CN111758572A - Method for tissue culture and rapid propagation of dendrobium nobile flower buds - Google Patents
Method for tissue culture and rapid propagation of dendrobium nobile flower buds Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 26
- 240000004638 Dendrobium nobile Species 0.000 title claims abstract description 17
- 230000006698 induction Effects 0.000 claims abstract description 31
- 241000196324 Embryophyta Species 0.000 claims abstract description 9
- 239000001963 growth medium Substances 0.000 claims description 78
- 238000005286 illumination Methods 0.000 claims description 36
- CGIDKJRJBMFXKV-UHFFFAOYSA-N 6-n'-benzylpurine-6,6-diamine Chemical compound N1=CN=C2N=CN=C2C1(N)NCC1=CC=CC=C1 CGIDKJRJBMFXKV-UHFFFAOYSA-N 0.000 claims description 35
- 229920001817 Agar Polymers 0.000 claims description 30
- 239000008272 agar Substances 0.000 claims description 30
- 238000005520 cutting process Methods 0.000 claims description 26
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 24
- 238000012258 culturing Methods 0.000 claims description 22
- 235000020415 coconut juice Nutrition 0.000 claims description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 18
- PRPINYUDVPFIRX-UHFFFAOYSA-N 1-naphthaleneacetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CC=CC2=C1 PRPINYUDVPFIRX-UHFFFAOYSA-N 0.000 claims description 17
- 239000002609 medium Substances 0.000 claims description 17
- 229930024421 Adenine Natural products 0.000 claims description 12
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 claims description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 12
- 229960000643 adenine Drugs 0.000 claims description 12
- 238000002791 soaking Methods 0.000 claims description 12
- 238000005406 washing Methods 0.000 claims description 12
- 239000012883 rooting culture medium Substances 0.000 claims description 11
- 239000012879 subculture medium Substances 0.000 claims description 9
- 238000000149 argon plasma sintering Methods 0.000 claims description 6
- 238000004140 cleaning Methods 0.000 claims description 6
- 235000020197 coconut milk Nutrition 0.000 claims description 6
- 238000001816 cooling Methods 0.000 claims description 6
- 201000010099 disease Diseases 0.000 claims description 6
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 6
- 239000012286 potassium permanganate Substances 0.000 claims description 6
- 230000002062 proliferating effect Effects 0.000 claims description 6
- 239000008223 sterile water Substances 0.000 claims description 6
- 239000008399 tap water Substances 0.000 claims description 6
- 235000020679 tap water Nutrition 0.000 claims description 6
- 238000012360 testing method Methods 0.000 claims description 6
- 230000035755 proliferation Effects 0.000 claims description 3
- 238000004659 sterilization and disinfection Methods 0.000 claims description 3
- 241001523681 Dendrobium Species 0.000 abstract description 10
- 230000002068 genetic effect Effects 0.000 abstract description 4
- 238000011031 large-scale manufacturing process Methods 0.000 abstract description 4
- 238000003912 environmental pollution Methods 0.000 abstract description 3
- 238000004519 manufacturing process Methods 0.000 abstract description 3
- 241001453636 Salvinia Species 0.000 description 5
- 241000233855 Orchidaceae Species 0.000 description 4
- 230000001954 sterilising effect Effects 0.000 description 4
- 230000004083 survival effect Effects 0.000 description 3
- 238000009423 ventilation Methods 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- 230000017260 vegetative to reproductive phase transition of meristem Effects 0.000 description 2
- 229920000742 Cotton Polymers 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- NWBJYWHLCVSVIJ-UHFFFAOYSA-N N-benzyladenine Chemical compound N=1C=NC=2NC=NC=2C=1NCC1=CC=CC=C1 NWBJYWHLCVSVIJ-UHFFFAOYSA-N 0.000 description 1
- 206010052428 Wound Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 150000002989 phenols Chemical class 0.000 description 1
- 230000001902 propagating effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000012882 rooting medium Substances 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
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Abstract
The invention provides a method for tissue culture and rapid propagation of flower buds of dendrobium nobile, which comprises the steps of sequentially carrying out induction culture of buds, enrichment culture of cluster buds, subculture of the cluster buds and rooting culture of strong seedlings to obtain complete plants, and then taking out of bottles, hardening seedlings and transplanting. The invention uses tender flower buds or side buds of pedicel as explants, greatly reduces the occurrence rate of browning or pollution easily caused by using stem buds, and the method has easy operation, low production cost and no environmental pollution, and can realize large-scale production. The dendrobium autumn seedling cultivated by the method has stable genetic character, maintains the characteristics of parents, and has the advantages of invariability, less investment, high yield, short period and the like.
Description
Technical Field
The invention belongs to the technical field of tissue culture and rapid propagation, and particularly relates to a method for tissue culture and rapid propagation of flower buds of dendrobium nobile.
Background
The Dendrobium nobile is evergreen (Dendrobium) and is a plant of Dendrobium of Orchidaceae. The flowers of the dendrobium are mainly grown on the top of stems, the branches of tall plants are 50-100cm long, the branches of small plants are 20-50cm long, more than ten or dozens of flowers are grown on inflorescences, the natural flowering phase is 8-12 months or longer, and the single-branch flowering phase is 1-2 months, so that the dendrobium is an important tropical orchid for both commodity cut flowers and pot flowers and is popular among people. At present, domestic and foreign researches on the fast propagation technology of dendrobium autumn are mainly focused on propagating and separating seedlings or germinating seeds by using stem buds as explants to obtain seedlings, but the stem buds as the explants are easy to brown and have higher pollution incidence rate because the stem buds are easy to undergo enzymatic browning and are caused by secretion of phenolic compounds at wounds, meanwhile, the stem buds are in contact with a matrix during growth, and have more bacteria, fungi and the like which are easy to be infected during the tissue culture process, but the flower buds grow from the top, are clean and have less browning and are easy to succeed in the tissue culture process; the progeny of the dendrobium nobile cultivated by the seeds has high character separation degree and large difference among individuals, and plants with consistent characters are difficult to realize. Therefore, the tissue propagation is carried out by using the dendrobium nobile orchid buds, plants with consistent characters are cultivated, and the method is very key in meeting the requirements of flower markets and realizing high-efficiency and large-scale production.
Disclosure of Invention
In view of the above, the technical problem to be solved by the present invention is to provide a method for tissue culture and rapid propagation of dendrobium autumn flower buds, a large number of test-tube plantlets with stable genetic characters can be obtained by tissue culture of dendrobium autumn flowers through a cluster bud approach, characteristics of parents are maintained, and advantages of invariability, low investment, high yield, short period and the like are provided.
The invention solves the technical problems through the following technical scheme:
a method for tissue culture and rapid propagation of dendrobium nobile flower buds is characterized by comprising the following steps:
1) and (3) disinfection of explants: selecting tender flower buds or flower stalks with the length of 2-3cm as explants, wherein the selected flower stalks need to be thick and strong without diseases, and cutting out the stem sections of the flower stalks with the long strips of 2-3cm and side buds. Washing tender flower bud or stem with tap water, cutting off bract, soaking in 75% ethanol for 30s, and adding 0.1% HgCl2And sterilizing the solution for 8-10 min, washing with sterile water for 5-8 times, and then sucking residual water by using sterile filter paper. For young flower buds, cutting bud tips of 0.5-1.0cm in length, and inoculating on an induction culture medium; cutting 0.5cm from each end of the pedicel stem segment, inoculating the remaining pedicel stem segment with lateral bud on the induction culture medium, culturing for 1-2 months until the lateral bud grows to 2-3cm, cutting 0.5-1.0cm bud tip, inoculating on the induction culture medium againOn a culture medium.
2) And (3) induction of buds: culturing for 30-60 days by adopting an induction culture medium, and enabling the bud tips to turn green and increase; the culture temperature is 26 +/-2 ℃, and the culture medium can be used for culturing the plant cells with scattered light;
3) and (3) multiplication of cluster buds: transferring the bud tip to a multiplication culture medium, and culturing for 50-60 days to obtain cluster buds, wherein the multiplication times can reach 3.0-5.0; the culture conditions were: the culture temperature is 26 +/-2 ℃, the illumination intensity is 2000 +/-100 lx, and the illumination time is 12 h/d;
4) subculturing: subculturing and proliferating for 1 time every 50-60 days, wherein the subculturing time is not more than 15 times generally; transferring the cells to a subculture medium for subculture, wherein the culture conditions are as follows: the culture temperature is 26 +/-2 ℃, the illumination intensity is 2000 +/-100 lx, and the illumination time is 12 h/d;
5) strong seedling culture: transferring cluster buds formed in the proliferation and subculture stages into a strong seedling culture medium; the culture conditions were: the culture temperature is 26 +/-2 ℃, the illumination intensity is 2000 +/-100 lx, and the illumination time is 12 h/d;
6) rooting culture: and (3) inoculating the plantlets formed in the strong seedling stage into a rooting culture medium for rooting culture, and culturing for about 60 days to obtain the plantlets with the height of 5-8 cm and 2-3 or more roots to form robust plantlets.
7) Transplanting test-tube seedlings: 3-5 months or 10-12 months, placing the test-tube plantlet in a greenhouse with natural light scattering, wherein the light intensity is 8000 lx-10000 lx, the temperature is 20-28 ℃, and the environment is clean; and (4) arranging the bottle seedlings in a straight line, placing the bottle seedlings on a culture shelf, hardening the seedlings for 15-20 days, and then taking out of the bottle for transplanting. Firstly, taking out a plantlet from a test tube, cleaning a culture medium at the root, then soaking in a 0.1% potassium permanganate solution for 5 minutes, taking out, and planting in a plastic cup with the caliber of 4.8cm by using water moss. Keeping the greenhouse ventilated, keeping the humidity at 70-80%, keeping the temperature above 15 ℃, and cooling by using a fan and a water curtain when the temperature is higher than 30 ℃.
Preferably, the induction culture medium is Hyponex (Huabao No. 1) 3g/L +6-BA (6-benzylamino adenine) 1.0-2.0 mg/g + NAA (naphthalene acetic acid) 0.1-0.5 mg/L + AC (activated carbon) 1.0-3.0 g/L + CW (coconut milk) 100.0-200.0 g/L, the culture medium contains 30g/L of sugar, 7g/L of agar, and the pH value is 5.8 +/-0.2.
Preferably, the multiplication medium Hyponex (Huabao No. 1) 3g/L +6-BA (6-benzylamino adenine) 1.0-3.0 mg/L + Ad (adenine) 1.0-3.0 mg/L + CW (coconut juice) 100.0-200.0 g/L, the medium contains 30g/L of sugar, 7g/L of agar and has a pH value of 5.8 +/-0.2, and the medium is cultured for 50-60 days to obtain cluster buds, wherein the multiplication times can reach 3.0-5.0.
Preferably, the subculture medium is Hyponex (Huabao 1) 3g/L +6-BA (6-benzylamino adenine) 1.0-3.0 mg/L + Ad (adenine) 1.0-3.0 mg/L + CW (coconut juice) 100.0-200.0 g/L, the medium contains 30g/L sugar, agar is 7g/L, and the pH value is 5.8 +/-0.2.
Preferably, the strong seedling culture medium is Hyponex (Huabao No. 1) 3g/L + CW (coconut milk) 100.0-200.0 g/L, the culture medium contains 30g/L of sugar, 7g/L of agar and the pH value is 5.8 +/-0.2.
Preferably, the rooting medium is Hyponex (Huabao No. 1) 3g/L + NAA 0.1-0.3 mg/L + CW 100.0-200.0 g/L, the medium contains sugar 30g/L, agar 7g/L, and the pH value is 5.8 +/-0.2.
Compared with the prior art, the method for tissue culture and rapid propagation of the flower buds of dendrobium nobile has the advantages that: the method has the advantages of easy operation, low production cost, no environmental pollution and large-scale production. The dendrobium autumn seedling cultivated by the method has stable genetic character, maintains the characteristics of parents, and has the advantages of invariability, less investment, high yield, short period and the like.
Detailed Description
In view of this, the method for tissue culture and rapid propagation of orchid buds of dendrobium nobile provided in the embodiment of the invention specifically comprises the following steps:
1) and (3) disinfection of explants: selecting tender flower buds or flower stalks with the length of 2-3cm as explants, wherein the selected flower stalks need to be thick and strong without diseases, and cutting out the stem sections of the flower stalks with the long strips of 2-3cm and side buds. Washing tender flower bud or stem with tap water, cutting off bract, soaking in 75% ethanol for 30s, and adding 0.1% HgCl2And sterilizing the solution for 8-10 min, washing with sterile water for 5-8 times, and then sucking residual water by using sterile filter paper. For young flower buds, cutting bud tips of 0.5-1.0cm in length, and inoculating on an induction culture medium; for the stem segment of the flower stalk, 0.5cm of each end thereof was cutAnd inoculating the stem segment of the flower stalk with the side bud on an induction culture medium for culture, wherein the side bud grows to 2-3cm after 1-2 months, and then cutting a bud tip with the length of 0.5-1.0cm and inoculating on the induction culture medium again.
2) And (3) induction of buds: culturing for 30-60 days by adopting an induction culture medium, and enabling the bud tips to turn green and increase; the culture temperature is 26 +/-2 ℃, and the culture medium can be used for culturing the plant cells with scattered light;
3) and (3) multiplication of cluster buds: transferring the bud tip to a multiplication culture medium, and culturing for 50-60 days to obtain cluster buds, wherein the multiplication times can reach 3.0-5.0; the culture conditions were: the culture temperature is 26 +/-2 ℃, the illumination intensity is 2000 +/-100 lx, and the illumination time is 12 h/d;
4) subculturing: subculturing and proliferating for 1 time every 50-60 days, wherein the subculturing time is not more than 15 times generally; transferring the cells to a subculture medium for subculture, wherein the culture conditions are as follows: the culture temperature is 26 +/-2 ℃, the illumination intensity is 2000 +/-100 lx, and the illumination time is 12 h/d;
5) strong seedling culture: transferring cluster buds formed in the proliferation and subculture stages into a strong seedling culture medium; the culture conditions were: the culture temperature is 26 +/-2 ℃, the illumination intensity is 2000 +/-100 lx, and the illumination time is 12 h/d;
6) rooting culture: and (3) inoculating the plantlets formed in the strong seedling stage into a rooting culture medium for rooting culture, and culturing for about 60 days to obtain the plantlets with the height of 5-8 cm and 2-3 or more roots to form robust plantlets.
7) Transplanting test-tube seedlings: 3-5 months or 10-12 months, placing the test-tube plantlet in a greenhouse with natural light scattering, wherein the light intensity is 8000 lx-10000 lx, the temperature is 20-28 ℃, and the environment is clean; and (4) arranging the bottle seedlings in a straight line, placing the bottle seedlings on a culture shelf, hardening the seedlings for 15-20 days, and then taking out of the bottle for transplanting. Firstly, taking out a plantlet from a test tube, cleaning a culture medium at the root, then soaking in a 0.1% potassium permanganate solution for 5 minutes, taking out, and planting in a plastic cup with the caliber of 4.8cm by using water moss. Keeping the greenhouse ventilated, keeping the humidity at 70-80%, keeping the temperature above 15 ℃, and cooling by using a fan and a water curtain when the temperature is higher than 30 ℃.
Wherein, each culture is under the condition of determined components, the content of each component used in the culture can be adjusted according to the actual culture condition, and the components of the culture medium and the content range of each component used in the method are as follows:
wherein the induction culture medium is Hyponex (Huabao No. 1) 3g/L +6-BA (6-benzylamino adenine) 1.0-2.0 mg/g + NAA (naphthalene acetic acid) 0.1-0.5 mg/L + AC (activated carbon) 1.0-3.0 g/L + CW (coconut milk) 100.0-200.0 g/L, the culture medium contains 30g/L of sugar, 7g/L of agar and the pH value is 5.8 +/-0.2.
Wherein the multiplication medium Hyponex (Huabao No. 1) 3g/L +6-BA (6-benzylamino adenine) 1.0-3.0 mg/L + Ad (adenine) 1.0-3.0 mg/L + CW (coconut juice) 100.0-200.0 g/L, the medium contains 30g/L of sugar, 7g/L of agar, the pH value is 5.8 +/-0.2, and the medium is cultured for 50-60 days to obtain cluster buds, wherein the multiplication times can reach 3.0-5.0.
Wherein the subculture medium is Hyponex (Huabao No. 1) 3g/L +6-BA (6-benzylaminopurine) 1.0-3.0 mg/L + Ad (adenine) 1.0-3.0 mg/L + CW (coconut juice) 100.0-200.0 g/L, the culture medium contains 30g/L of sugar, 7g/L of agar and the pH value is 5.8 +/-0.2.
Wherein the strong seedling culture medium is Hyponex (Huabao No. 1) 3g/L + CW (coconut milk) 100.0-200.0 g/L, the culture medium contains 30g/L of sugar, 7g/L of agar and the pH value is 5.8 +/-0.2.
Wherein the rooting culture medium is Hyponex (Huabao No. 1) 3g/L, NAA 0.1-0.3 mg/L, CW 100.0-200.0 g/L, the culture medium contains sugar 30g/L, agar 7g/L and the pH value is 5.8 +/-0.2.
In addition, since the cultivation process is affected by various factors such as temperature, light, humidity, etc., the treatment method, the cultivation conditions, and the cultivation time are appropriately adjusted according to the specific needs in each step of the present invention.
In order that the invention may be more readily understood, specific embodiments thereof will be described further below.
Example 1:
selecting tender flower buds or flower stalks with the length of 2-3cm as explants, wherein the selected flower stalks need to be thick and strong without diseases, and cutting out the stem sections of the flower stalks with the long strips of 2-3cm and side buds. Washing tender flower bud or stem with tap water, cutting off bract, soaking in 75% ethanol for 30s, and adding 0.1% HgCl2Sterilizing the solution for 8-10 min, washing with sterile water for 5-8 times, and sterilizingThe residual water was blotted dry with bacteria filter paper. For young flower buds, cutting bud tips of 0.5-1.0cm in length, and inoculating on an induction culture medium; for the pedicel stem segment, cutting 0.5cm from each end, inoculating the remaining pedicel stem segment with lateral bud on the induction culture medium, culturing until the lateral bud grows to 2-3cm after 1-2 months, and inoculating the bud tip with length of 0.5-1.0cm on the induction culture medium again. Wherein the induction culture medium is Hyponex (Huabao No. 1) 3g/L +6-BA (6-benzylamino adenine) 1.0mg/g + NAA (naphthalene acetic acid) 0.1mg/L + AC (activated carbon) 1.0g/L + CW (coconut juice) 100.0g/L, the culture medium contains 30g/L of sugar, 7g/L of agar and has a pH value of 5.8 +/-0.2, and the bud tip is cultured for 30-60 days and turns green and increases. The culture temperature is 26 +/-2 ℃, and the culture medium is only required to have scattered light. Transferring bud tips obtained by culturing flower buds to a cluster bud multiplication medium: hyponex (Huabao No. 1) 3g/L +6-BA (6-benzylamino adenine) 1.0 mg/L + Ad (adenine) 1.0 mg/L + CW (coconut juice) 100.0g/L, a culture medium containing 30g/L sugar, agar 7g/L and a pH value of 5.8 +/-0.2, and culturing for 50-60 days to obtain cluster buds, wherein the multiplication times can reach 3.0. The culture conditions were: the culture temperature is 26 +/-2 ℃, the illumination intensity is 2000 +/-100 lx, and the illumination time is 12 h/d. Subculturing and proliferating for 1 time every 50-60 days, and generally subculturing times are not more than 15 times. The subculture medium is Hyponex (Huabao No. 1) 3g/L +6-BA (6-benzylamino adenine) 1.0 mg/L + Ad (adenine) 1.0 mg/L + CW (coconut juice) 100.0g/L, the medium contains 30g/L sugar, agar is 7g/L, and the pH value is 5.8 +/-0.2. The culture conditions were: the culture temperature is 26 +/-2 ℃, the illumination intensity is 2000 +/-100 lx, and the illumination time is 12 h/d. And transferring the cluster buds formed in the propagation subculture stage into a strong seedling culture medium. The strong seedling culture medium is Hyponex (Huabao No. 1) 3g/L + CW (coconut juice) 100.0g/L, the culture medium contains 30g/L sugar, 7g/L agar and the pH value is 5.8 +/-0.2. The culture conditions were: the culture temperature is 26 +/-2 ℃, the illumination intensity is 2000 +/-100 lx, and the illumination time is 12 h/d. The plantlets formed in the strong seedling stage are inoculated into a rooting culture medium, wherein the rooting culture medium is Hyponex (Huabao No. 1) 3g/L, NAA 0.1mg/L and CW100.0 g/L, the culture medium contains sugar 30g/L, agar 7g/L and the pH value is 5.8 +/-0.2. After about 60 days of culture, the height of the seedling can reach 5-8 cm, 2-3 or more roots are formed, and a robust plantlet is formed. 3-5 months or 10-12 months, placing the test-tube plantlet in a greenhouse with natural light scattering, wherein the light intensity is 8000 lx-10000 lx, the temperature is 20-28 ℃, and the environment is clean; the bottle seedlings are arranged in a row,placing on a culture shelf, hardening seedlings for 15-20 days, and taking out of bottles for transplanting. Firstly, taking out a plantlet from a test tube, cleaning a culture medium at the root, then soaking in a 0.1% potassium permanganate solution for 5 minutes, taking out, and planting in a plastic cup with the caliber of 4.8cm by using water moss. Keeping the greenhouse ventilation, keeping the humidity at 70-80%, keeping the temperature above 15 ℃, cooling by using a fan and a water curtain when the temperature is higher than 30 ℃, and ensuring that the transplanting survival rate can reach 95%.
Example 2:
selecting tender flower buds or flower stalks with the length of 2-3cm as explants, wherein the selected flower stalks need to be thick and strong without diseases, and cutting out the stem sections of the flower stalks with the long strips of 2-3cm and side buds. Washing tender flower bud or stem with tap water, cutting off bract, soaking in 75% ethanol for 30s, and adding 0.1% HgCl2And sterilizing the solution for 8-10 min, washing with sterile water for 5-8 times, and then sucking residual water by using sterile filter paper. For young flower buds, cutting bud tips of 0.5-1.0cm in length, and inoculating on an induction culture medium; for the pedicel stem segment, cutting 0.5cm from each end, inoculating the remaining pedicel stem segment with lateral bud on the induction culture medium, culturing until the lateral bud grows to 2-3cm after 1-2 months, and inoculating the bud tip with length of 0.5-1.0cm on the induction culture medium again. Wherein the induction culture medium is Hyponex (Huabao No. 1) 3g/L +6-BA (6-benzylamino adenine) 1.5mg/g + NAA (naphthalene acetic acid) 0.3mg/L + AC (activated carbon) 2.0g/L + CW (coconut juice) 150.0g/L, the culture medium contains 30g/L of sugar, 7g/L of agar, the pH value is 5.8 +/-0.2, the culture lasts for 30-60 days, and the bud tips become green and increase. The culture temperature is 26 +/-2 ℃, and the culture medium is only required to have scattered light. Transferring bud tips obtained by culturing flower buds to a cluster bud multiplication medium: hyponex (Huabao No. 1) 3g/L +6-BA (6-benzylamino adenine) 2.0 mg/L + Ad (adenine) 2.0 mg/L + CW (coconut juice) 150.0g/L, a culture medium containing sugar 30g/L, agar 7g/L and a pH value of 5.8 +/-0.2, and culturing for 50-60 days to obtain cluster buds, wherein the multiplication times can reach 4.0. The culture conditions were: the culture temperature is 26 +/-2 ℃, the illumination intensity is 2000 +/-100 lx, and the illumination time is 12 h/d. Subculturing and proliferating for 1 time every 50-60 days, and generally subculturing times are not more than 15 times. The subculture medium is Hyponex (Huabao No. 1) 3g/L +6-BA (6-benzylamino adenine) 2.0 mg/L + Ad (adenine) 2.0 mg/L + CW (coconut juice) 150.0g/L, the medium contains 30g/L sugar, agar is 7g/L, and the pH value is 5.8 plus or minus 0.2. The culture conditions were: the culture temperature is 26 +/-2 ℃, the illumination intensity is 2000 +/-100 lx, and the illumination time is 12 h/d. Transferring the cluster buds formed in the multiplication subculture stage into a strong seedling culture medium, wherein the strong seedling culture medium is Hyponex (Huabao No. 1) 3g/L + CW (coconut juice) 150.0g/L, the culture medium contains 30g/L of sugar, 7g/L of agar and the pH value is 5.8 +/-0.2. The culture conditions were: the culture temperature is 26 +/-2 ℃, the illumination intensity is 2000 +/-100 lx, and the illumination time is 12 h/d. The plantlets formed in the strong seedling stage are inoculated into a rooting culture medium, wherein the rooting culture medium is Hyponex (Huabao No. 1) 3g/L + NAA0.2mg/L + CW 150.0g/L, the culture medium contains 30g/L of sugar, 7g/L of agar and has the pH value of 5.8 +/-0.2. After about 60 days of culture, the height of the seedling can reach 5-8 cm, 2-3 or more roots are formed, and a robust plantlet is formed. 3-5 months or 10-12 months, placing the test-tube plantlet in a greenhouse with natural light scattering, wherein the light intensity is 8000 lx-10000 lx, the temperature is 20-28 ℃, and the environment is clean; and (4) arranging the bottle seedlings in a straight line, placing the bottle seedlings on a culture shelf, hardening the seedlings for 15-20 days, and then taking out of the bottle for transplanting. Firstly, taking out a plantlet from a test tube, cleaning a culture medium at the root, then soaking in a 0.1% potassium permanganate solution for 5 minutes, taking out, and planting in a plastic cup with the caliber of 4.8cm by using water moss. Keeping the greenhouse ventilation, keeping the humidity at 70-80%, keeping the temperature above 15 ℃, cooling by using a fan and a water curtain when the temperature is higher than 30 ℃, and ensuring that the transplanting survival rate can reach 95%.
Example 3:
selecting tender flower buds or flower stalks with the length of 2-3cm as explants, wherein the selected flower stalks need to be thick and strong without diseases, and cutting out the stem sections of the flower stalks with the long strips of 2-3cm and side buds. Washing tender flower bud or stem with tap water, cutting off bract, soaking in 75% ethanol for 30s, and adding 0.1% HgCl2And sterilizing the solution for 8-10 min, washing with sterile water for 5-8 times, and then sucking residual water by using sterile filter paper. For young flower buds, cutting bud tips of 0.5-1.0cm in length, and inoculating on an induction culture medium; for the pedicel stem segment, cutting 0.5cm from each end, inoculating the remaining pedicel stem segment with lateral bud on the induction culture medium, culturing until the lateral bud grows to 2-3cm after 1-2 months, and inoculating the bud tip with length of 0.5-1.0cm on the induction culture medium again. Wherein the induction culture medium is Hyponex (Huabao No. 1) 3g/L +6-BA (6-benzylamino adenine))2.0mg/g + NAA (naphthylacetic acid) 0.5mg/L + AC (activated carbon) 3.0g/L + CW (coconut milk) 200.0g/L, the culture medium contains 30g/L of sugar, 7g/L of agar, the pH value is 5.8 +/-0.2, the culture lasts for 30-60 days, and the bud tip turns green and increases. The culture temperature is 26 +/-2 ℃, and the culture medium is only required to have scattered light. Transferring bud tips obtained by culturing flower buds to a cluster bud multiplication medium: 3g/L of Hyponex (Huabao No. 1), 3.0 mg/L of 6-BA (6-benzylamino adenine), 3.0 mg/L of Ad (adenine), 200.0g/L of light cotton and 200.0g/L of CW (coconut juice), wherein the culture medium contains 30g/L of sugar, 7g/L of agar and 5.8 +/-0.2 of pH value, and the cluster buds are obtained after being cultured for 50-60 days, and the multiplication times can reach 5.0. The culture conditions were: the culture temperature is 26 +/-2 ℃, the illumination intensity is 2000 +/-100 lx, and the illumination time is 12 h/d. Subculturing and proliferating for 1 time every 50-60 days, and generally subculturing times are not more than 15 times. The subculture medium is Hyponex (Huabao No. 1) 3g/L +6-BA (6-benzylamino adenine) 3.0 mg/L + Ad (adenine) 3.0 mg/L + CW (coconut juice) 200.0g/L, the medium contains 30g/L sugar, agar is 7g/L, and the pH value is 5.8 +/-0.2. The culture conditions were: the culture temperature is 26 +/-2 ℃, the illumination intensity is 2000 +/-100 lx, and the illumination time is 12 h/d. Transferring the cluster buds formed in the multiplication subculture stage into a strong seedling culture medium, wherein the strong seedling culture medium is Hyponex (Huabao No. 1) 3g/L + CW (coconut juice) 200.0g/L, the culture medium contains 30g/L of sugar, 7g/L of agar and the pH value is 5.8 +/-0.2. The culture conditions were: the culture temperature is 26 +/-2 ℃, the illumination intensity is 2000 +/-100 lx, and the illumination time is 12 h/d. The plantlets formed in the strong seedling stage are inoculated into a rooting culture medium, wherein the rooting culture medium is Hyponex (Huabao No. 1) 3g/L, NAA 0.3mg/L and CW 200.0g/L, the culture medium contains sugar 30g/L, agar 7g/L and the pH value is 5.8 +/-0.2. After about 60 days of culture, the height of the seedling can reach 5-8 cm, 2-3 or more roots are formed, and a robust plantlet is formed. 3-5 months or 10-12 months, placing the test-tube plantlet in a greenhouse with natural light scattering, wherein the light intensity is 8000 lx-10000 lx, the temperature is 20-28 ℃, and the environment is clean; and (4) arranging the bottle seedlings in a straight line, placing the bottle seedlings on a culture shelf, hardening the seedlings for 15-20 days, and then taking out of the bottle for transplanting. Firstly, taking out a plantlet from a test tube, cleaning a culture medium at the root, then soaking in a 0.1% potassium permanganate solution for 5 minutes, taking out, and planting in a plastic cup with the caliber of 4.8cm by using water moss. Keeping the greenhouse ventilation, keeping the humidity at 70-80%, keeping the temperature above 15 ℃, cooling by using a fan and a water curtain when the temperature is higher than 30 ℃, and ensuring that the transplanting survival rate can reach 95%.
Compared with the prior art, the method for tissue culture and rapid propagation of the orchid buds of dendrobium nobile disclosed in the mode has the advantages of easiness in operation, low production cost, no environmental pollution and capability of realizing large-scale production. The dendrobium autumn seedling cultivated by the method has stable genetic character, maintains the characteristics of parents, and has the advantages of invariability, less investment, high yield, short period and the like.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (6)
1. A method for tissue culture and rapid propagation of dendrobium nobile flower buds is characterized by comprising the following steps:
1) and (3) disinfection of explants: selecting tender flower buds or flower stalks with the length of 2-3cm as explants, wherein the selected flower stalks need to be thick and strong without diseases, and cutting off the stem sections of the flower stalks with the long strips of 2-3cm and side buds; washing tender flower bud or stem with tap water, cutting off bract, soaking in 75% ethanol for 30s, and adding 0.1% HgCl2Sterilizing the solution for 8-10 min, washing with sterile water for 5-8 times, and then sucking out residual water by using sterile filter paper; for young flower buds, cutting bud tips of 0.5-1.0cm in length, and inoculating on an induction culture medium; for the pedicel stem segment, cutting 0.5cm from each end, inoculating the rest pedicel stem segment with side bud on the induction culture medium for culturing, after 1-2 months, the side bud grows to 2-3cm, and then cutting 0.5-1.0cm bud tip to inoculate on the induction culture medium again;
2) and (3) induction of buds: culturing for 30-60 days by adopting an induction culture medium, and enabling the bud tips to turn green and increase; the culture temperature is 26 +/-2 ℃, and the culture medium can be used for culturing the plant cells with scattered light;
3) and (3) multiplication of cluster buds: transferring the bud tip to a multiplication culture medium, and culturing for 50-60 days to obtain cluster buds, wherein the multiplication times can reach 3.0-5.0; the culture conditions were: the culture temperature is 26 +/-2 ℃, the illumination intensity is 2000 +/-100 lx, and the illumination time is 12 h/d;
4) subculturing: subculturing and proliferating for 1 time every 50-60 days, wherein the subculturing time is not more than 15 times generally; transferring the cells to a subculture medium for subculture, wherein the culture conditions are as follows: the culture temperature is 26 +/-2 ℃, the illumination intensity is 2000 +/-100 lx, and the illumination time is 12 h/d;
5) strong seedling culture: transferring cluster buds formed in the proliferation and subculture stages into a strong seedling culture medium; the culture conditions were: the culture temperature is 26 +/-2 ℃, the illumination intensity is 2000 +/-100 lx, and the illumination time is 12 h/d;
6) rooting culture: and (3) inoculating the plantlets formed in the strong seedling stage into a rooting culture medium for rooting culture, and culturing for about 60 days to obtain the plantlets with the height of 5-8 cm and 2-3 or more roots to form robust plantlets.
7) Transplanting test-tube seedlings: 3-5 months or 10-12 months, placing the test-tube plantlet in a greenhouse with natural light scattering, wherein the light intensity is 8000 lx-10000 lx, the temperature is 20-28 ℃, and the environment is clean; arranging the bottle seedlings in a straight line, placing the bottle seedlings on a culture shelf, hardening the seedlings for 15-20 days, and then taking out the bottles for transplanting; firstly, taking out a plantlet from a test tube, cleaning a culture medium at the root, then soaking in a 0.1% potassium permanganate solution for 5 minutes, taking out, and planting in a plastic cup with the caliber of 4.8cm by using moss; keeping the greenhouse ventilated, keeping the humidity at 70-80%, keeping the temperature above 15 ℃, and cooling by using a fan and a water curtain when the temperature is higher than 30 ℃.
2. The method for tissue culture and rapid propagation of dendrobium nobile flower buds as claimed in claim 1, which is characterized in that: the induction culture medium is Hyponex (Huabao No. 1) 3g/L +6-BA (6-benzylamino adenine) 1.0-2.0 mg/g + NAA (naphthalene acetic acid) 0.1-0.5 mg/L + AC (activated carbon) 1.0-3.0 g/L + CW (coconut milk) 100.0-200.0 g/L, the culture medium contains 30g/L of sugar, 7g/L of agar and the pH value is 5.8 +/-0.2.
3. The method for tissue culture and rapid propagation of dendrobium nobile flower buds as claimed in claim 1, which is characterized in that: the multiplication medium Hyponex (Huabao No. 1) 3g/L +6-BA (6-benzylamino adenine) 1.0-3.0 mg/L + Ad (adenine) 1.0-3.0 mg/L + CW (coconut juice) 100.0-200.0 g/L, the medium contains 30g/L of sugar, 7g/L of agar, the pH value is 5.8 +/-0.2, and the medium is cultured for 50-60 days to obtain cluster buds, wherein the multiplication times can reach 3.0-5.0.
4. The method for tissue culture and rapid propagation of dendrobium nobile flower buds as claimed in claim 1, which is characterized in that: the subculture medium is Hyponex (Huabao No. 1) 3g/L +6-BA (6-benzylamino adenine) 1.0-3.0 mg/L + Ad (adenine) 1.0-3.0 mg/L + CW (coconut juice) 100.0-200.0 g/L, the culture medium contains 30g/L of sugar, 7g/L of agar and the pH value is 5.8 +/-0.2.
5. The method for tissue culture and rapid propagation of dendrobium nobile flower buds as claimed in claim 1, which is characterized in that: the strong seedling culture medium is Hyponex (Huabao No. 1) 3g/L + CW (coconut juice) 100.0-200.0 g/L, the culture medium contains 30g/L of sugar, 7g/L of agar and the pH value is 5.8 +/-0.2.
6. The method for tissue culture and rapid propagation of dendrobium nobile flower buds as claimed in claim 1, which is characterized in that: the rooting culture medium is Hyponex (Huabao No. 1) 3g/L, NAA 0.1-0.3 mg/L, CW 100.0-200.0 g/L, the culture medium contains sugar 30g/L, agar 7g/L and the pH value is 5.8 +/-0.2.
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