CN117089509B - Tissue culture medium and method for inducing primordium by drynaria fortunei spores and application - Google Patents
Tissue culture medium and method for inducing primordium by drynaria fortunei spores and application Download PDFInfo
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0018—Culture media for cell or tissue culture
- C12N5/0025—Culture media for plant cell or plant tissue culture
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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
- A01H4/002—Culture media for tissue culture
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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
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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Abstract
The invention belongs to the technical field of medicinal plant spore breeding, and particularly relates to a tissue culture medium for inducing primordium by utilizing drynaria fortunei spores, a method and application. The invention provides a tissue culture medium for inducing protophylls by drynaria fortunei spores, which comprises a spore germination liquid medium and a solid proliferation medium; in the tissue culture medium provided by the invention, 6-BA (6-benzylaminopurine) stimulates cell division to promote spore germination and formation of protoleaf bodies; NAA (1-naphthylacetic acid) promotes spore germination and primordial leaf growth; GA 3 (gibberellin) promotes spore germination, so that spore germination time is shortened; 2,4-D (2, 4-dichlorophenoxyacetic acid) promotes the formation of protoleaf bodies. Sucrose provides an energy source for spore germination and formation of protophylls. The method is simple and easy to operate, and can reduce the damage to the original plants caused by the adoption of the split propagation and reduce the production cost.
Description
Technical Field
The invention belongs to the technical field of medicinal plant spore breeding, and particularly relates to a tissue culture medium for inducing primordium by utilizing drynaria fortunei spores, a method and application.
Background
Rhizoma Drynariae, also called "Drynariae" (Drynaria roosii Nakaike), is a plant of the genus Drynanthaceae (DRYNARIACEAE) of the genus Drynanthaceae (Drynaria). The total of the plants of the genus 8 is about 100, the plants of the family drynariaceae planted in China are about more than 5, about 30, distributed in tropical to ocean tropical areas, and mainly produced in Zhejiang, hubei, guangdong, guangxi, sichuan and other places in China.
Rhizoma Drynariae root is used as medicine, is rich in flavone, alkaloid, phenols, etc., and has effects of dispelling blood stasis, relieving pain, promoting reunion of bone, and treating toothache, lumbago, chronic diarrhea, etc. In recent years, natural resources are gradually exhausted due to blind excavation, and wild resources are seriously damaged. In 2002, dryopteris has been suggested to be listed in the national endangered protection plant directory. The drynariaceae plants are attached to rocks or tall trunks, the requirements on environmental conditions are high, the suitable temperature range, humidity range and soil pH value range are narrow, survival is difficult in introduction and cultivation, and great difficulty is brought to germplasm preservation and utilization.
At present, no artificial large-scale drynaria rhizome is planted, so that the adoption of the tissue culture, induced breeding and seedling of drynaria rhizome spores is urgently needed to be quickened to realize artificial cultivation, and the problem of shortage of sources of traditional Chinese medicine drynaria rhizome raw materials is solved.
The artificial seedling propagation of the drynaria fortunei in the prior art mainly comprises drynaria fortunei tubers, drynaria fortunei gametophytes and drynaria fortunei callus, and the drynaria fortunei spores are used as explants for carrying out the study of the artificial seedling culture of the drynaria fortunei, so that the cultivation period is long. For the artificial seedling raising of drynaria fortunei by taking drynaria fortunei spores as explants, the generation of the primordium leaf body is very important to the spore tissue culture of the drynaria fortunei, so that the development of a tissue culture medium for the drynaria fortunei spores to induce the primordium leaf body is urgently needed to shorten the cultivation period.
Disclosure of Invention
In order to solve the problems, the invention aims to provide a tissue culture medium and a method for inducing the primordium of the dryopteris fragrans spores and application thereof, which can effectively promote the formation of the primordium of the dryopteris fragrans and shorten the period of culturing the dryopteris fragrans spores into the primordium.
The invention provides a tissue culture medium for inducing protophylls by drynaria fortunei spores, which comprises a spore germination liquid medium and a solid proliferation medium;
The spore germination liquid medium is based on 1/2MS, and further comprises: 0.4-0.6 mg/L6-BA, 0.01-0.15 mg/L GA 3, 0.05-0.15 mg/L NAA, 25-35 g/L sucrose and 90-110U/mL antibiotics;
the solid multiplication medium is based on MS, and further comprises: 0.4-0.6 mg/L6-BA, 0.4-0.6 mg/L NAA, 0.9-1.1 mg/L2, 4-D, 25-35 g/L sucrose, 6.5-7.5 g/L agar and 90-110U/mL antibiotics.
The invention provides a method for inducing primordia by drynaria fortunei spores, which comprises the following steps: inoculating the sterilized dryopteris fragrans spores into the spore germination liquid culture medium in the technical scheme, and culturing until the spores germinate to form flocculus, so as to obtain flocculus tissues;
And transferring the flocculent tissue into the solid multiplication medium according to the technical scheme, and performing induction culture to generate a protoleaf body.
Preferably, the method for obtaining the drynaria fortunei spores comprises the following steps: collecting wild fresh dryopteris fragrans leaves, and scraping sporangia groups on the leaves by a sterile tool.
Preferably, the method for sterilizing the dryopteris spores comprises the following steps: sterilizing with 70% alcohol for 9-11 s, and washing with sterile water for more than 10 times; then sterilizing with 0.1% HgCl 2 -7 min, and washing with sterile water for more than 10 times.
Preferably, the temperature of the culture is 18-22 ℃; the culture time is 15-23 d.
Preferably, the culturing is performed in an alternate light and dark environment; the time of illumination culture in each light-dark alternation period is 12-14 h/d, and the time of dark culture is 8-12 h/d; the illumination intensity is 1500-2500 lx during illumination culture.
Preferably, the temperature of the induction culture is 18-22 ℃; the time of the induction culture is 20-25 d.
Preferably, the induction culture is performed in an alternate light and dark environment; the time of illumination culture in each light-dark alternation period is 12-14 h/d, and the time of dark culture is 8-12 h/d; the illumination intensity is 1500-2500 lx during illumination culture.
The invention provides application of the tissue culture medium or the method in at least one of the following technical scheme:
(1) Shortening the time for the drynaria fortunei spores to germinate to form flocculent tissues;
(2) Shortens the time for forming protoleaf bodies by the proliferation of the flocculent tissue of the drynaria fortunei.
The invention has the beneficial effects that: the invention provides a tissue culture medium for inducing protophylls by drynaria fortunei spores, which comprises a spore germination liquid medium and a solid proliferation medium; the spore germination liquid medium is based on 1/2MS, and further comprises: 0.4-0.6 mg/L6-BA, 0.01-0.15 mg/L GA 3, 0.05-0.15 mg/L NAA, 25-35 g/L sucrose and 90-110U/mL antibiotics; the solid multiplication medium is based on MS, and further comprises: 0.4-0.6 mg/L6-BA, 0.4-0.6 mg/L NAA, 0.9-1.1 mg/L2, 4-D, 25-35 g/L sucrose, 6.5-7.5 g/L agar and 90-110U/mL antibiotics. In the tissue culture medium provided by the invention, 6-BA (6-benzylaminopurine) stimulates cell division to promote spore germination and formation of protoleaf bodies; NAA (1-naphthylacetic acid) promotes spore germination and primordial leaf growth; GA 3 (gibberellin) promotes spore germination, so that spore germination time is shortened; 2,4-D (2, 4-dichlorophenoxyacetic acid) promotes the formation of protoleaf bodies. Sucrose provides an energy source for spore germination and formation of protophylls. The results of the embodiment show that the drynaria fortunei spores can germinate into green flocculent tissue for 15-23 days, the flocculent tissue obtained can form protoleaf bodies for 20 days, the time for forming the protoleaf bodies is shorter than that of a conventional culture medium, and the pollution rate of the protoleaf bodies is lower. The method is simple and easy to operate, and can reduce the damage to the original plants caused by the adoption of the split propagation and reduce the production cost.
Drawings
FIG. 1 is a diagram of a floc tissue of a dryopteris crassifolia group induced by germination of liquid culture spores;
FIG. 2 is a schematic diagram of a first group of parallel experimental drynaria fortunei sporulation primordium cultures of example 4, 20 d;
FIG. 3 is a schematic diagram of a second group of parallel experiment of a group of drynaria fortunei floc tissue to form a protophyll culture 20d of example 4;
FIG. 4 is a schematic diagram of the first group of parallel experiments of example 4 showing the flocculent tissue formation of the dryopteris crassifolia culture at 20d under a stereoscopic microscope.
Detailed Description
The invention provides a tissue culture medium for inducing primordium by drynaria fortunei spores, which comprises a spore germination liquid medium and a solid proliferation medium;
The spore germination liquid medium is based on 1/2MS, and further comprises: 0.4-0.6 mg/L6-BA, 0.01-0.15 mg/L GA 3, 0.05-0.15 mg/L NAA, 25-35 g/L sucrose and 90-110U/mL antibiotics;
the solid multiplication medium is based on MS, and further comprises: 0.4-0.6 mg/L6-BA, 0.4-0.6 mg/L NAA, 0.9-1.1 mg/L2, 4-D, 25-35 g/L sucrose, 6.5-7.5 g/L agar and 90-110U/mL antibiotics.
In the invention, the spore germination liquid culture medium is based on 1/2MS, and further comprises: 0.4-0.6 mg/L6-BA, 0.01-0.15 mg/L GA 3, 0.05-0.15 mg/LNAA, 25-35 g/L sucrose and 90-110U/mL antibiotics; preferably, the medium is based on 1/2MS and further comprises only: 0.4-0.6 mg/L6-BA, 0.01-0.15 mg/L GA 3, 0.05-0.15 mg/L NAA, 25-35 g/L sucrose and 90-110U/mL antibiotics. The concentration of 6-BA in the spore germination liquid culture medium provided by the invention is 0.4-0.6 mg/L, and more preferably 0.5mg/L. The concentration of GA 3 in the spore germination liquid culture medium provided by the invention is 0.01-0.15 mg/L, more preferably 0.05-0.12 mg/L, and even more preferably 0.1mg/L. The concentration of NAA in the spore germination liquid culture medium provided by the invention is 0.05-0.15 mg/L, and more preferably 0.1mg/L. The concentration of sucrose in the spore germination liquid culture medium provided by the invention is 25-35 g/L, and more preferably 30g/L.
The concentration of the antibiotics in the spore germination liquid culture medium provided by the invention is 90-110U/mL, and more preferably 100U/mL.
The spore germination liquid medium provided by the invention has the advantage that the 6-BA and GA 3 with higher concentration and NAA with lower concentration are matched, so that the germination of spores can be promoted. According to the spore germination liquid culture medium, agar is not added, so that spores can be completely immersed in the culture medium, germination time is shortened, and meanwhile, 100U/mL of antibiotics with final concentration are added into the culture medium, so that bacterial pollution caused by experimental operation can be effectively prevented.
In the present invention, the solid multiplication medium is an MS-based medium, further comprising: 0.4-0.6 mg/L6-BA, 0.4-0.6 mg/L NAA, 0.9-1.1 mg/L2, 4-D, 25-35 g/L sucrose, 6.5-7.5 g/L agar and 90-110U/mL antibiotics; preferably, the MS-based medium further comprises only: 0.4-0.6 mg/L6-BA, 0.4-0.6 mg/L NAA, 0.9-1.1 mg/L2, 4-D, 25-35 g/L sucrose, 6.5-7.5 g/L agar and 90-110U/mL antibiotics.
The concentration of 6-BA in the solid multiplication medium provided by the invention is 0.4-0.6 mg/L, more preferably 0.5mg/L; NAA concentration in the solid value-added culture medium provided by the invention is 0.4-0.6 mg/L, and more preferably 0.5mg/L; the concentration of 2,4-D in the solid proliferation culture medium provided by the invention is 0.9-1.1 mg/L, more preferably 1mg/L; the concentration of sucrose in the solid value-added culture medium provided by the invention is 25-35 g/L sucrose, more preferably 30g/L sucrose; the concentration of agar in the solid value-added culture medium provided by the invention is 6.5-7.5 g/L agar, more preferably 7g/L agar; the concentration of the antibiotics in the solid value-added culture medium provided by the invention is 90-110U/mL, and more preferably 100U/mL.
The interaction of 6-BA, NAA and 2,4-D in the solid multiplication medium provided by the invention can effectively promote the growth of the protoleaf body, and shortens the time of the induction culture of the protoleaf body. In addition, the antibiotic with the final concentration of 100U/mL is added into the solid multiplication medium, so that bacterial pollution caused by experimental operation can be effectively prevented, and the success rate of culture is increased.
The sources of GA 3, 2,4-D, 6-BA, NAA, sucrose and agar are not particularly limited, and conventional commercial products can be adopted.
In the present invention, the antibiotic is preferably a penicillin-streptomycin solution comprising 100000 units of penicillin and 10mg of streptomycin per 1mL of active ingredient. The penicillin-streptomycin solution is diluted by 100 times for use. The source of the penicillin-streptomycin solution is not limited, and conventional commercial products are adopted, and biosharp biotechnological products are taken as examples in the invention.
The invention also provides a method for inducing the protophyll by the drynaria fortunei spores, which comprises the following steps: inoculating the sterilized dryopteris fragrans spores into the spore germination liquid culture medium in the technical scheme, and culturing until the spores germinate to form flocculus, so as to obtain flocculus tissues;
And transferring the flocculent tissue into the solid multiplication medium according to the technical scheme, and performing induction culture to generate a protoleaf body.
The invention inoculates the sterilized drynaria fortunei spores into the spore germination liquid culture medium in the tissue culture medium, and cultures until the spores germinate and form flocculus, thus obtaining flocculus tissue. The means of inoculation according to the invention is preferably sterile forceps.
In the present invention, the method for obtaining the drynaria fortunei spores preferably comprises: collecting fresh leaves of wild drynaria fortunei with sporangia groups, and scraping the sporangia groups on the back surfaces of the fresh leaves. The scraping according to the invention is preferably accomplished with a sterile tool. In an embodiment of the invention, the sterile tool is preferably a sterile scalpel. The scraping according to the invention is preferably carried out in a sterile bench. In the embodiment of the invention, the sterile workbench is preferably an ultra-clean workbench.
After obtaining the sporangia group, the invention preferably disinfects said sporangia group. The method for sterilizing the dryopteris spores of the invention preferably comprises the following steps: sterilizing with 70% alcohol for 9-11 s, and washing with sterile water for more than 10 times; then sterilizing by using HgCl 2 solution with the mass concentration of 0.1% for 5-7 min, and flushing by using sterile water for more than 10 times. The disinfection mode of the invention firstly uses alcohol with the volume concentration of 70 percent to disinfect for 9-11 s, more preferably 10s; more preferably, the washing is performed with sterile water more than 10 times, and more preferably 10 times; soaking in 0.1% mercuric chloride solution for 5-7 min, preferably 6min; finally, the washing is preferably performed with sterile water more than 10 times, more preferably 10 times.
The invention is not particularly limited to tools used in sterilization, and conventional tools are used. In embodiments of the present invention, the means for sanitizing applications preferably comprise a cell filter screen and a cell culture plate. The invention also preferably comprises sterilizing a 40 μm cell filter screen and a cell culture plate; the sterilization is carried out by immersing 40 μm cell filter screen and cell culture plate in 70-75% alcohol for 5min, sterilizing, and sterilizing by ultraviolet irradiation on ultra-clean bench for 30min.
After the sterilized drynaria fortunei spores are obtained, the sterilized drynaria fortunei spores are preferably inoculated into a spore germination liquid medium for culture, so that flocculent tissues are obtained. The temperature of the culture in the spore germination liquid culture medium is preferably 18-22 ℃, more preferably 20 ℃; the time of the cultivation is preferably 15 to 23 days, more preferably 15 to 18 days, still more preferably 15 days; the cultivation is preferably performed under an alternate light and dark environment, and the time of the light cultivation in each alternate light and dark period is preferably 12-14 h/d, more preferably 12h/d; the time of the dark culture is preferably 8 to 12 hours/d, more preferably 12 hours/d; the illumination intensity in the illumination culture of the invention is preferably 1500-2500 lx, more preferably 2000lx; the culture of the invention is preferably carried out until the spores germinate into green floccules. The green flocculent tissue is formed by aggregating a plurality of spores after germination.
The method comprises the steps of culturing to obtain flocculent tissue, transferring the flocculent tissue into the solid proliferation culture medium according to the technical scheme, and performing induction culture to obtain the protoleaf body.
The transfer method of the present invention preferably uses forceps to inoculate the mass of flocculent tissue onto the solid multiplication medium. According to the invention, after the floccule tissue is inoculated to the solid multiplication medium, the floccule material can be contacted with oxygen in the air while absorbing nutrient substances in the medium, so that the floccule tissue can be effectively promoted to grow into a protophyll body.
The induction culture temperature on the solid multiplication medium is preferably 18-22 ℃, more preferably 20-21 ℃; the time of the induction culture is preferably 20 to 25 days, more preferably 20 to 23 days, and still more preferably 20 days; the induction culture is preferably carried out in an environment with alternate light and dark, and the time of illumination culture in each alternate light and dark period is preferably 12-14 h/d, more preferably 12h/d; the time of the dark culture is preferably 8 to 12 hours/d, more preferably 12 hours/d; the illumination intensity during the illumination culture is preferably 1500 to 2500lx, more preferably 2000lx.
The invention also provides application of the tissue culture medium or the method in at least one of the following technical scheme:
(1) Shortening the time for the drynaria fortunei spores to germinate to form flocculent tissues;
(2) Shortens the time for forming protoleaf bodies by the proliferation of the flocculent tissue of the drynaria fortunei.
In the embodiment of the invention, the time for the dryopteris crassifolia spores to germinate to form the flocculent tissue is 15-23 d, the time for the dryopteris crassifolia flocculent tissue to proliferate to form the protoleaf body is 20d, and compared with the conventional seedling culture, the tissue culture time is shortened. The method is simple and easy to operate, and can reduce the damage to the original plants caused by the adoption of the split propagation and reduce the production cost.
The technical solutions provided by the present invention are described in detail below with reference to the drawings and examples for further illustrating the present invention, but they should not be construed as limiting the scope of the present invention.
The antibiotics in the following examples and comparative examples were penicillin-streptomycin solutions comprising 100000 units of penicillin and 10mg of streptomycin per 1mL of active ingredient. The penicillin-streptomycin solution is diluted by 100 times for use.
Examples 1 to 3 and comparative examples 1 to 8 used sporangia groups obtained from wild fresh drynaria leaves collected in the same batch.
Example 1
A method for inducing spore germination by adopting drynaria fortunei spores comprises the following steps:
(1) Collecting wild fresh leaf of Dryopteris, and growing sporangium group on leaf. The collected dryopteris fragrans leaves are put into kraft paper bags to be carried back, the leaves are placed on an ultra-clean workbench, and a sterile scalpel is used for carefully scraping off sporangia groups on the back surfaces of the leaves.
(2) Placing 40 μm cell filter screen and cell culture plate in 75% alcohol, soaking for 5min, taking out, placing on ultra-clean bench, and sterilizing with ultraviolet light for 30min.
(3) 3 Wells of the 6-well cell culture plate are numbered 1, 2,3, respectively. Placing the scraped-off drynaria fortunei spore cyst group in the step (1) into the sterilized cell filter screen in the step (2), placing the drynaria fortunei spore cyst group into a hole with the mark of 1 of a cell culture plate, pouring 70% alcohol into the hole with the volume concentration of 1, sterilizing for 10 seconds, and flushing spores with sterile water for 10 times. Then placing a cell filter screen containing sporangia groups into holes with the mark of 2 of a cell culture plate, then adding HgCl 2 solution with the mass concentration of 0.1% for disinfection for 5-7 min, and washing with sterile water for 10 times again. Then the cell filter screen containing sporangia is placed in the well labeled 3 of the cell culture plate (a small amount of sterile water is added dropwise).
(4) The sporangia group in the well labeled 3 of step (3) was inoculated into spore germination liquid medium with sterile forceps. The spore germination liquid culture medium comprises the following components: the 1/2 MS-based medium also contained only 0.5 mg/L6-BA, 0.1mg/L GA 3, 0.1mg/L NAA, 30g/L sucrose and 100U/mL antibiotics. The inoculated spore germination liquid culture medium is placed in a biochemical incubator for culture, the temperature is controlled to be 20 ℃, the illumination intensity is 2000lx, the illumination time is 12 hours, and the culture is carried out until the spores germinate into green floccules (see figure 1). The time and contamination rate of spore germination were recorded.
Example 2
The only difference is that the spore germination liquid medium is replaced with example 1: the basal medium based on 1/2MS also contains only: 0.5 mg/L6-BA, 0.15mg/L GA 3, 0.1mg/L NAA, 30g/L sucrose and 100U/mL antibiotic.
Example 3
The only difference is that the spore germination liquid medium is replaced with example 1: the basal medium based on 1/2MS also contains only: 0.5 mg/L6-BA, 0.05mg/L GA 3, 0.1mg/L NAA, 30g/L sucrose and 100U/mL antibiotic.
Comparative example 1
The only difference is that the spore germination solid medium is replaced with the spore germination liquid medium as in example 1, and the spore germination solid medium comprises the following components: the 1/2 MS-based medium also contained only 0.5mg/L6-BA, 0.1mg/L GA 3, 0.1mg/L NAA, 30g/L sucrose, 100U/mL antibiotics and 7g/L agar.
Comparative example 2
The only difference is that the spore germination liquid medium is replaced with example 1: the basal medium was 1/4 MS-based and contained only 0.5 mg/L6-BA, 0.1mg/L GA 3, 0.1mg/L NAA, 30g/L sucrose and 100U/mL antibiotics.
Comparative example 3
The only difference is that the spore germination liquid medium is replaced with example 1: the basal medium was 1/8 MS-based and contained only 0.5 mg/L6-BA, 0.1mg/L GA 3, 0.1mg/L NAA, 30g/L sucrose and 100U/mL antibiotics.
Comparative example 4
The only difference is that the spore germination liquid medium is replaced with example 1: the basal medium was 1/10 MS-based and contained only 0.5 mg/L6-BA, 0.1mg/L GA 3, 0.1mg/L NAA, 30g/L sucrose and 100U/mL antibiotics.
Comparative example 5
The only difference is that the spore germination liquid medium is replaced with example 1: MS-based medium also contained only 0.5 mg/L6-BA, 0.1mg/L GA 3, 0.1mg/L NAA, 30g/L sucrose and 100U/mL antibiotics.
Comparative example 6
The only difference is that the spore germination liquid medium is replaced with example 1: the B5-based medium also contained only 0.5 mg/L6-BA, 0.1mg/L GA 3, 0.1mg/L NAA, 30g/L sucrose and 100U/mL antibiotics.
Comparative example 7
The only difference is that the spore germination liquid medium is replaced with example 1: WPM-based medium also contained only 0.5 mg/L6-BA, 0.1mg/L GA 3, 0.1mg/L NAA, 30g/L sucrose and 100U/mL antibiotics.
Comparative example 8
The only difference is that the spore germination liquid medium is replaced with example 1: the 1/2 MS-based medium also contained only 0.5 mg/L6-BA, 0.1mg/L NAA, 30g/L sucrose and 100U/mL antibiotics.
The green mass floc time for the spores of the dryopteris crassifolia of examples 1 to 3 and comparative examples 1 to 8 to germinate is shown in Table 1.
TABLE 1 time and contamination Rate of germination of biological Dryopteris spores of examples 1-3 and comparative examples 1-8
Note that: spore germination was calculated from inoculation into the spore germination liquid medium.
As shown in the results of Table 1, the spore germination liquid medium and the culture method provided by the invention can effectively reduce the germination time and pollution rate of the dryopteris fragrans spores, can obtain the dryopteris fragrans spore germination flocculent tissue more quickly and effectively, and the culture process is simple and easy to implement, easy to produce and can greatly shorten the production time cost.
The same batch of the floc tissue obtained in example 1 was used in the experiments of example 4 and comparative examples 10 to 15 as follows.
Example 4
A method for inducing primordium by using drynaria fortunei spores comprises the following steps:
the flocculent mass obtained in example 1 was inoculated with forceps onto a solid multiplication medium consisting of MS-based medium, and containing only 0.5 mg/L6-BA, 0.5mg/L NAA, 1.0 mg/L2, 4-D, 30g/L sucrose, 7g/L agar and 100U/mL antibiotic for cultivation. Culturing in biochemical incubator at 20deg.C under 2000lx illumination for 12 hr to obtain herba Drynariae leaf body, and recording germination time as shown in Table 2.
Example 4 three parallel experiments were performed, and the primordium obtained by inoculating the group flocculent tissue onto a solid multiplication medium for culturing for 20d is shown in fig. 2 to 4, and fig. 2 is a schematic diagram of the primordium formed by culturing the group flocculent tissue of the first parallel experiment dryopteris fragrans for 20 d; FIG. 3 is a schematic diagram of a second group of parallel experimental drynaria fortunei spore group flocculent tissue formation protoleaf culture 20 d; the pictures of the primordia obtained by the first group of parallel experimental cultures 20d are shown in FIG. 4.
Comparative example 9
The only difference is that the solid multiplication medium is replaced by example 4: the medium was based on 1/2MS and contained only 0.1 mg/L6-BA, 0.05mg/L IAA, 30g/L sucrose and 7g/L agar.
Comparative example 10
The only difference is that the solid multiplication medium is replaced by example 4: the medium was based on 1/2MS and contained only 2,4-D1.0mg/L, 6-B0.5 mg/L, 30g/L sucrose and 7g/L agar.
Comparative example 11
The only difference is that the solid multiplication medium is replaced by example 4: 1/2MS based medium also contained only 0.5 mg/L6-BA, 0.1mg/L IBA, 0.1mg/L NAA, 30g/L sucrose, 7g/L agar and 100U/mL antibiotics.
Comparative example 12
The only difference is that the solid multiplication medium is replaced by example 4: 1/2MS based medium also contained only 0.5 mg/L6-BA, 0.1mg/L IBA, 0.1mg/L NAA, 30g/L sucrose, 7g/L agar and 100U/mL antibiotics.
Comparative example 13
The only difference is that the solid multiplication medium is replaced by example 4: 1/4MS based medium also contained only 0.5 mg/L6-BA, 0.5mg/L NAA, 1.0 mg/L2, 4-D, 30g/L sucrose, 7g/L agar and 100U/mL antibiotics.
Comparative example 14
The only difference is that the solid multiplication medium is replaced by example 4: the basal medium was 1/8 MS-based and contained only 0.5 mg/L6-BA, 0.5mg/L NAA, 1.0 mg/L2, 4-D, 30g/L sucrose, 7g/L agar and 100U/mL antibiotics.
Comparative example 15
The only difference is that the solid multiplication medium is replaced by example 4: the basal medium was 1/10 MS-based and contained only 0.5 mg/L6-BA, 0.5mg/L NAA, 1.0 mg/L2, 4-D, 30g/L sucrose, 7g/L agar and 100U/mL antibiotics.
The time and contamination rate of the formation of the protoleaf bodies from the spores of dryopteris fragrans of example 4 and comparative examples 9 to 15 are shown in Table 2.
TABLE 2 time and contamination Rate of formation of protoleaf bodies by floc tissue of Dryopteris sporidium in example 4 and comparative examples 9 to 15
Note that: the time to form protoleaf body was calculated from the inoculation of the mass of flocculent tissue.
As shown in the results of Table 2, the solid multiplication medium and the culture method provided by the invention effectively reduce the time and pollution rate of forming the original leaf body of the drynaria fortunei spores, and the culture process is simple and easy to implement, and easy to produce, and can greatly reduce the damage of the plant division propagation to the original plant and reduce the production cost.
In conclusion, the spore germination liquid medium and the solid proliferation medium provided by the invention can obviously reduce the time for forming the protophyll by the spores of the drynaria fortunei, the sporangium groups 15-23 d can germinate into green floccule tissues, and the obtained floccule tissues 20d can form the protophyll. The method is simple and easy to operate, and greatly reduces the damage and cost of the original plants caused by the adoption of the split propagation in production.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.
Claims (8)
1. A method for inducing a protophyll by a drynaria fortunei spore, comprising: inoculating the sterilized dryopteris fragrans spores into a spore germination liquid culture medium, and culturing until the spores germinate to form flocculus, thereby obtaining flocculus tissues; the spore germination liquid medium is based on 1/2MS, and further comprises: 0.4-0.6 mg/L6-BA, 0.01-0.15 mg/L GA 3, 0.05-0.15 mg/LNAA, 25-35 g/L sucrose and 90-110U/mL antibiotics; the illumination intensity of the culture is 1500-2500 lx;
Transferring the flocculent tissue into a solid proliferation culture medium, and performing induction culture to generate a protoleaf body; the solid multiplication medium is based on MS, and further comprises: 0.4-0.6 mg/L6-BA, 0.4-0.6 mg/L NAA, 0.9-1.1 mg/L2, 4-D, 25-35 g/L sucrose, 6.5-7.5 g/L agar and 90-110U/mL antibiotics;
The method for sterilizing the drynaria fortunei spores comprises the following steps: sterilizing with 70% alcohol for 9-11 s, and washing with sterile water for more than 10 times; sterilizing with HgCl 2 solution of 0.1% concentration for 5-7 min, and flushing with sterile water for over 10 times; the illumination intensity of the induced culture is 1500-2500 lx.
2. The method of claim 1, wherein the spore germination liquid medium is 1/2 MS-based medium further comprising only 0.5 mg/L6-BA, 0.1mg/LGA 3, 0.1mg/L NAA, 30g/L sucrose and 100U/mL antibiotic;
Or alternatively
The spore germination liquid culture medium takes 1/2MS as a basic culture medium and also only contains 0.5 mg/L6-BA, 0.15mg/L GA 3, 0.1mg/L NAA, 30g/L sucrose and 100U/mL antibiotics;
Or alternatively
The spore germination liquid culture medium takes 1/2MS as a basic culture medium and also only contains 0.5 mg/L6-BA, 0.05mg/L GA 3, 0.1mg/L NAA, 30g/L sucrose and 100U/mL antibiotics;
The solid multiplication medium is composed of MS-based medium, and also contains only 0.5 mg/L6-BA, 0.5mg/L NAA, 1.0 mg/L2, 4-D, 30g/L sucrose, 7g/L agar and 100U/mL antibiotics.
3. The method of claim 1, wherein the method of obtaining the drynaria spores comprises: collecting wild fresh dryopteris fragrans leaves, and scraping sporangia groups on the leaves by a sterile tool.
4. The method according to claim 1, wherein the temperature of the cultivation is 18-22 ℃; the culture time is 15-23 d.
5. The method according to claim 1 or 4, wherein the culturing is performed in an alternate light and dark environment; the time of light culture in each light-dark alternation period is 12-14 h/d, and the time of dark culture is 8-12 h/d.
6. The method according to claim 1, wherein the temperature of the induction culture is 18-22 ℃; the time of the induction culture is 20-25 d.
7. The method according to claim 1 or 6, wherein the induction culture is performed in an alternate light-dark environment; the time of light culture in each light-dark alternation period is 12-14 h/d, and the time of dark culture is 8-12 h/d.
8. Use of the method of any one of claims 1 to 7 in at least one of:
(1) Shortening the time for the drynaria fortunei spores to germinate to form flocculent tissues;
(2) Shortens the time for forming protoleaf bodies by the proliferation of the flocculent tissue of the drynaria fortunei.
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| 消毒方式、无机盐浓度及光照强度对槲蕨孢子繁殖的影响;张银丽;杜红红;李杨;李东;季梦成;姜闯道;石雷;;园艺学报;20090525(第05期);711-716 * |
| 蕨类植物繁殖研究进展;韩敬, 赵莉;安徽农业科学;20060225(第07期);1261-1263 * |
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