CN111406652A - Tissue culture and rapid propagation method of drynaria fortunei seedlings by green spheroid (GGB) approach - Google Patents
Tissue culture and rapid propagation method of drynaria fortunei seedlings by green spheroid (GGB) approach Download PDFInfo
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- 238000013459 approach Methods 0.000 title description 5
- 230000000366 juvenile effect Effects 0.000 claims abstract description 46
- 230000004069 differentiation Effects 0.000 claims abstract description 40
- 230000035755 proliferation Effects 0.000 claims abstract description 27
- 238000012258 culturing Methods 0.000 claims abstract description 25
- 230000006698 induction Effects 0.000 claims abstract description 16
- 230000001954 sterilising effect Effects 0.000 claims abstract description 7
- 238000005286 illumination Methods 0.000 claims abstract description 5
- 239000001963 growth medium Substances 0.000 claims description 52
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 40
- 229930006000 Sucrose Natural products 0.000 claims description 40
- 239000005720 sucrose Substances 0.000 claims description 40
- 229920001817 Agar Polymers 0.000 claims description 30
- 239000008272 agar Substances 0.000 claims description 30
- 239000002609 medium Substances 0.000 claims description 21
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- 239000003599 detergent Substances 0.000 claims description 4
- 239000004744 fabric Substances 0.000 claims description 4
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 claims description 4
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- 239000006260 foam Substances 0.000 claims description 3
- 229960002523 mercuric chloride Drugs 0.000 claims description 3
- 230000034303 cell budding Effects 0.000 abstract description 4
- UQHKFADEQIVWID-UHFFFAOYSA-N cytokinin Natural products C1=NC=2C(NCC=C(CO)C)=NC=NC=2N1C1CC(O)C(CO)O1 UQHKFADEQIVWID-UHFFFAOYSA-N 0.000 abstract description 2
- 239000004062 cytokinin Substances 0.000 abstract description 2
- 241001116742 Drynaria Species 0.000 description 3
- 230000001939 inductive effect Effects 0.000 description 3
- 241000894007 species Species 0.000 description 3
- 241000350742 Aglaomorpha fortunei Species 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 241000985694 Polypodiopsida Species 0.000 description 2
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- 235000009936 Pteridium aquilinum Nutrition 0.000 description 2
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- DFPMSGMNTNDNHN-ZPHOTFPESA-N naringin Chemical compound O[C@@H]1[C@H](O)[C@@H](O)[C@H](C)O[C@H]1O[C@H]1[C@H](OC=2C=C3O[C@@H](CC(=O)C3=C(O)C=2)C=2C=CC(O)=CC=2)O[C@H](CO)[C@@H](O)[C@@H]1O DFPMSGMNTNDNHN-ZPHOTFPESA-N 0.000 description 1
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- 238000004161 plant tissue culture 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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Abstract
The invention provides a drynaria fortunei germchit tissue culture and rapid propagation method in a green spheroid (GGB) way, which is characterized by comprising the following steps: sterilizing explants; GGB induction; GGB proliferation culture; GGB differentiation culture; cutting a bud cluster; culturing juvenile sporophytes. After the drynaria fortunei GGB is obtained, in the propagation culture of GGB, the reduction of the propagation efficiency caused by the differentiation of GGB under the condition of illumination is obviously improved by reducing the use of cytokinin and using a dark condition, the propagation speed of GGB is increased, and the use cost of light energy is reduced. After the drynaria fortunei GGB is differentiated to generate a budding, the rhizome of the tender sporophyte obtained by splitting the budding is extremely small, and the time required for culturing the rhizome to reach the volume capable of being taken out of a bottle is long.
Description
Technical Field
The invention belongs to the technical field of plant tissue culture, and particularly relates to a method for obtaining drynaria fortunei tissue culture seedlings through a green spheroid approach.
Background
Green spherical bodies (GGBs) are a special structure found in fern tissue culture systems, usually spherical bodies consisting of numerous Green granules, and are therefore named. GGB can be continuously proliferated in the induction culture medium, and the culture medium can be rapidly differentiated into young seedlings by changing the components of the culture medium, and has the characteristics of high proliferation rate, rapid differentiation and short seedling period. Therefore, GGB has great development prospect for propagation of fern seedlings. At present, GGB tissue culture rapid propagation approaches are established for various ferns, such as platycerium (leaf beautiful and immortal, 2020), penholder tree (Zhang Yan, etc., 2020), osmunda japonica (pottery pellin and Gaokui, 2019), pteridium aquilinum (hujinyao, etc., 2018), and pteridium aquilinum (He jun, etc., 2016).
Drynaria roosii (Drynaria roosii), also called Shiyanjiang, Yanlian ginger, Hedychium spicatum, belongs to the middle-sized epiphytic fern of Drynariaceae (Drynariaceae) Drynariae. In China, the method is mainly distributed in Zhejiang, Fujian, Taiwan, Guangdong, Guangxi, Jiangxi, Hubei, Sichuan, Guizhou, Yunnan and other places. Drynaria rhizome is used as the rhizome of drynaria and is collected in the "Chinese pharmacopoeia" (Zhou Tong Shu and Zhou Rong Han, 1998). The content of main active ingredients (naringin and total flavone) in the rhizome of drynaria fortunei is higher than that of other similar plants (Zhoukui, etc., 1997; Zhouyanghan and kokuan, 2005), 70% of the drynaria rhizome medicinal materials sold on the market at present are from the rhizome of drynaria fortunei (Zhoukui, Zhouyanghan, 1998), however, the traditional drynaria fortunei medicinal materials are completely from the digging of wild drynaria fortunei, and the work of artificial propagation and cultivation of drynaria fortunei at home and abroad is still in the beginning stage. Due to the large market demand, wild drynaria fortunei resources are excessively exploited, and the living ecological environment of drynaria fortunei is also seriously damaged. In 2002, drynaria fortunei has been proposed to be listed in the national endangered list of protected plants (Zhang Xianchun, 2002). The research on the artificial breeding technology of the drynaria fortunei is helpful to promote the development of drynaria fortunei industry, protect drynaria fortunei wild resources and relieve the deterioration of ecological environment.
GGB of various ferns have been obtained by induction, but GGB of different species has certain differences in size, growth rate, external morphology, and suitable conditions for proliferation and differentiation vary from species to species. Work on artificial propagation of drynaria fortunei has been advanced, and patent "A propagation method of drynaria fortunei" (Shilei and xu Yan, 2008) reports a method for obtaining aseptic seedlings by sowing sterilized drynaria fortunei spores on a culture medium, which can rapidly obtain drynaria fortunei sporophytes but is limited by the activity of the spores, and still needs to collect fertile sporophylls of drynaria fortunei and properly store the collected spores. The GGB breeding method for establishing the drynaria fortunei germchit can break the limitation, but the method has not been reported yet. In addition, the drynaria fortunei tissue culture seedlings need to be transplanted out of bottles when the rhizome grows to be about 0.6-0.8 cm, the operation is time-consuming due to the fact that the rhizome is too small, the survival rate is low, and a technology for promoting rapid growth of the drynaria fortunei juvenile sporophyte in the bottles is not available at present.
Disclosure of Invention
In order to solve the problems, the invention provides a drynaria fortunei seedling tissue culture rapid propagation method in a green spheroid (GGB) way.
The invention relates to a drynaria fortunei germchit tissue culture rapid propagation method of a green spheroid (GGB) approach, which comprises the following steps: sterilizing explants; GGB induction; GGB proliferation culture; GGB differentiation culture; cutting a bud cluster; culturing juvenile sporophytes.
The explant used in the invention is a strong drynaria fortunei rhizome, preferably a rhizome which grows in the current season.
The method for disinfecting the drynaria fortunei rhizome explant comprises the steps of placing the rhizome in a detergent solution with the concentration of 5 g/L for soaking for 5-10 min, washing for 30-60 min under running water, taking out the rhizome after no foam is generated, sucking surface water with filter paper, soaking for 30s with 75% alcohol solution, quickly placing the rhizome in 0.1% mercury bichloride solution for surface disinfection for 6-8 min, and finally washing for 3-5 times with sterile water.
The induction method of the drynaria fortunei GGB in the invention comprises the steps of cutting the sterilized rhizome into small segments with the length of 0.5cm, inoculating the small segments into an induction culture medium, placing the culture medium in a culture room for culture, adding 2.0 mg/L6-BA, 30 g/L sucrose and 7 g/L agar into the induction culture medium by using 1/2MS as a basic culture medium, and adjusting the pH to 5.8-5.9 before high-temperature and high-pressure sterilization, wherein the explant generates obvious GGB in about 2 months;
the proliferation culture method of drynaria fortunei GGB comprises the steps of transferring GGB obtained by induction into a GGB proliferation culture medium, placing the culture medium in a culture chamber for light-proof culture, wherein the proliferation culture medium is 1/2MS + 1.0-2.0 mg/L6-BA + 3% sucrose + 0.7% agar, cutting GGB after 60-80 days, re-inoculating the cut and re-inoculated culture medium, and the proliferation culture time is overlong GGB, so that the proliferation rate is reduced, and the differentiation rate is increased.
The differentiation culture and bud cluster segmentation method of drynaria fortunei GGB in the invention comprises the following steps: GGB in a proliferation state is inoculated into a differentiation medium which is 1/2MS + 3% sucrose + 0.7% agar and placed in a culture chamber for culture. After GGB has differentiated to generate a large number of cluster buds, the cluster buds are divided and subcultured in a differentiation medium. GGB is in proliferation state for a long time, the cluster bud generated by first differentiation has small volume and is not easy to separate, and also has small amount of proliferation growth, and independent juvenile sporophyte can be obtained by multiple division culture.
The method for culturing the drynaria fortunei juvenile sporophyte comprises the following steps: inoculating the obtained independent juvenile sporophyte into an MS + 3% sucrose + 0.7% agar culture medium, transferring to a basic culture medium containing 6-8% sucrose after 20-40 days, placing in a culture chamber for culturing for 20-30 days, then transferring the juvenile sporophyte into an MS + 3% sucrose + 0.7% agar culture medium, carrying out generation once after 2 months, and after 4 months, the juvenile sporophyte rhizome is about 0.6-0.8 cm long, and the juvenile sporophyte has leaves and adventitious roots and can be taken out of a bottle.
The culture conditions in the invention are that the temperature of the culture room is 24 +/-1 ℃, and the illumination intensity is 50-60 mu mo L/(m)2S) photoperiod 14/10h, relative humidity 30%. In GGB proliferation culture, the light-proof treatment requires that the culture dish is covered with tinfoil paper or double-layer black cloth.
In the invention, the independent juvenile sporophytes are obtained from 3-4 times of cutting subculture after GGB differentiation in a proliferation state to generate cluster buds, and the period of 80-120 days is about 150-170 days for the independent juvenile sporophytes to develop into seedlings capable of coming out of bottles. After the drynaria fortunei GGB is obtained, in the propagation culture of GGB, the reduction of the propagation efficiency caused by the differentiation of GGB under the condition of illumination is obviously improved by reducing the use of cytokinin and using a dark condition, the propagation speed of GGB is increased, and the use cost of light energy is reduced.
Drynaria fortunei grows stolonically, the rhizome has growing points and stores nutrient substances, and the seedlings in bottles can survive without leaves or roots, but the rhizome is essential. After the drynaria fortunei GGB is differentiated to generate a budding, the rhizome of the tender sporophyte obtained by cutting the budding is extremely small, and the time for culturing the rhizome to reach the volume which can be taken out of a bottle is long. According to the invention, the juvenile sporophytes are treated by adding sucrose with higher concentration into the culture medium, so that the growth speed of the rhizomes at the later stage is greatly increased, and the propagation period of the seedlings is further shortened.
Detailed Description
The method of the invention comprises the following steps: sterilizing explants; GGB induction; GGB proliferation culture; GGB differentiation culture; cutting a bud cluster; culturing juvenile sporophytes.
(1) And (3) disinfection of the explant, namely selecting the strong drynaria fortunei rhizome as the explant, soaking the rhizome in a detergent solution with the concentration of 5 g/L for 5-10 min, washing the rhizome for 30-60 min under running water, taking the rhizome out after no foam is generated, sucking surface water by using filter paper, soaking the rhizome in a 75% alcohol solution for 30s, quickly placing the rhizome in a 0.1% mercuric chloride solution for surface disinfection for 6-8 min, and finally washing the rhizome for 3-5 times by using sterile water.
(2) GGB inducing, cutting sterilized rhizome into 0.5cm pieces, inoculating to the inducing culture medium, and culturing in culture room, wherein 1/2MS +2.0 mg/L6-BA + 3% sucrose + 0.7% agar is used as inducing culture medium.
(3) GGB transferring the induced GGB to a proliferation culture medium, and culturing in a culture room in a dark place, wherein the proliferation culture medium is 1/2MS + 1.0-2.0 mg/L6-BA + 3% sucrose + 0.7% agar.
(4) GGB differentiation culture and shoot division: GGB was inoculated into a differentiation medium 1/2MS + 3% sucrose + 0.7% agar and cultured in a culture chamber. After GGB differentiation produces a large number of cluster buds, the cluster buds are repeatedly cut and subcultured to a differentiation medium.
(5) Culturing juvenile sporophytes: inoculating the obtained independent juvenile sporophyte into an MS + 3% sucrose + 0.7% agar culture medium, transferring to a basic culture medium containing 6-8% sucrose after 30 days, placing in a culture room for culturing for 20 days, and then transferring the juvenile sporophyte to the MS + 3% sucrose + 0.7% agar culture medium for continuous culture.
The conditions of the culture room comprise that the temperature is 24 +/-1 ℃, and the illumination intensity is 50-60 mu mo L/(m)2S), photoperiod 14/10h, relative humidity 30%. In GGB proliferation culture, the light-proof treatment requires that the culture dish is covered with tinfoil paper or double-layer black cloth.
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Example 1
(1) And (3) sterilizing the explant, namely selecting the strong drynaria fortunei rhizome as the explant, soaking the rhizome in a detergent solution with the concentration of 5 g/L for 8min, washing the rhizome under running water for 60min, taking out the rhizome, sucking surface water by using filter paper, soaking the rhizome in a 75% alcohol solution for 30s, quickly placing the rhizome in a 0.1% mercuric chloride solution for surface sterilization for 6min, and finally washing the rhizome with sterile water for 5 times.
(2) GGB, using 1/2MS +2.0 mg/L6-BA + 3% sucrose + 0.7% agar as an induction culture medium, cutting the sterilized rhizome in the step (1) into small sections with the length of 0.5cm, inoculating the small sections into the induction culture medium, and placing the small sections into a culture room for culture, wherein a small amount of GGB with the diameter of 0.1-0.2 cm can be seen in 55d, and the induction rate of the GGB is 35%.
(3) GGB, transferring the GGB obtained in the step (2) into a GGB proliferation culture medium, and placing the culture medium in a culture room for light-proof culture, wherein the light-proof culture condition is that a tin foil paper is covered on a culture dish, the proliferation culture medium is 1/2MS +1.5 mg/L6-BA + 3% sucrose + 0.7% agar, the average fresh weight of GGB is 50.2mg after 30 days, the differentiation rate is about 28%, and the average fresh weight of GGB is 81.3mg after 60 days, the differentiation rate is about 45%.
(4) GGB differentiation culture and shoot division: inoculating GGB proliferated in the step (3) into a differentiation medium, and culturing in a culture chamber, wherein the differentiation medium is 1/2MS + 3% sucrose + 0.7% agar. GGB differentiate to generate a large number of cluster buds after 30 days, wherein the cluster buds are different in size, and each bud cluster is cut into 4-5 clusters and subcultured in a differentiation medium. Every 30 days, it was again split and subcultured. Obtaining independent juvenile sporophytes after 3 times of segmentation.
(5) Culturing juvenile sporophytes: inoculating the independent juvenile sporophytes obtained in the step (4) into an MS + 3% sucrose + 0.7% agar culture medium, transferring into a minimal medium containing 6% sucrose after 30 days, placing in a culture chamber for culturing for 20 days, then transferring the juvenile sporophytes into an MS + 3% sucrose + 0.7% agar culture medium, subculturing into the same culture medium after 2 months, and after 4 months, the average fresh weight of the juvenile sporophytes is 0.64g, wherein the average fresh weight of the rhizomes is about 0.35 g.
Example 2
(1) Same as example 1, step (1).
(2) Same as example 1, step (2).
(3) GGB culturing under dark condition by transferring GGB obtained in step (2) to GGB proliferation culture medium, covering double-layer black cloth on the culture dish, wherein the proliferation culture medium is 1/2MS +2.0 mg/L6-BA + 3% sucrose + 0.7% agar, the average fresh weight of GGB is 51.6mg after 30 days, the differentiation rate is about 27%, and the average fresh weight of GGB is 88.4mg after 60 days, the differentiation rate is about 32%.
(4) GGB differentiation culture and shoot division: inoculating GGB proliferated in the step (3) into a differentiation medium, and culturing in a culture chamber, wherein the differentiation medium is 1/2MS + 3% sucrose + 0.7% agar. GGB differentiate to generate a large number of cluster buds after 30 days, wherein the cluster buds are different in size, and each bud cluster is cut into 4-5 clusters and subcultured in a differentiation medium. Every 30 days, it was again split and subcultured. Obtaining independent juvenile sporophytes after 4 times of segmentation.
(5) Culturing juvenile sporophytes: inoculating the independent juvenile sporophytes obtained in the step (4) into an MS + 3% sucrose + 0.7% agar culture medium, transferring into a basic culture medium containing 8% sucrose after 30 days, placing in a culture chamber for culturing for 20 days, then transferring the juvenile sporophytes into an MS + 3% sucrose + 0.7% agar culture medium, subculturing into the same culture medium after 2 months, and after 4 months, the average fresh weight of the juvenile sporophytes is 0.77g, wherein the average fresh weight of the rhizomes is about 0.42 g.
Comparative example 1
(1) Same as example 1, step (1).
(2) Same as example 1, step (2).
(3) GGB proliferation culture, wherein the GGB obtained in the step (2) is transferred to GGB proliferation culture medium, and the proliferation culture medium is 1/2MS +1.5 mg/L6-BA + 3% sucrose + 0.7% agar, the average fresh weight of GGB is 58.2mg after 30 days, the differentiation rate is about 78%, the average fresh weight of GGB.1 mg after 60 days, and the differentiation rate is 100%.
(4) GGB differentiation culture and shoot division: inoculating GGB proliferated in the step (3) into a differentiation medium, and culturing in a culture chamber, wherein the differentiation medium is 1/2MS + 3% sucrose + 0.7% agar. GGB differentiate to generate a large number of cluster buds after 30 days, wherein the cluster buds are different in size, and each bud cluster is cut into 4-5 clusters and subcultured in a differentiation medium. Every 30 days, it was again split and subcultured. Independent juvenile sporophytes are obtained after 4 times of segmentation, juvenile sporophytes can be obtained during each segmentation, and the more the segmentation times are, the more the number of the obtained juvenile sporophytes is.
(5) Culturing juvenile sporophytes: and (3) inoculating the independent juvenile sporophytes obtained in the step (4) into an MS + 3% sucrose + 0.7% agar culture medium, transferring to the MS + 3% sucrose + 0.7% agar culture medium after 30 days, subculturing to the same culture medium after 3 months, wherein the average fresh weight of the juvenile sporophytes is 0.49g after 4 months, and the average fresh weight of the rhizomes is about 0.24 g.
Comparative example 2
(1) Same as example 1, step (1).
(2) Same as example 1, step (2).
(3) Same as example 2, step (3).
(4) Same as example 2, step (4).
(5) Culturing juvenile sporophytes: and (3) inoculating the independent juvenile sporophytes obtained in the step (4) into MS + 3% sucrose + 0.7% agar culture medium, respectively inoculating the juvenile sporophytes into MS + 0.7% agar culture medium containing 3%, 6% and 9% sucrose after 30 days, placing the inoculated juvenile sporophytes into a culture chamber for culture without subculture, weighing the fresh weight of the juvenile sporophytes in each culture medium by using an analytical balance after 80 days, and then removing the leaves and roots of the juvenile sporophytes to weigh the fresh weight of the rhizome. As a result, it was found that the fresh weight of juvenile sporophytes in the medium containing 3% sucrose was 223.8mg on average, wherein the fresh weight of the rootstock was about 72.8mg on average; the average fresh weight of juvenile sporophytes in the 6% sucrose containing medium was 84.0mg, and the average fresh weight of rhizomes was about 30.3 mg; the average fresh weight of juvenile sporophytes in the 9% sucrose medium was 43.2mg and the average fresh weight of rhizomes was about 19.2 mg. The results show that juvenile sporophytes grow stunted and the rhizomes grow slowly in the medium containing sucrose at a higher concentration for a long period of time.
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 technical principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (8)
1. A tissue culture and rapid propagation method of drynaria fortunei seedlings by a green spheroid (GGB) way is characterized by comprising the following steps: sterilizing explants; GGB induction; GGB proliferation culture; GGB differentiation culture; cutting a bud cluster; culturing juvenile sporophytes.
2. The tissue culture and rapid propagation method of drynaria fortunei seedlings as claimed in claim 1, wherein the disinfection of the explant comprises the steps of: selecting a strong drynaria fortunei rhizome as an explant, placing the rhizome in a detergent solution for soaking for 5-10 min, washing for 30-60 min under running water, taking out after no foam is generated, sucking surface water by using filter paper, soaking for 30s by using a 75% alcohol solution, quickly placing in a 0.1% mercuric chloride solution for surface disinfection for 6-8 min, and finally washing for 3-5 times by using sterile water.
3. The drynaria fortunei germchit tissue culture and rapid propagation method of claim 2, wherein the drynaria fortunei rhizome as the explant is the annual rhizome in the growing season.
4. The tissue culture and rapid propagation method of drynaria fortunei germchit of claim 1, wherein the induction of GGB comprises the steps of cutting the sterilized rhizome into 0.5cm long segments with 1/2MS +2.0 mg/L6-BA + 3% sucrose + 0.7% agar as the induction culture medium, inoculating to the induction culture medium, and culturing in a culture room.
5. The tissue culture and rapid propagation method of drynaria fortunei germchit of claim 1, wherein the propagation culture of GGB comprises the steps of transferring the induced GGB into a propagation culture medium, and culturing in a culture room in the absence of light, wherein the propagation culture medium is 1/2MS + 1.0-2.0 mg/L6-BA + 3% sucrose + 0.7% agar.
6. The drynaria fortunei germchit tissue culture and rapid propagation method of claim 1, wherein the differentiation culture and bud cluster segmentation of GGB comprises the following steps: GGB is inoculated in a differentiation medium, the differentiation medium is 1/2MS + 3% sucrose + 0.7% agar, after GGB differentiates to generate a large amount of cluster buds, the cluster buds are repeatedly cut and subcultured in the differentiation medium.
7. The tissue culture and rapid propagation method of drynaria fortunei seedlings as claimed in claim 1, wherein the juvenile sporophyte culture comprises the following steps: inoculating the obtained independent juvenile sporophyte into an MS + 3% sucrose + 0.7% agar culture medium, transferring to a basic culture medium containing 6-8% sucrose after 20-40 days, placing in a culture chamber for culturing for 20-30 days, and then transferring the juvenile sporophyte into an MS + 3% sucrose + 0.7% agar culture medium for continuous culture.
8. The tissue culture and rapid propagation method of drynaria fortunei seedlings according to any of claims 1 to 7, characterized in that the culture conditions are that the temperature of the culture room is 24 ± 1 ℃, and the illumination intensity is 50 to 60 μmo L/(m)2S) with a light cycle of 14/10h and a relative humidity of 30%, the culture dish is covered with tinfoil paper or double-layer black cloth in the light-shielding treatment in GGB proliferation culture.
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