CN115644058A - Method for promoting orchidaceae plant seeds to quickly grow seedlings - Google Patents
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- 239000001963 growth medium Substances 0.000 claims abstract description 37
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- 239000012883 rooting culture medium Substances 0.000 claims abstract description 16
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- 238000005507 spraying Methods 0.000 claims abstract description 12
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- 239000012535 impurity Substances 0.000 claims description 5
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- 239000007640 basal medium Substances 0.000 claims description 4
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- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 4
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- ATNHDLDRLWWWCB-AENOIHSZSA-M chlorophyll a Chemical compound C1([C@@H](C(=O)OC)C(=O)C2=C3C)=C2N2C3=CC(C(CC)=C3C)=[N+]4C3=CC3=C(C=C)C(C)=C5N3[Mg-2]42[N+]2=C1[C@@H](CCC(=O)OC\C=C(/C)CCC[C@H](C)CCC[C@H](C)CCCC(C)C)[C@H](C)C2=C5 ATNHDLDRLWWWCB-AENOIHSZSA-M 0.000 description 1
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/20—Reduction of greenhouse gas [GHG] emissions in agriculture, e.g. CO2
- Y02P60/21—Dinitrogen oxide [N2O], e.g. using aquaponics, hydroponics or efficiency measures
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Abstract
The invention discloses a method for promoting orchid seeds to quickly grow seedlings, which comprises the steps of sterilizing fruit pods, splitting the fruit pods, uniformly spraying seeds in the fruit pods on a basic culture medium, absorbing a strong seedling culture medium after the seeds germinate and uniformly spreading the strong seedling culture medium on buds, absorbing a rooting culture medium when the buds grow to have 3-5 leaves and the plant height reaches 3-4cm, uniformly spreading the rooting culture medium on the bases of the buds, spraying nutrient solution on the leaves of the buds, and transplanting and planting the buds after the roots grow. The method only needs to perform inoculation for 1 time in the whole process, does not need bottle rotation, saves time, reduces pollution, improves working efficiency, has short growth time of tissue culture seedlings, only needs 270 days from sowing to seedling formation, greatly shortens the breeding time compared with the traditional 1-year breeding time, and can accelerate the breeding process of new varieties.
Description
Technical Field
The invention belongs to the technical field of plant tissue culture, and particularly relates to a method for promoting orchids to quickly grow seedlings.
Background
The plant tissue culture technology is widely applied to the production of orchid seedlings, and particularly for phalaenopsis, dendrobe, oncidium, anoectochilus roxburghii and other plants, the rapid popularization of new varieties of the phalaenopsis, dendrobe, oncidium, anoectochilus roxburghii and the like depends on the rapid breeding of tissue culture. In the hybrid cultivation of new species, the aseptic seeding technology of the seeds of the orchids is the basis for breeding the new species, and the prior art is to broadcast the hybrid seeds into a germination induction culture medium, transfer the seeds into a strong seedling culture medium after the seeds germinate, strengthen the seedlings for several times, and finally insert a rooting culture medium to finish the breeding of the hybrid seedlings. The technology has the disadvantages of long time consumption, more than 1 year from sowing to seedling formation, labor consumption, at least more than 3 times of transfer in the whole process, culture medium consumption and more than 3 generations of culture medium consumption.
The invention patent application 'a method for once-forming bletilla striata tissue culture seedling' (CN 108739380A) discloses a method for completing the production of the tissue culture seedling from seeds to the stage of rooting and bulb-forming by only preparing a culture medium once without transferring. The practice of the patent is equivalent to adopting a liquid culture medium, which easily causes that the seedlings are difficult to breathe and die due to excessive moisture.
The information disclosed in this background section is only for enhancement of understanding of the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art that is already known to a person skilled in the art.
Disclosure of Invention
Aiming at the technical problems, the invention provides a method for promoting the orchid seeds to quickly grow seedlings, which comprises the following steps:
step S1, pod sterilization treatment: selecting green, full and disease and pest-free pods of orchids which are not cracked yet, wiping impurities on the surfaces of the pods by alcohol cotton balls, and sterilizing on a super-clean workbench;
step S2, aseptic seeding: splitting the fruit pods, and uniformly spraying seeds in the fruit pods on a basic culture medium in a culture bottle; the basic culture medium comprises the following components in percentage by weight: MS +6-BA 3.0mg/L + NAA0.1mg/L + banana 50g/L + sucrose 30g/L + agar 4g/L;
step S3, strong seedling culture: after sowing for 90 days, germinating seeds, sterilizing the surface of a culture bottle, opening a bottle cap, sucking a strong seedling culture medium by using a sterile needle tube, and uniformly scattering the strong seedling culture medium on buds; the formula of the strong seedling culture medium is as follows: MS +6-BA1.0mg/L + NAA0.5mg/L + banana 100g/L + cane sugar 30g/L + agar 4g/L;
step S4, rooting culture: after culturing for 90 days for strong seedlings, sterilizing the surface of a culture bottle when 3-5 leaves grow out from the buds and the plant height reaches 3-4cm, opening the bottle cap, absorbing a rooting culture medium by using a sterile needle tube, uniformly scattering the rooting culture medium on the base part of the seedlings, absorbing a nutrient solution by using an atomizer, and spraying the nutrient solution on the leaves of the seedlings; the formula of the rooting culture medium is as follows: MS, 1.0mg/L NAA, 100g/L banana, 30g/L sucrose and 4g/L agar; the formula of the nutrient solution is as follows: MS + triacontanol 0.05g/L + trehalose 0.1g/L;
and step S5, transplanting: after rooting culture for 90 days, the root system grows out, and then transplanting and planting can be carried out.
Wherein the Orchidaceae plant is tree orchid (Epidendrum) or herba Dendrobii (Dendrobium nobile Lindl.).
The sterilization treatment method in the step S1 comprises the following steps: sterilizing with 75% alcohol for 60s, washing with sterile water for 3 times, sterilizing with 0.1% mercuric chloride for 15min, and washing with sterile water for 4 times.
In step S2, the culture flask was filled with 110mL of the basal medium.
In step S2, cutting off two ends of the fruit pod, longitudinally cutting and splitting the fruit pod, putting the fruit pod into a bottle filled with 20mL of sterile water, slightly shaking the bottle to enable seeds on the fruit pod to fall into the sterile water, and then sucking 0.5-1mL of sterile water containing the seeds by a suction pipe to uniformly spread the sterile water on a basic culture medium uniformly sprinkled in a culture bottle.
Wherein, in the step S3 and the step S4, the method for sterilizing the surface of the culture bottle comprises the following steps: the surface of the flask was wiped with 75% alcohol and sterilized.
In step S3, 10mL of strong seedling culture medium is sucked by a sterile needle tube and uniformly sprinkled on the buds.
Wherein, in step S4, 10mL of rooting medium is sucked by a sterile needle tube and uniformly sprinkled on the base of the plantlet.
Wherein, the culture conditions of the step S2-the step S4 are as follows: the temperature is 25 +/-2 ℃, the illumination intensity is 1500-2000 lx, and the illumination time is 12h/d.
Compared with the prior art, the invention has the following beneficial effects:
(1) The method provided by the invention only needs to perform inoculation for 1 time in the whole process, does not need bottle rotation, saves time, reduces pollution, improves working efficiency, has short growth time of tissue culture seedlings, only needs 270 days from sowing to seedling formation, greatly shortens the breeding time compared with the traditional breeding time of 1 year, and can accelerate the breeding process of new varieties;
(2) Compared with a method for one-time seedling formation of bletilla striata tissue culture seedlings (CN 108739380A), the method adopts a solid culture medium for spraying, adopts a liquid culture medium for the patent, uses a needle tube for sucking and spraying the solid culture medium, can form a certain gap at the base of the plantlet, ensures the breathing of the plantlet, and easily leads the death of the plantlet due to excessive moisture of the liquid culture medium;
(3) According to the invention, in the rooting stage of the tissue culture seedlings, nutrient solution is sprayed by a spraying machine, so that the absorption of the leaves of the plantlets is effectively promoted, the color of the leaves of the plantlets is more dark green, the plantlets are robust, and the transplanting survival rate is higher. Triacontanol is a plant growth promoter with a wide application range, can be absorbed by stems and leaves of plants, then promotes the growth of the plants, increases the accumulation of dry substances, improves the permeability of cell membranes, increases the content of chlorophyll, improves the photosynthetic strength, and enhances the activities of amylase, polyoxase and peroxidase. Trehalose has a certain effect on improving the resistance of the tissue culture seedlings, and the transplanting survival rate can be improved by spraying the trehalose on the leaf surfaces before transplanting. The trehalose in the plant body can protect the plant from being damaged by the outside under the severe environment condition, thereby prolonging the life of the plant.
Detailed Description
The following detailed description is to be read in connection with specific embodiments, but it should be understood that the scope of the invention is not limited to the specific embodiments. The raw materials used in the examples were all commercially available unless otherwise specified.
Example 1 promotion of fast seedling establishment of orchid seeds
Step S1, pod sterilization treatment: selecting green, plump and disease and pest free tree orchid fruit pods which are not cracked, wiping impurities on the surfaces of the tree orchid fruit pods with alcohol cotton balls, sterilizing the tree orchid fruit pods for 60s with 75% alcohol on a super clean workbench, washing the tree orchid fruit pods with sterile water for 3 times, sterilizing the tree orchid fruit pods with 0.1% mercuric chloride for 15min, and washing the tree orchid fruit pods with the sterile water for 4 times;
step S2, aseptic seeding: cutting off two ends of a fruit pod, longitudinally cutting and splitting the fruit pod, putting the fruit pod into a bottle filled with 20mL of sterile water, slightly shaking to enable seeds on the fruit pod to fall into the sterile water, sucking 1mL of mixed liquid of the sterile water and the seeds by a suction pipe, uniformly scattering the mixed liquid on a basal medium uniformly scattered in a culture bottle, and inoculating 20 bottles of each fruit pod; the basic culture medium comprises the following components in percentage by weight: MS +6-BA 3.0mg/L + NAA0.1mg/L + banana 50g/L + cane sugar 30g/L + agar 4g/L;
step S3, strong seedling culture: after sowing for 90 days, germinating seeds, wiping the surface of a culture bottle with 75% alcohol for sterilization, opening a bottle cap, sucking 10mL of strong seedling culture medium by using a sterile needle tube, and uniformly scattering the strong seedling culture medium on buds; the formula of the strong seedling culture medium is as follows: MS +6-BA1.0mg/L + NAA0.5mg/L + banana 100g/L + sucrose 30g/L + agar 4g/L;
step S4, rooting culture: after culturing strong seedlings for 90 days, wiping the surface of a culture bottle with 75% alcohol for sterilization when 3-5 leaves grow out from the buds and the plant height reaches 3-4cm, opening a bottle cap, sucking 10mL of rooting culture medium by using a sterile needle tube, uniformly scattering the rooting culture medium on the base part of the seedlings, sucking nutrient solution by using an atomizer, and spraying the nutrient solution on the leaves of the seedlings; the formula of the rooting culture medium is as follows: MS + NAA 1.0mg/L + banana 100g/L + sucrose 30g/L + agar 4g/L; the formula of the nutrient solution is as follows: MS + triacontanol 0.05g/L + trehalose 0.1g/L;
and step S5, transplanting: after rooting culture for 90 days, the roots grow out, and then transplanting and planting can be carried out, wherein the total culture time is 270 days.
Example 2 promoting fast seedling formation of Dendrobium nobile seeds
Step S1, pod sterilization treatment: selecting green and plump dendrobium nobile fruit pods which are not cracked yet and have no diseases or insect pests, wiping impurities on the surfaces of the fruit pods by alcohol cotton balls, sterilizing the fruit pods for 60s by using 75% alcohol on a super-clean workbench, washing the fruit pods for 3 times in an aseptic manner, sterilizing the fruit pods for 15min by using 0.1% mercuric chloride, and washing the fruit pods for 4 times in an aseptic manner;
step S2, aseptic seeding: cutting off two ends of a fruit pod, longitudinally cutting and splitting the fruit pod, putting the fruit pod into a bottle filled with 10mL of sterile water, slightly shaking to enable seeds on the fruit pod to fall into the sterile water, sucking 1mL of mixed liquid of the sterile water and the seeds by a suction pipe, uniformly scattering the mixed liquid on a basal medium uniformly scattered in a culture bottle, and inoculating 10 bottles of each fruit pod; the basic culture medium comprises the following components in percentage by weight: MS +6-BA 3.0mg/L + NAA0.1mg/L + banana 50g/L + cane sugar 30g/L + agar 4g/L;
step S3, strong seedling culture: after sowing for 90 days, germinating seeds, wiping the surface of a culture bottle with 75% alcohol for sterilization, opening a bottle cap, sucking 10mL of strong seedling culture medium by using a sterile needle tube, and uniformly scattering the strong seedling culture medium on buds; the formula of the strong seedling culture medium is as follows: MS +6-BA1.0mg/L + NAA0.5mg/L + banana 100g/L + sucrose 30g/L + agar 4g/L;
step S4, rooting culture: after culturing strong seedlings for 90 days, wiping the surface of a culture bottle with 75% alcohol for sterilization when 3-5 leaves grow out from the buds and the plant height reaches 3-4cm, opening a bottle cap, sucking 10mL of rooting culture medium by using a sterile needle tube, uniformly scattering the rooting culture medium on the base part of the seedlings, sucking nutrient solution by using an atomizer, and spraying the nutrient solution on the leaves of the seedlings; the formula of the rooting culture medium is as follows: MS + NAA 1.0mg/L + banana 100g/L + sucrose 30g/L + agar 4g/L; the formula of the nutrient solution is as follows: MS + triacontanol 0.05g/L + trehalose 0.1g/L;
and step S5, transplanting: after rooting culture for 90 days, the root systems grow out, and transplanting and planting can be carried out, wherein the total culture time is 270 days.
Comparative example 3 traditional cultivation method of tree orchid seed
Step S1, pod sterilization treatment: selecting unraveled, green, full and pest-free tree orchid fruit pods, wiping impurities on the surfaces of the tree orchid fruit pods with alcohol cotton balls, sterilizing the tree orchid fruit pods for 60s with 75% alcohol on a super-clean workbench, washing the tree orchid fruit pods with sterile water for 3 times, sterilizing the tree orchid fruit pods with 0.1% mercuric chloride for 15min, and washing the tree orchid fruit pods with sterile water for 4 times;
step S2, aseptic seeding: cutting off two ends of a fruit pod, longitudinally cutting open the fruit pod, taking out seeds by using forceps, and uniformly sowing the seeds in a germination induction medium (1/2MS + NAA0.1mg/L + 50ml/L of coconut juice + 30g/L of sucrose + 4.2g/L of agar). The temperature of the culture room is 23-27 ℃, the illumination intensity is 2400 lx-3200 lx, and the photoperiod is 10-12 h (the same below);
step S3, culturing strong seedlings for one time: after sowing for 90 days, germinating seeds, selecting germinated robust cymbidium seedlings, and inserting the seedlings into a strong seedling culture medium (MS +6-BA1.0mg/L + NAA0.5mg/L + banana 100g/L + sucrose 30g/L + agar 4 g/L);
step S4, secondary strong seedling culture: after the seedlings are strong for 80 days at one time, the orchid seedlings grow tall, and the seedlings are taken out and inserted into a new strong seedling culture medium (MS +6-BA1.0mg/L + NAA0.5mg/L + banana 100g/L + sucrose 30g/L + agar 4 g/L);
step S5, rooting culture: after the second strong seedling is carried out for 80 days, cutting the aseptic small seedling of the cymbidium into single plants, and inoculating the single plants into a rooting culture medium (1/2MS + NAA0.5mg/L + banana 50g/L + white granulated sugar 25g/L + agar 4.2g/L, pH is 5.5-5.8);
step S6, transplanting: after rooting culture for 80 days, the roots grow out, and then transplanting and planting can be carried out, wherein the total culture time is 330 days.
The labor time and the number of flasks consumed in example 1 and comparative example 3 were investigated, and the results are shown in Table 1:
TABLE 1 labor time and number of flasks consumed for example 1 and comparative example 3
As can be seen from Table 1, the method of the present invention consumes less labor time for treating each flask, and has high work efficiency and low production cost.
The average plant height, leaf number and transplanting survival rate of 50 rooted seedlings of orchids of example 1 and comparative example 3 after being transplanted for 90 days are respectively counted, and the results are shown in table 2:
TABLE 2 comparison of transplanting effects of the rooted seedlings of denudata in example 1 and comparative example 3
As can be seen from Table 2, the rooted seedlings obtained in example 1 were superior to those obtained in comparative example 3 in terms of plant height, leaf number and transplanting survival rate after 90 days of transplanting.
The foregoing descriptions of specific exemplary embodiments of the present invention have been presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain certain principles of the invention and its practical application to enable one skilled in the art to make and use various exemplary embodiments of the invention and various alternatives and modifications. It is intended that the scope of the invention be defined by the claims and their equivalents.
Claims (9)
1. A method for promoting rapid seedling formation of orchid seeds is characterized by comprising the following steps:
step S1, pod sterilization treatment: selecting non-cracked, green, full and disease and pest free fruit pods of orchids, wiping impurities on the surfaces of the fruit pods by alcohol cotton balls, and performing sterilization treatment on the ultra-clean workbench;
step S2, aseptic seeding: splitting the fruit pod, and uniformly spraying seeds in the fruit pod on a basic culture medium in a culture bottle; the basic culture medium comprises the following components in percentage by weight: MS +6-BA 3.0mg/L + NAA0.1mg/L + banana 50g/L + cane sugar 30g/L + agar 4g/L;
step S3, strong seedling culture: after sowing for 90 days, germinating seeds, sterilizing the surface of a culture bottle, opening a bottle cap, sucking a strong seedling culture medium by using a sterile needle tube, and uniformly scattering the strong seedling culture medium on buds; the formula of the strong seedling culture medium is as follows: MS +6-BA1.0mg/L + NAA0.5mg/L + banana 100g/L + cane sugar 30g/L + agar 4g/L;
step S4, rooting culture: after culturing strong seedlings for 90 days, when 3-5 leaves grow out from the buds and the plant height reaches 3-4cm, sterilizing the surface of a culture bottle, opening a bottle cap, sucking a rooting culture medium by using a sterile needle tube, uniformly scattering the rooting culture medium on the base part of the seedlings, sucking a nutrient solution by using an atomizer, and spraying the nutrient solution on the leaves of the seedlings; the formula of the rooting culture medium is as follows: MS, 1.0mg/L NAA, 100g/L banana, 30g/L sucrose and 4g/L agar; the formula of the nutrient solution is as follows: MS + triacontanol 0.05g/L + trehalose 0.1g/L;
and step S5, transplanting: after rooting culture for 90 days, the root system grows out, and then transplanting and planting can be carried out.
2. The method of claim 1, wherein: the Orchidaceae plant is tree orchid or dendrobium.
3. The method according to claim 1, wherein the sterilization treatment in step S1 is performed by: sterilizing with 75% alcohol for 60s, washing with sterile water for 3 times, sterilizing with 0.1% mercuric chloride for 15min, and washing with sterile water for 4 times.
4. The method of claim 1, wherein: in step S2, the flask was filled with 110mL of basal medium.
5. The method of claim 1, wherein: in step S2, cutting off two ends of the fruit pod, longitudinally cutting open the fruit pod, putting the fruit pod into a bottle filled with 20mL of sterile water, slightly shaking to enable the seeds on the fruit pod to fall into the sterile water, and then sucking 0.5-1mL of sterile water containing the seeds by a suction pipe and uniformly scattering the sterile water on the basic culture medium uniformly sprayed in the culture bottle.
6. The method of claim 1, wherein: in step S3 and step S4, the method for sterilizing the surface of the culture flask comprises: the surface of the flask was wiped with 75% alcohol and sterilized.
7. The method of claim 1, wherein: in step S3, 10mL of strong seedling culture medium is sucked by a sterile needle tube and uniformly sprinkled on the buds.
8. The method of claim 1, wherein: in step S4, 10mL of rooting medium is aspirated by a sterile needle tube and uniformly scattered on the base of the plantlet.
9. The method of claim 1, wherein: the culture conditions of step S2-step S4 are as follows: the temperature is 25 +/-2 ℃, the illumination intensity is 1500-2000 lx, and the illumination time is 12h/d.
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