CN112136687A - Rapid propagation and in-vitro preservation method of saprophytic orchid - Google Patents
Rapid propagation and in-vitro preservation method of saprophytic orchid Download PDFInfo
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- CN112136687A CN112136687A CN202011172244.XA CN202011172244A CN112136687A CN 112136687 A CN112136687 A CN 112136687A CN 202011172244 A CN202011172244 A CN 202011172244A CN 112136687 A CN112136687 A CN 112136687A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/02—Methods or apparatus for hybridisation; Artificial pollination ; Fertility
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- 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
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N3/00—Preservation of plants or parts thereof, e.g. inhibiting evaporation, improvement of the appearance of leaves or protection against physical influences such as UV radiation using chemical compositions; Grafting wax
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Abstract
The invention provides a rapid propagation and in-vitro preservation method of saprophytic orchid, and relates to the technical field of plant propagation. The rapid propagation and in-vitro preservation method takes a fruit pod with seeds obtained after artificial pollination as an object, obtains protocorms through disinfection and sterile germination of the seeds, induces the protocorms to obtain rhizomes, further establishes an in-vitro preservation system of the large rhizomes by taking the rhizomes as the object, and has a propagation coefficient within half a year of 1: 4, the in vitro preservation subculture period reaches about 2 years. The rapid propagation and in vitro preservation method effectively solves the problem of tissue culture and rapid propagation of saprophytic orchid, prolongs the in vitro preservation period, is favorable for further preservation, development and utilization of saprophytic orchid germplasm resources, and lays a foundation for future resource mining.
Description
Technical Field
The invention belongs to the technical field of plant breeding, and particularly relates to a rapid propagation and in-vitro preservation method of saprophytic orchid.
Background
The Cymbidium macrocarpizum (Cymbidium macrocrorhizon) is a Cymbidium plant of orchidaceae, does not have green leaves, is the only saprophytic species in the Cymbidium plant, and is distributed under the forest or at the forest margin of the elevation of 400-2100 meters in the four Sichuan, Yunnan, Guizhou and Guangxi provinces of China. Generally, the seeds of orchid plants are very fine, about 0.05-6.0 mm long and about 0.01-0.9 mm wide, and the number of seeds contained in each fruit varies from thousands of seeds to millions of seeds, but because the seeds do not contain endosperm, the germination rate is very low under natural conditions. Traditionally, orchid species are propagated in a plant division mode mostly, but the efficiency is not high. Since the saprophytic orchid needs to grow by means of nutrient symbiosis with fungi, the separate propagation is difficult. The rotten orchid has important medicinal value, but the field resource storage amount is not large due to the rotten property of the rotten orchid. At present, only gastrodia elata among saprophytic species of orchidaceae realizes artificial propagation and creates great value, and artificial culture of other saprophytic orchids is not successful. With the change of ecological environment and the aggravation of interference of human activities, the habitat of the saprophytic orchid is extremely easy to be damaged, so that the saprophytic orchid is extremely easy to be extinct. "cymbidium" generally refers to species of cymbidium, such as cymbidium goeringii, cymbidium kanran, cymbidium ensifolium and the like, and the rhizobia macrorrhiza is the only saprophytic cymbidium in cymbidium which cannot be photoautotrophed, and the related research on the rhizobia macrorrhiza has important significance for further and deeply researching the species evolution relationship, the gene function defect, the new species breeding and the resource utilization of the cymbidium. The method solves the problems of resource protection and utilization of saprophytic orchids and the like, and the key is to solve the problems of non-symbiotic germination, multiplication culture, in-vitro preservation and the like of seeds of the saprophytic orchids.
Disclosure of Invention
In view of the above, the present invention aims to provide a rapid propagation and in vitro preservation method for saprophytic orchid, which realizes rapid propagation by artificial tissue culture and in vitro preservation of germplasm resources, and provides technical support for subsequent resource research, development, sustainable utilization, germplasm resource exchange, and the like.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a rapid propagation and in vitro preservation method of saprophytic orchid, which comprises the following steps: (1) carrying out artificial cross pollination on the flowering saprophytic orchid, and selecting an uncracked pod which is 160-200 days after pollination as an explant;
(2) stripping off the washed and disinfected explants, spreading the seeds on the surface of a sterile germination culture medium for dark culture, and growing rhizoids to obtain germinated seeds; the sterile germination medium comprises an aqueous solution of: 3g/L Huabao 1, 60.5mg/L vitamin B, 10.1mg/L vitamin B, 0.5mg/L nicotinic acid, 2mg/L glycine, 100mg/L, NAA 0.1.1-0.5 mg/L inositol, 50-100 mL/L coconut juice, 20g/L sucrose and 5.6g/L agar;
(3) when the rhizome of the germinated seed grows to 0.8-1.2 cm, transferring the rhizome to a rhizome induction and multiplication culture medium for dark culture to obtain a rapid propagation seedling; the rhizome induction and proliferation culture medium comprises an aqueous solution of the following components: 3g/L Huabao 1, 60.5mg/L vitamin B, 10.1mg/L vitamin B, 0.5mg/L nicotinic acid, 2mg/L glycine, 100mg/L inositol, 5-10 mg/L, KT 2 to 5 mg/L6-BA, 100 to 200mL/L coconut juice, 30g/L sucrose, 5.6g/L agar and 1g/L active carbon;
(4) transplanting the rhizomes of the germinated seeds or the rhizomes of the rapid propagation seedlings into an in vitro preservation culture medium for in vitro preservation, wherein the in vitro preservation culture medium comprises: 3g/L Huabao No. 1, 60.5mg/L vitamin B, 10.1mg/L vitamin B, 0.5mg/L nicotinic acid, 2mg/L glycine, 100mg/L, NAA 0.1.1-0.5 mg/L inositol, 20g/L sucrose and 5.6g/L agar.
Preferably, the saprophytic orchid in the step (1) comprises Cymbidium macrocarphizon.
Preferably, the cleaning and disinfecting of step (2) comprises: scrubbing the surface of the explant with gauze, soaking in 75 vol.% ethanol for 1min in sterile environment, and adding 0.2 wt.% HgCl2Soaking in water solution for 10min, and washing with sterile water for 5 times.
Preferably, the dark culture time in the step (2) is 50-120 d.
Preferably, the temperature of the dark culture in step (2) and step (3) is 25 +/-2 ℃.
Preferably, the temperature for the ex vivo preservation in step (4) is 25 ± 2 ℃.
The invention provides a rapid propagation and in vitro preservation method of saprophytic orchid, which takes a fruit pod obtained after artificial pollination as an object, obtains protocorm through disinfection and aseptic germination of seeds, induces the protocorm to obtain a pseudoroot (rhizome), further establishes an in vitro preservation system of the large cymbidium by taking the rhizome as the object, and the propagation coefficient in half a year reaches 1: 4, the in vitro preservation subculture period reaches about 2 years. The rapid propagation and in vitro preservation method effectively solves the problem of tissue culture and rapid propagation of saprophytic orchid, prolongs the in vitro preservation period, is favorable for further preservation, development and utilization of saprophytic orchid germplasm resources, and lays a foundation for future resource mining.
Drawings
FIG. 1 is a process of aseptic germination of Rohdea macrorrhiza seeds, wherein A represents Rohdea macrorrhiza seeds; b shows that the embryos of the cymbidium giganteum seeds begin to expand when the cymbidium giganteum seeds are cultured for 26 days; c represents that the embryo breaks through the seed coat when the embryo is cultured for 43 d; D/E shows that when cultured for 54D, rhizoid appears; f, differentiating a rhizome when the culture lasts for 120 days;
FIG. 2 shows sterile rhizomes of Rohdea macrorrhiza obtained by proliferation induction.
Detailed Description
The invention provides a rapid propagation and in vitro preservation method of saprophytic orchid, which comprises the following steps: (1) carrying out artificial cross pollination on the flowering saprophytic orchid, and selecting an uncracked pod which is 160-200 days after pollination as an explant;
(2) stripping off the washed and disinfected explants, spreading the seeds on the surface of a sterile germination culture medium for dark culture, and growing rhizoids to obtain germinated seeds; the sterile germination medium comprises an aqueous solution of: 3g/L Huabao 1, 60.5mg/L vitamin B, 10.1mg/L vitamin B, 0.5mg/L nicotinic acid, 2mg/L glycine, 100mg/L, NAA 0.1.1-0.5 mg/L inositol, 50-100 mL/L coconut juice, 20g/L sucrose and 5.6g/L agar;
(3) when the rhizome of the germinated seed grows to 0.8-1.2 cm, transferring the rhizome to a rhizome induction and multiplication culture medium for dark culture to obtain a rapid propagation seedling; the rhizome induction and proliferation culture medium comprises an aqueous solution of the following components: 3g/L Huabao 1, 60.5mg/L vitamin B, 10.1mg/L vitamin B, 0.5mg/L nicotinic acid, 2mg/L glycine, 100mg/L inositol, 5-10 mg/L, KT 2 to 5 mg/L6-BA, 100 to 200mL/L coconut juice, 30g/L sucrose, 5.6g/L agar and 1g/L active carbon;
(4) transplanting the rhizomes of the germinated seeds or the rhizomes of the rapid propagation seedlings into an in vitro preservation culture medium for in vitro preservation, wherein the in vitro preservation culture medium comprises: 3g/L Huabao No. 1, 60.5mg/L vitamin B, 10.1mg/L vitamin B, 0.5mg/L nicotinic acid, 2mg/L glycine, 100mg/L, NAA 0.1.1-0.5 mg/L inositol, 20g/L sucrose and 5.6g/L agar.
The method carries out artificial cross pollination on the flowering saprophytic orchid, and selects an uncracked pod which is 160-200 days after pollination as an explant. The saprophytic orchid of the present invention preferably comprises Cymbidium macrocarpozon. The method preferably selects strong Cymbidium macrocephalum (Cymbidium macrocephalon) flowering plants in the field for artificial cross pollination, more preferably selects flowers which are completely expanded for 1-2 d for pollination, picks pollen grains by adopting a sterilized bamboo toothpick to be adhered to the stigma of another flower, and then marks the pollen grains. In the invention, the fruit pod of the large-rooted orchid turns into light yellow green but does not crack after about 180 days after pollination, a large number of completely developed seeds are contained in the fruit pod, and the fruit pod at the period is taken as an explant of a subsequent experiment.
The method peels off the washed and disinfected explants, so that seeds are spread on the surface of a sterile germination culture medium for dark culture, and the germinated seeds are obtained after rhizoids grow; the sterile germination medium comprises an aqueous solution of: 3g/L Huabao No. 1, 60.5mg/L vitamin B, 10.1mg/L vitamin B, 0.5mg/L nicotinic acid, 2mg/L glycine, 100mg/L, NAA 0.1.1-0.5 mg/L inositol, 50-100 mL/L coconut juice, 20g/L sucrose and 5.6g/L agar. The cleaning and disinfecting of the present invention preferably comprises: scrubbing the surface of the explant with gauze, soaking in 75 vol.% ethanol for 1min in sterile environment, and adding 0.2 wt.% HgCl2Soaking in water solution for 10min, and washing with sterile water for 5 times. The sterile environment of the present invention is preferably an ultra clean bench. The invention preferably uses filter paper to absorb the surface moisture of the disinfected fruit pod, uses scissors to longitudinally split the fruit pod, shakes the fruit shell to spread the seeds on the surface of the prepared sterile germination culture mediumAnd (5) kneading. In the sterile germination culture medium, the coconut juice contains rich nutrition required by orchid seed germination, and can further promote seed germination and subsequent growth of rhizome by matching with macroelements such as Huabao No. 1 and the like and the use of the cell auxin NAA with the concentration. The method comprises the following steps of carrying out dark culture on seeds on the sterile germination culture medium, wherein the temperature of the dark culture is 25 +/-2 ℃, the dark culture is carried out by taking the cymbidium sinense as an example in the embodiment of the invention, and when the seeds are cultured for 26d, the cymbidium sinense starts to develop without breaking the skin; when the seeds are cultured for 43d, the seeds of the cymbidium giganteum begin to break the skin; when the cultivation lasts for 54 days, the large-rooted orchid seeds with broken peels begin to grow rhizoids, the germination rate is about 60%, and when the cultivation continues to last for 120 days, the protocorms are gradually differentiated into rootstocks.
When the rootstock of the germinated seed grows to 0.8-1.2 cm, transferring the rootstock to a rootstock induction and multiplication culture medium for dark culture to obtain a rapid propagation seedling; the rhizome induction and proliferation culture medium comprises an aqueous solution of the following components: 3g/L Huabao No. 1, 60.5mg/L vitamin B, 10.1mg/L vitamin B, 0.5mg/L nicotinic acid, 2mg/L glycine, 100mg/L inositol, 5-10 mg/L, KT 2 to 5 mg/L6-BA, 100 to 200mL/L coconut juice, 30g/L sucrose, 5.6g/L agar and 1g/L active carbon. The invention preferably transfers 1cm long rhizome to the induction and proliferation culture medium of the rhizome to carry out dark culture, and the temperature of the dark culture is preferably 25 +/-2 ℃. In the embodiment of the invention, the rhizomes of the cymbidium macrocephalum with the length of 1cm are transferred to a rhizomes induction and proliferation culture medium for about 1 month, so that the obvious extension of the rhizomes can be observed, the growth of bifurcate proliferation begins to appear, when the rhizomes are cultured for 6 months, the proliferation multiple of the rhizomes can reach more than 4 times, and at the moment, the rhizomes grow well and are not browned or withered. In the rhizoid induction and proliferation culture medium, two cytokinins KT and 6-BA with higher concentration are used and are matched with a large amount of coconut juice, so that rich nutrient requirements are provided for proliferation and elongation of rhizoids of cymbidium macrorrhizae.
The invention transplants the rhizomes of the germinating seeds or the rhizomes of the rapid propagation seedlings into an in vitro preservation culture medium for in vitro preservation, and the in vitro preservation culture medium comprises: 3g/L Huabao No. 1, 60.5mg/L vitamin B, 10.1mg/L vitamin B, 0.5mg/L nicotinic acid, 2mg/L glycine, 100mg/L, NAA 0.1.1-0.5 mg/L inositol, 20g/L sucrose and 5.6g/L agar.
The temperature of the in vitro preservation is 25 +/-2 ℃, the in vitro preservation is carried out by using the method, the survival rhizome can be transferred to an induction and multiplication culture medium of the rhizome after being preserved for 2 years, and a large amount of rhizome materials can be obtained.
The method for rapid propagation and in vitro preservation of saprophytic orchid provided by the invention is described in detail with reference to the following examples, but the method is not to be construed as limiting the scope of the invention.
Example 1
1. Obtaining of explants
In the field, flowering plants of strong Cymbidium macrocarpozon are selected for artificial cross pollination. Selecting flowers which are completely unfolded for 1-2 days for pollination in about the middle ten days of 7 months in the flowering period of the cymbidium macrocephalum in Kunming area, picking up pollen grains by adopting a sterilized bamboo toothpick, adhering the pollen grains to the stigma of another flower strain, and then marking. And after about 180 days after pollination, the fruit pod of the large-rooted orchid turns into light yellow green but does not crack, a large number of completely developed seeds are contained in the fruit pod, and the fruit pod at the period is taken as an explant of a subsequent experiment.
2. Sterilization and sterile germination of explants
Scrubbing the surface of the large root orchid pod which is not cracked with clean gauze, transferring the large root orchid pod into a superclean workbench, sterilizing the large root orchid pod for 1 minute by using 75% alcohol (volume fraction), and then sterilizing the large root orchid pod by using 0.2% (mass fraction) of HgCl2Soaking in the solution for 10min, shaking the container to sterilize, washing with sterile water for 5 times, drying the surface water of the sterilized fruit pod with filter paper, splitting the pod longitudinally with scissors, shaking the fruit shell to make the seed spread on the surface of the prepared seed sterile germination culture medium (containing Huabao No. 1 3g, vitamin B60.5 mg, vitamin B10.1mg, nicotinic acid 0.5mg, glycine 2.0mg, inositol 100mg, NAA 0.5mg, coconut juice 50mL, sucrose 20g, and agar 5.6g per liter). Culturing at 25 + -2 deg.C in dark, and culturing for 26 days until the seed of cymbidium macrocephalum begins to develop without breaking the peel; when the seeds are cultured for 43d, the seeds of the cymbidium giganteum begin to break the skin;when the seeds are cultured for 54 days, the broken rhizoid orchid seeds begin to grow rhizoid, and the germination rate is about 60 percent. When the cultivation is continued to 120 days, the protocorm gradually differentiates into a rhizome (FIG. 1).
3. Induction and proliferation of rhizomes
The rootstock of large orchid about 1cm long obtained by aseptic germination and growth of seeds is transferred to a rootstock induction and proliferation culture medium (containing Huabao No. 1 3.0g, vitamin B60.5 mg, vitamin B10.1mg, nicotinic acid 0.5mg, glycine 2.0mg, inositol 100mg, 6-BA 8.0mg, KT 2.0mg, 100.0mL of coconut milk, 3% sucrose, agar 5.6g and activated carbon 1.0g per liter). Around 1 month after transfer, significant elongation of the rootstock was observed and bifurcate proliferative growth began to occur (FIG. 2). When the culture is carried out for 6 months, the multiplication multiple of the rhizome can reach more than 4 times, and the rhizome grows well without browning or withering. Culturing at 25 + -2 deg.C in dark.
4. In vitro preservation
The rhizomes with the length of about 0.5cm are inoculated on an in vitro preservation culture medium (each liter contains No. 1 Huabao 3.0g, vitamin B60.5 mg, vitamin B10.1mg, nicotinic acid 0.5mg, glycine 2.0mg, inositol 100mg, NAA 0.5mg, cane sugar 20g and agar 5.6g), one end with the length of the rhizomes can continue to grow, but the other end can be browned. After 2 years of storage, the surviving rootstock is transferred to induction and propagation medium of the rootstock, and a large amount of rootstock material is obtained. Culturing at 25 + -2 deg.C in dark.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (6)
1. A fast propagation and in vitro preservation method of saprophytic orchid is characterized by comprising the following steps: (1) carrying out artificial cross pollination on the flowering saprophytic orchid, and selecting an uncracked pod which is 160-200 days after pollination as an explant;
(2) stripping off the washed and disinfected explants, spreading the seeds on the surface of a sterile germination culture medium for dark culture, and growing rhizoids to obtain germinated seeds; the sterile germination medium comprises an aqueous solution of: 3g/L Huabao 1, 60.5mg/L vitamin B, 10.1mg/L vitamin B, 0.5mg/L nicotinic acid, 2mg/L glycine, 100mg/L, NAA 0.1.1-0.5 mg/L inositol, 50-100 mL/L coconut juice, 20g/L sucrose and 5.6g/L agar;
(3) when the rhizome of the germinated seed grows to 0.8-1.2 cm, transferring the rhizome to a rhizome induction and multiplication culture medium for dark culture to obtain a rapid propagation seedling; the rhizome induction and proliferation culture medium comprises an aqueous solution of the following components: 3g/L Huabao 1, 60.5mg/L vitamin B, 10.1mg/L vitamin B, 0.5mg/L nicotinic acid, 2mg/L glycine, 100mg/L inositol, 5-10 mg/L, KT 2 to 5 mg/L6-BA, 100 to 200mL/L coconut juice, 30g/L sucrose, 5.6g/L agar and 1g/L active carbon;
(4) transplanting the rhizomes of the germinated seeds or the rhizomes of the rapid propagation seedlings into an in vitro preservation culture medium for in vitro preservation, wherein the in vitro preservation culture medium comprises: 3g/L Huabao No. 1, 60.5mg/L vitamin B, 10.1mg/L vitamin B, 0.5mg/L nicotinic acid, 2mg/L glycine, 100mg/L, NAA 0.1.1-0.5 mg/L inositol, 20g/L sucrose and 5.6g/L agar.
2. The rapid propagation and ex vivo preservation method according to claim 1, wherein the saprophytic orchid of step (1) comprises Cymbidium macrocarpazon.
3. The rapid propagation and ex vivo preservation method according to claim 1, wherein the washing and sterilizing of step (2) comprises: scrubbing the surface of the explant with gauze, soaking in 75 vol.% ethanol for 1min in sterile environment, and adding 0.2 wt.% HgCl2Soaking in water solution for 10min, and washing with sterile water for 5 times.
4. The rapid propagation and in vitro preservation method according to claim 1, wherein the dark culture time in step (2) is 50-120 d.
5. The rapid propagation and ex vivo preservation method according to claim 1, wherein the temperature of the dark culture in step (2) and step (3) is 25 ± 2 ℃.
6. The rapid propagation and ex vivo preservation method according to claim 1, wherein the temperature of the ex vivo preservation in the step (4) is 25 ± 2 ℃.
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| CN114190277A (en) * | 2021-12-07 | 2022-03-18 | 中国科学院昆明植物研究所 | Method for promoting blooming and fructification of large root orchid test tube |
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| GODO, TOSHINARI等: "The effects of illumination, temperature and 6-benzylaminoprine on asymbiotic seed germination andprotocorm development in vitro in the achlorophyllous orchid Gastrodia pubilabiata Sawa", 《THE EFFECTS OF ILLUMINATION, TEMPERATURE AND 6-BENZYLAMINOPRINE ON ASYMBIOTIC SEED GERMINATION ANDPROTOCORM DEVELOPMENT IN VITRO IN THE ACHLOROPHYLLOUS ORCHID GASTRODIA PUBILABIATA SAWA》 * |
| 孙崇波等: "蕙兰种子无菌萌发及植株再生 ", 《浙江农业学报》 * |
| 庞惠仙等: "大花蕙兰与奇花虎头兰杂交种子无菌萌发及成苗途径", 《西南林学院学报》 * |
| 曾宋君等: "墨兰及其杂种的组织培养与快速繁殖 ", 《广西植物》 * |
| 王卜琼等: "兰花育种研究进展", 《园艺学报》 * |
| 陈瑶瑶等: "杂交兰‘韩国桃花’×蕙兰种间杂交种子无菌萌发特征研究 ", 《园艺学报》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114190277A (en) * | 2021-12-07 | 2022-03-18 | 中国科学院昆明植物研究所 | Method for promoting blooming and fructification of large root orchid test tube |
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| Publication number | Publication date |
|---|---|
| CN112136687B (en) | 2022-02-18 |
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