insect pests, robust in individual and typical in variety as a donor stock plant, selecting an annual robust branch with the diameter of 5mm and an axillary bud from the donor stock plant at 10 am in a sunny spring day, washing the explant for 2 hours in running water, cutting the branch into a stem section with the length of 2cm and leaf marks by using a dissecting scissors, soaking the stem section in 75% alcohol for 30 seconds, taking out, soaking in 0.1% mercury bichloride for 9 minutes, continuously shaking a small beaker to ensure that each explant is thoroughly sterilized, and finally washing with sterile water for 3 times to obtain an aseptic explant;
(2) obtaining of sterile stock Strain
Inoculating the sterile explants onto a sterile stock screening medium (the formula is MS + BA0-3.0mg/L + 3% sucrose + 0.8% agar, and the pH is 6.0) according to the morphological upper and lower ends, inoculating one sterile explant in each bottle, setting the photoperiod to 16h illumination/8 h dark, the illumination intensity to 2000Lx, and performing illumination culture for 10 days, wherein the growth and pollution conditions of the sterile explants are observed during the period, and the polluted and ungerminated materials are discarded, so as to obtain the sterile stock;
(3) multiplication of sterile seedlings
Cutting aseptic buds of an aseptic parent plant to form aseptic seedlings with wedge-shaped bases, then inoculating the aseptic seedlings to a full-energy culture medium (the formula is MS + BA 3.0mg/L + IBA2.0mg/L + 3% sucrose + 0.8% agar, and the pH value is 6.0) for illumination culture, observing the proliferation condition of the aseptic seedlings, selecting aseptic seedling single plants which grow rapidly, robustly and obviously and determining the aseptic seedlings as single bud clones after 4 weeks, and then carrying out subculture with the same formula once every 4 weeks to obtain the single bud clone aseptic seedlings;
(4) induction of young calli
1mm petiole of a single bud clone aseptic seedling with the size of 5mm multiplied by 10mm is cut, 3 knives are cut along the vertical direction of the main vein of each leaf, then the back of the leaf is downwards inoculated on a high-concentration 2,4-D callus induction culture medium (the formula is MS +2, 4-D10.0 mg/L) for dark culture, the formation of callus is observed after 1 month, and young and tender callus is cut in time;
(5) induction of embryogenic callus
Inoculating the tender callus to a low-concentration 2,4-D embryogenic induction culture medium (the formula is MS +2, 4-D5.0 mg/L) for dark culture, carrying out subculture once in the same formula every 2 weeks, observing the formation of granular somatic embryos after twice subculture, and immediately inoculating the tender callus to an all-purpose culture medium for light culture (for somatic embryo proliferation induction) to obtain somatic embryos;
(6) multiplication of somatic embryos
Inoculating the somatic embryos into a totipotent culture medium for subculture with the same formula, moderately cutting 5mm somatic embryos, and aggregating 3 individual embryos into clusters to obtain clustered somatic embryos;
(7) germination of somatic embryos
Separating the cluster somatic embryos to obtain single cotyledon somatic embryos with the size of 7mm, then inoculating the single cotyledon somatic embryos into an all-round culture medium for illumination culture, germinating the somatic embryos after 3 weeks, cutting the somatic embryos, inoculating the somatic embryos into an MS solid culture medium for illumination culture, and obtaining the Chinese rose rooting seedlings after 3 weeks.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (10)
1. A high-efficiency plant regeneration method for inducing germination through a Chinese rose somatic embryo is characterized by comprising the following steps:
(1) obtaining of sterile explants
Selecting a robust branch with axillary buds from a Chinese rose plant as an explant, and then washing, shearing and sterilizing the explant to obtain a sterile explant;
(2) obtaining of sterile stock Strain
Inoculating the sterile explant to a sterile stock screening culture medium for illumination culture to obtain a sterile stock;
(3) multiplication of sterile seedlings
Cutting aseptic buds of an aseptic stock plant to form aseptic seedlings with wedge-shaped bases, inoculating the aseptic seedlings to an all-round culture medium for illumination culture, selecting the aseptic seedlings which grow rapidly, are strong and obviously proliferate after 4 weeks, and performing subculture with the same formula once every 4 weeks to obtain single-bud clone aseptic seedlings;
(4) induction of young calli
Cutting off the leaf with the petiole of the single-bud clone aseptic seedling, cutting 2-3 knives along the vertical direction of the main vein of the leaf, then inoculating the leaf back downwards to a high-concentration 2,4-D callus induction culture medium for dark culture, and cutting off the young callus after 1 month;
(5) induction of embryogenic callus
Inoculating the tender callus onto a low-concentration 2,4-D embryogenic induction culture medium for dark culture, carrying out subculture with the same formula once every 2 weeks, observing the formation of granular somatic embryos after twice subculture, and then inoculating the granular somatic embryos into an all-round culture medium for light culture to obtain somatic embryos;
(6) multiplication of somatic embryos
Inoculating the somatic embryos into an all-purpose culture medium for subculture in the same formula, appropriately cutting, and aggregating into clusters to obtain clustered somatic embryos;
(7) germination of somatic embryos
Separating the cluster somatic embryos to obtain single cotyledon somatic embryos, inoculating the single cotyledon somatic embryos into a totipotent culture medium for illumination culture, germinating the somatic embryos after 3 weeks, cutting the somatic embryos, inoculating the somatic embryos into the totipotent culture medium for illumination culture, and obtaining monthly rose regeneration plants germinated by the somatic embryos after 3 weeks.
2. The method for efficient plant regeneration through induction of germination by a Chinese rose somatic embryo according to claim 1, wherein in step (1), the Chinese rose plant is 4-5 years old, free from plant diseases and insect pests, robust, and has variety typicality; the branches grow for 1 year and have the diameter of 3-5 mm; the flushing time is 1-2 h; the length of the shearing is 1-2 cm.
3. The method for regenerating a plant with high efficiency by inducing germination through a Chinese rose somatic embryo according to claim 1, wherein in the step (1), the step of sterilizing is specifically: firstly, putting the explant into 75% alcohol by mass concentration to be soaked for 30s, taking out the explant, putting the explant into 0.1% mercury bichloride by mass concentration to be soaked for 7-9min, and finally, washing the explant with sterile water for 3 times.
4. The method for high-efficiency plant regeneration through induction of germination of Chinese rose embryos of claim 1, wherein in the step (2), the formulation of the sterile mother plant screening medium is as follows: MS + BA0-3.0mg/L + 3% sucrose + 0.8% agar, pH 6.0;
the photoperiod of the illumination culture is 16h of illumination/8 h of darkness, the illumination intensity is 2000Lx, and the culture time is 10 days.
5. The method for efficient plant regeneration through induction of germination by Chinese rose somatic embryos of claim 1, wherein in the step (3), the formula of the totipotent medium is as follows: MS + BA 0.1-3.0mg/L + IBA0.1-2.0mg/L + 3% sucrose + 0.8% agar, and pH is 6.0.
6. The method for regenerating a plant having high efficiency through germination induction by a Chinese rose somatic embryo according to claim 1, wherein in the step (4), the size of the leaf is 5mm x 10 mm; the length of the petiole is 1 mm;
the formula of the high-concentration 2,4-D callus induction culture medium is as follows: MS +2, 4-D1.0-10.0 mg/L.
7. The method for high-efficiency plant regeneration through induction of germination by Chinese rose somatic embryos of claim 1, wherein in the step (5), the formula of the low-concentration 2,4-D embryogenesis induction medium is as follows: MS +2, 4-D0.1-5.0 mg/L.
8. The method for efficient plant regeneration through induction of germination by Chinese rose somatic embryos of claim 1, wherein in step (6), the clustered somatic embryos comprise 3-5 somatic embryos with a size of 3-5 mm.
9. The method for efficient plant regeneration by induction of germination of a Chinese rose somatic embryo according to claim 1, wherein the size of the single cotyledon embryo in step (7) is 4-7 mm.
10. The method for efficiently regenerating plants through inducing germination of Chinese rose embryos of claim 1, wherein in the step (7), if rooting is needed, the Chinese rose embryos are cut and inoculated into an MS solid culture medium for illumination culture, and Chinese rose rooted seedlings are obtained after 3 weeks.
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| CN118006674A (en) * | 2024-04-09 | 2024-05-10 | 云南省农业科学院花卉研究所 | Application of RcWUS gene in regulation of China rose regeneration |
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| CN108308026A (en) * | 2018-03-09 | 2018-07-24 | 无锡南村花卉苗木专业合作社 | A kind of Growth anddevelopment tissue culture propagation |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108308026A (en) * | 2018-03-09 | 2018-07-24 | 无锡南村花卉苗木专业合作社 | A kind of Growth anddevelopment tissue culture propagation |
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| CN118006674A (en) * | 2024-04-09 | 2024-05-10 | 云南省农业科学院花卉研究所 | Application of RcWUS gene in regulation of China rose regeneration |
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