CN115644058B - Method for promoting quick seedling formation of orchid seeds - Google Patents
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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 quick seedling formation of orchid seeds, which comprises the steps of sterilizing fruit pods, cutting the fruit pods, uniformly spraying seeds in the fruit pods on a basic culture medium, germinating the seeds, sucking a strong seedling culture medium, uniformly spraying the strong seedling culture medium on small buds, uniformly spraying a rooting culture medium on the basal part of the small seedlings when the small buds grow into 3-5 leaves and the plant height reaches 3-4cm, spraying nutrient solution on the leaves of the small seedlings, and transplanting and planting after the root system grows out. The method of the invention only needs to inoculate 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 quick seedling formation of orchid seeds.
Background
The plant tissue culture technology is widely applied to orchid plant seedling production, and particularly aims at the quick propagation of new varieties of butterfly orchid, dendrobium, oncidium, anoectochilus roxburghii and the like, namely the quick propagation of tissue culture. In the process of breeding new varieties by hybridization, the sterile sowing technology of orchid seeds is the basis for breeding the new varieties, the existing technology is to broadcast the hybridized seeds into a germination induction culture medium, after the seeds germinate, the seeds are transferred into a seedling strengthening culture medium, the seedlings are strengthened for several times, and finally the seedlings are inoculated into a rooting culture medium, so that the breeding of the hybridized seedlings is completed. The technology has the defects of long time consumption, more than 1 year of time from sowing to seedling formation, labor consumption, at least 3 times of transfer in the whole process, medium consumption and more than 3 generations of medium consumption.
The invention patent application (CN 108739380A) discloses a method for forming tissue culture seedlings of bletilla striata at one time, which is characterized in that the tissue culture seedlings are produced from the seeds to the rooting bulb stage of the bletilla striata by preparing a culture medium once without transferring, and the technical scheme is that the seeds of the bletilla striata are scattered on the surface of an MS culture medium, and after the seeds of the bletilla striata bud, liquid inducers with different formulas are added into a culture bottle at different culture stages. The practice of this patent is equivalent to the use of liquid culture medium, which can easily lead to the difficulty of respiration of the young seedlings and death 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 already known to a person of ordinary skill in the art.
Disclosure of Invention
Aiming at the technical problems, the invention discloses a method for promoting quick seedling formation of orchid seeds, which comprises the following steps:
step S1, sterilization treatment of fruit pods: selecting orchid pods which are not cracked, green, full and free of diseases and insect pests, wiping impurities on the surfaces of the pods with alcohol cotton balls, and sterilizing on an ultra-clean workbench;
step S2, aseptic seeding: splitting 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 formula: MS+6-BA 3.0mg/L+NAA0.1 mg/L+banana 50 g/L+sucrose 30 g/L+agar 4g/L;
step S3, strong seedling cultivation: after 90d of sowing, the seeds germinate, the surface of the culture flask is sterilized, the bottle cap is opened, and a sterile needle tube is used for sucking the strong seedling culture medium and uniformly scattering the strong seedling culture medium on the small buds; the formula of the strong seedling culture medium is as follows: MS+6-BA1.0mg/L+NAA 0.5 mg/L+banana 100 g/L+sucrose 30 g/L+agar 4g/L;
step S4, rooting culture: after the strong seedlings are cultivated for 90 days, when 3-5 leaves grow out 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 of the seedling, sucking a nutrient solution by using an atomizer, and spraying the nutrient solution on the leaves of the seedling; the rooting culture medium comprises the following formula: MS+NAA 1.0 mg/L+banana 100 g/L+sucrose 30 g/L+agar 4g/L; the formula of the nutrient solution is as follows: MS+triacontanol 0.05 g/L+trehalose 0.1g/L;
step S5, transplanting: after rooting culture for 90d, the root system grows out, and transplanting and planting can be performed.
Wherein the orchid is tree orchid (epidemic) or dendrobium (Dendrobium nobile Lindl.).
The sterilization treatment method in the step S1 comprises the following steps: sterilizing with 75% alcohol on an ultra clean bench for 60s, sterilizing with sterile water for 3 times, sterilizing with 0.1% mercuric chloride for 15min, and sterilizing with sterile water for 4 times.
Wherein, in step S2, the culture flask is filled with 110mL of basal medium.
In the step S2, two ends of the fruit pod are cut off, the fruit pod is longitudinally cut, the fruit pod is placed into a bottle filled with 20mL of sterile water, the bottle is gently shaken, seeds on the fruit pod fall into the sterile water, and then 0.5-1mL of sterile water containing the seeds is sucked by a suction pipe and uniformly scattered on a basic culture medium uniformly scattered in a culture bottle.
In the step S3 and the step S4, the surface sterilization method of the culture bottle comprises the following steps: the flask surface was wiped with 75% alcohol and sterilized.
Wherein, in step S3, 10mL of strong seedling culture medium is sucked by a sterile needle tube and uniformly scattered on the buds.
Wherein, in step S4, 10mL of rooting culture medium is sucked by a sterile needle tube and uniformly scattered on the base of the young seedling.
Wherein, the culture conditions of the step S2 to 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 1 inoculation 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 compared with the traditional 1-year breeding time, and can accelerate the breeding process of new varieties;
(2) Compared with the method of disposable seedling formation of bletilla tissue culture seedling (CN 108739380A), the invention adopts solid culture medium spraying, and the patent adopts liquid culture medium, and the invention adopts needle tube to suck and spray the solid culture medium, so that a certain gap can be formed at the base of the seedling, the respiration of the seedling is ensured, and the liquid culture medium easily causes death of the seedling due to excessive water content;
(3) The nutrition is absorbed only through the basal part in the traditional growth process of the tissue culture seedling, and the nutrition liquid is sprayed through the sprayer in the rooting stage of the tissue culture seedling, so that the absorption of the leaves of the seedling is effectively promoted, the leaf color of the seedling is more greener, the seedling is stronger, and the transplanting survival rate is higher. Triacontanol is a plant growth promoter with a quite wide application range, and can be absorbed by stems and leaves of plants, and then promote the growth of the plants, increase the accumulation of dry matters, improve the permeability of cell membranes, increase the content of chlorophyll, improve the photosynthetic strength, and enhance the activities of amylase, polyoxidase and peroxidase. Trehalose plays a certain role in improving the resistance of tissue culture seedlings, and the transplanting survival rate can be improved by spraying on leaf surfaces before transplanting. Trehalose in the plant body can protect the plant from external injury under severe environmental conditions, thereby prolonging the life of the plant.
Detailed Description
The following detailed description is, therefore, to be taken in conjunction with the specific embodiments, it is to be understood that the scope of the invention is not limited to the specific embodiments. The raw materials used in the examples were commercially available unless otherwise specified.
Example 1 promotion of quick seedling formation of Dendrocala seed
Step S1, sterilization treatment of fruit pods: selecting a tree orchid pod which is not cracked, green, full and free of diseases and insect pests, wiping impurities on the surface of the pod with alcohol cotton balls, sterilizing for 60s with 75% alcohol on an ultra-clean workbench, sterilizing for 15min with 0.1% mercuric chloride for 3 times with sterile water, and sterilizing for 4 times with sterile water;
step S2, aseptic seeding: cutting off two ends of the fruit pod, longitudinally cutting the fruit pod, putting the fruit pod into a bottle filled with 20mL of sterile water, gently shaking to enable seeds on the fruit pod to fall into the sterile water, sucking 1mL of mixed solution of the sterile water and the seeds by using a suction pipe, uniformly scattering the mixed solution on a basic culture medium uniformly sprayed in a culture bottle, and inoculating 20 bottles per fruit pod; the basic culture medium comprises the following formula: MS+6-BA 3.0mg/L+NAA0.1 mg/L+banana 50 g/L+sucrose 30 g/L+agar 4g/L;
step S3, strong seedling cultivation: after 90d of sowing, the seeds germinate, the surface of the culture flask is wiped with 75% alcohol for sterilization, the bottle cap is opened, 10mL of strong seedling culture medium is sucked by a sterile needle tube and uniformly spread on the small buds; the formula of the strong seedling culture medium is as follows: MS+6-BA1.0 mg/L+NAA0.5mg/L+banana 100 g/L+sucrose 30 g/L+agar 4g/L;
step S4, rooting culture: after the strong seedlings are cultivated for 90 days, when 3-5 leaves grow out and the plant height reaches 3-4cm, wiping the surface of a culture bottle with 75% alcohol for sterilization, opening a bottle cap, sucking 10mL of rooting culture medium with a sterile needle tube, uniformly scattering the rooting culture medium on the base of the seedling, sucking nutrient solution with an atomizer, and spraying the nutrient solution on the leaves of the seedling; the rooting culture medium comprises the following formula: MS+NAA 1.0 mg/L+banana 100 g/L+sucrose 30 g/L+agar 4g/L; the formula of the nutrient solution is as follows: MS+triacontanol 0.05 g/L+trehalose 0.1g/L;
step S5, transplanting: after rooting culture for 90d, the root system grows out, and then transplanting and planting can be carried out, and the total culture time is 270d.
Example 2 promotion of rapid seedling formation of Dendrobium nobile seeds
Step S1, sterilization treatment of fruit pods: selecting a dendrobe pod which is not cracked, green, full and free of diseases and insect pests, wiping impurities on the surface of the pod by using alcohol cotton balls, sterilizing for 60 seconds by using 75% alcohol on an ultra-clean workbench, sterilizing for 15 minutes by using 0.1% mercuric chloride for 3 times by using sterile water, and washing for 4 times by using sterile water;
step S2, aseptic seeding: cutting off two ends of the fruit pod, longitudinally cutting the fruit pod, putting the fruit pod into a bottle filled with 10mL of sterile water, gently shaking to enable seeds on the fruit pod to fall into the sterile water, sucking 1mL of mixed solution of the sterile water and the seeds by using a suction pipe, uniformly scattering the mixed solution on a basic culture medium uniformly sprayed in a culture bottle, and inoculating 10 bottles on each fruit pod; the basic culture medium comprises the following formula: MS+6-BA 3.0mg/L+NAA0.1 mg/L+banana 50 g/L+sucrose 30 g/L+agar 4g/L;
step S3, strong seedling cultivation: after 90d of sowing, the seeds germinate, the surface of the culture flask is wiped with 75% alcohol for sterilization, the bottle cap is opened, 10mL of strong seedling culture medium is sucked by a sterile needle tube and uniformly spread on the small buds; the formula of the strong seedling culture medium is as follows: MS+6-BA1.0 mg/L+NAA0.5mg/L+banana 100 g/L+sucrose 30 g/L+agar 4g/L;
step S4, rooting culture: after the strong seedlings are cultivated for 90 days, when 3-5 leaves grow out and the plant height reaches 3-4cm, wiping the surface of a culture bottle with 75% alcohol for sterilization, opening a bottle cap, sucking 10mL of rooting culture medium with a sterile needle tube, uniformly scattering the rooting culture medium on the base of the seedling, sucking nutrient solution with an atomizer, and spraying the nutrient solution on the leaves of the seedling; the rooting culture medium comprises the following formula: MS+NAA 1.0 mg/L+banana 100 g/L+sucrose 30 g/L+agar 4g/L; the formula of the nutrient solution is as follows: MS+triacontanol 0.05 g/L+trehalose 0.1g/L;
step S5, transplanting: after rooting culture for 90d, the root system grows out, and then transplanting and planting can be carried out, and the total culture time is 270d.
Comparative example 3 traditional culture method of cymbidium seeds
Step S1, sterilization treatment of fruit pods: selecting a tree orchid pod which is not cracked, green, full and free of diseases and insect pests, wiping impurities on the surface of the pod with alcohol cotton balls, sterilizing for 60s with 75% alcohol on an ultra-clean workbench, sterilizing for 15min with 0.1% mercuric chloride for 3 times with sterile water, and sterilizing for 4 times with sterile water;
step S2, aseptic seeding: the pod was cut at both ends, split longitudinally, and the seeds were removed with forceps and evenly spread on germination induction medium (1/2MS+NAA0.1 mg/L+coconut juice 50 ml/L+sucrose 30 g/L+agar 4.2 g/L). 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 applies below);
step S3, strong seedling cultivation is carried out once: after 90d of sowing, the seeds germinate, and the germinated robust tree blue seedlings are selected and inserted into a strong seedling culture medium (MS+6-BA 1.0mg/L+NAA0.5 mg/L+banana 100 g/L+sucrose 30 g/L+agar 4 g/L);
step S4, secondary strong seedling culture: after 80d of strong seedlings, the seedlings of the tree orchid grow high, and the seedlings are taken out and inserted into a new strong seedling culture medium (MS+6-BA 1.0mg/L+NAA0.5 mg/L+banana 100 g/L+sucrose 30 g/L+agar 4 g/L);
step S5, rooting culture: after 80d of secondary strong seedling, cutting the aseptic seedling of the cymbidium into single plants, and inoculating the single plants into a rooting culture medium (1/2MS+NAA0.5 mg/L+banana 50 g/L+white granulated sugar 25 g/L+agar 4.2g/L, pH 5.5-5.8);
step S6, transplanting: after rooting culture for 80d, the root system grows out, and then transplanting and planting can be carried out, and the total culture time is 330d.
The manual time and the number of flasks consumed for example 1 and comparative example 3 were investigated and the results are shown in Table 1:
TABLE 1 time taken by the labor and number of flasks for example 1 and comparative example 3
As can be seen from Table 1, the method of the present invention has the advantages of shorter labor time for processing each culture bottle, higher working efficiency and lower production cost.
The average plant height, the number of leaves and the transplanting survival rate of 50 tree orchid rooting seedlings of example 1 and comparative example 3 after 90 days of transplanting are respectively counted, and the results are shown in table 2:
table 2 comparison of the transplanting Effect of the rooted seedlings of the cymbidium goeringii of example 1 and comparative example 3
As can be seen from Table 2, the rooting seedlings obtained in example 1 were superior to the rooting seedlings of comparative example 3 in terms of plant height, number of leaves, and transplanting survival rate after 90d of transplanting.
The foregoing descriptions of specific exemplary embodiments of the present invention are 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 the specific principles of the invention and its practical application to thereby enable one skilled in the art to make and utilize the invention in various exemplary embodiments and with various modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.
Claims (8)
1. A method for promoting rapid seedling formation of orchid seeds, comprising the following steps:
step S1, sterilization treatment of fruit pods: selecting orchid pods which are not cracked, green, full and free of diseases and insect pests, wiping impurities on the surfaces of the pods with alcohol cotton balls, and sterilizing on an ultra-clean workbench;
step S2, aseptic seeding: splitting 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 formula: MS+6-BA 3.0mg/L+NAA0.1 mg/L+banana 50 g/L+sucrose 30 g/L+agar 4g/L;
step S3, strong seedling cultivation: after 90d of sowing, the seeds germinate, the surface of the culture flask is sterilized, the bottle cap is opened, and a sterile needle tube is used for sucking the strong seedling culture medium and uniformly scattering the strong seedling culture medium on the small buds; the formula of the strong seedling culture medium is as follows: MS+6-BA1.0 mg/L+NAA0.5mg/L+banana 100 g/L+sucrose 30 g/L+agar 4g/L;
step S4, rooting culture: after the strong seedlings are cultivated for 90 days, when 3-5 leaves grow out 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 of the seedling, sucking a nutrient solution by using an atomizer, and spraying the nutrient solution on the leaves of the seedling; the rooting culture medium comprises the following formula: MS+NAA 1.0 mg/L+banana 100 g/L+sucrose 30 g/L+agar 4g/L; the formula of the nutrient solution is as follows: MS+triacontanol 0.05 g/L+trehalose 0.1g/L;
step S5, transplanting: after rooting culture for 90d, root system grows out, and transplanting and planting can be performed;
wherein the orchid is cymbidium goeringii.
2. The method according to claim 1, wherein the sterilization process in step S1 comprises: sterilizing with 75% alcohol on an ultra clean bench for 60s, sterilizing with sterile water for 3 times, sterilizing with 0.1% mercuric chloride for 15min, and sterilizing with sterile water for 4 times.
3. A method according to claim 1, characterized in that: in step S2, the flask is filled with 110mL of basal medium.
4. A method according to claim 1, characterized in that: in the step S2, two ends of the fruit pod are cut off, the fruit pod is longitudinally cut off, then the fruit pod is put into a bottle filled with 20mL of sterile water, the bottle is gently shaken to enable seeds on the fruit pod to fall into the sterile water, and then 0.5-1mL of sterile water containing the seeds is sucked by a suction pipe and uniformly sprayed on a basic culture medium in a culture bottle.
5. A method according to claim 1, characterized in that: in the step S3 and the step S4, the surface sterilization method of the culture bottle comprises the following steps: the flask surface was wiped with 75% alcohol and sterilized.
6. A method according to claim 1, characterized in that: in step S3, 10mL of the strong seedling medium is sucked by a sterile needle tube and uniformly spread on the buds.
7. A method according to claim 1, characterized in that: in step S4, 10mL of rooting medium is sucked by a sterile needle tube and uniformly scattered on the base of the young seedling.
8. A method according to claim 1, characterized in that: 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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| CN202211164286.8A CN115644058B (en) | 2022-09-23 | 2022-09-23 | Method for promoting quick seedling formation of orchid seeds |
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