CN112021179A - Tissue culture method of Dryopteris fragrans - Google Patents
Tissue culture method of Dryopteris fragrans Download PDFInfo
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- 244000115361 Dryopteris fragrans Species 0.000 title claims abstract description 86
- 235000005075 Dryopteris fragrans Nutrition 0.000 title claims abstract description 85
- 238000012136 culture method Methods 0.000 title claims abstract description 19
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- 238000012258 culturing Methods 0.000 claims abstract description 32
- 230000035784 germination Effects 0.000 claims abstract description 28
- 230000035755 proliferation Effects 0.000 claims abstract description 11
- 230000006698 induction Effects 0.000 claims abstract description 8
- 238000005728 strengthening Methods 0.000 claims abstract description 7
- 230000001939 inductive effect Effects 0.000 claims abstract description 6
- 239000012879 subculture medium Substances 0.000 claims abstract description 5
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 3
- 238000004659 sterilization and disinfection Methods 0.000 claims description 18
- 241000736285 Sphagnum Species 0.000 claims description 13
- TWFZGCMQGLPBSX-UHFFFAOYSA-N Carbendazim Natural products C1=CC=C2NC(NC(=O)OC)=NC2=C1 TWFZGCMQGLPBSX-UHFFFAOYSA-N 0.000 claims description 12
- JNPZQRQPIHJYNM-UHFFFAOYSA-N carbendazim Chemical compound C1=C[CH]C2=NC(NC(=O)OC)=NC2=C1 JNPZQRQPIHJYNM-UHFFFAOYSA-N 0.000 claims description 12
- 239000006013 carbendazim Substances 0.000 claims description 12
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- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 claims description 8
- 238000000034 method Methods 0.000 claims description 8
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- 238000011081 inoculation Methods 0.000 claims description 2
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- 235000016709 nutrition Nutrition 0.000 description 3
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- 238000004161 plant tissue culture Methods 0.000 description 3
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- 241001148499 Ceratopteris Species 0.000 description 1
- 241001148765 Dryopteridaceae Species 0.000 description 1
- 241000196133 Dryopteris Species 0.000 description 1
- 240000002834 Paulownia tomentosa Species 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
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G24/00—Growth substrates; Culture media; Apparatus or methods therefor
- A01G24/20—Growth substrates; Culture media; Apparatus or methods therefor based on or containing natural organic material
- A01G24/28—Growth substrates; Culture media; Apparatus or methods therefor based on or containing natural organic material containing peat, moss or sphagnum
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a tissue culture method of Dryopteris fragrans, which comprises the following steps: (1) germination of Dryopteris fragrans spores; inoculating Dryopteris fragrans spores into a germination culture medium for culturing and germinating to obtain clumpy gametophytes; (2) inducing to grow seedlings; inoculating the gametophyte into an induction culture medium for culturing to obtain a sporophyte; (3) proliferation; inoculating the sporophyte into a subculture medium for culturing to obtain a cluster seedling; (4) strengthening seedlings; inoculating the cluster seedlings into 1/2MS culture medium for culture to obtain rootless seedlings; (5) transplanting and hardening seedlings; transplanting and hardening the rootless seedlings to obtain the seedling-hardening agent. The invention has the advantages that: successfully constructs a high-efficiency seedling propagation system of the Dryopteris fragrans, realizes the artificial rapid propagation of the Dryopteris fragrans, and provides technical support for the protection, development and utilization of the Dryopteris fragrans.
Description
Technical Field
The invention belongs to the technical field of plant tissue culture, and particularly relates to a tissue culture method of Dryopteris fragrans.
Background
Dryopteris (Polysticum glaciale Christ) is a perennial small herb fern of the genus Ceratopteris of the family Dryopteridaceae, and is a specific alpine species in our country. The Dryopteris fragrans is rare in quantity and endangered to be extinct, is listed as a national grade I key protection plant, a very small population plant, and is listed as a near-danger (NT) grade plant in Red catalogue of endangered species (IUCN) of world nature protection alliance. The Wanglong fern is only distributed in Yunnan, Sichuan and Tibet, etc., and grows in mountain glacier cave, cliff and rock cracks with altitude of 3200-flavored and 4700 m. The scales of the Dryopteris fragrans are reddish brown at first and are pale in color when old. The plants of the Dryopteris fragrans are beautiful, can be developed as micro-landscape plants for miniascapes or ornamental stone mountains in plateau areas, have good ornamental value, but are difficult to reproduce in the traditional mode due to special habitat, so that the Dryopteris fragrans plants cannot be developed and utilized.
In addition, effective protection, development and utilization are carried out on endangered plants, and an effective method is to establish a high-efficiency propagation technical system. The plant tissue culture technology needs less materials and has little influence on the normal growth of plants, thus being a commonly used propagation method at present. In order to better protect the Dryopteris fragrans and to develop and utilize the Dryopteris fragrans later, the problem of difficult propagation is solved, and no propagation report of Dryopteris fragrans is found at present. Therefore, the modern plant tissue culture technology is adopted to establish the high-efficiency seedling propagation system of the Dryopteris fragrans, and the method has important significance for the protection, scientific research, development and utilization of the Dryopteris fragrans.
Disclosure of Invention
In view of the above problems, the present invention provides a tissue culture method of Dryopteris fragrans.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a tissue culture method of Dryopteris fragrans comprises the following steps:
(1) germination of Dryopteris fragrans spores; inoculating Dryopteris fragrans spores into a germination culture medium for culturing and germinating to obtain clumpy gametophytes;
(2) inducing to grow seedlings; inoculating the gametophyte into an induction culture medium for culturing to obtain a sporophyte;
(3) proliferation; inoculating the sporophyte into a subculture medium for culturing to obtain a cluster seedling;
(4) strengthening seedlings; inoculating the cluster seedlings into 1/2MS culture medium for culture to obtain rootless seedlings;
(5) transplanting and hardening seedlings; transplanting and hardening the rootless seedlings to obtain the seedling-hardening agent.
Preferably, the spore of Dryopteris fragrans in the step (1) further comprises a step of disinfection before inoculation; the disinfection is carried out by adopting 0.1% mercury bichloride solution by mass concentration, and the disinfection time is 4-7 minutes.
Particularly preferably, the time for disinfection is 5 to 6 minutes.
Preferably, the temperature for culturing and germinating in the step (1) is 5-25 ℃, the illumination intensity for culturing and germinating is 1500-2000 Lx, and the illumination time for culturing and germinating is 8-14 hours/day.
Particularly preferably, the temperature for culturing germination is 15 ℃ to 20 ℃.
Preferably, the germination medium in the step (1) adopts one of MS medium, 1/2MS medium, 1/4MS medium or 1/8MS medium.
Preferably, the induction medium in the step (2) is 1/2MS medium.
Preferably, the subculture medium in step (3) is 1/2MS medium.
Preferably, before transplanting, the rootless seedlings in the step (5) are firstly placed in a room under natural light conditions and sealed for hardening seedlings; cleaning the culture medium of the root-free seedling base part during transplanting, removing 40-50% of leaves above the base part, and sterilizing with carbendazim; transplanting is carried out by adopting a substrate comprising sphagnum and river sand.
Particularly preferably, the time for closed-bottle seedling exercising is 10-20 days; the carbendazim with the mass percentage concentration of one thousandth is adopted for disinfection, and then the sterilized carbendazim is cooled for 15-30 minutes; the matrix is prepared from sphagnum and river sand, and the mass ratio of the sphagnum to the river sand is 2: 1; the temperature during transplanting is kept between 20 ℃ and 30 ℃, and the humidity is kept above 80%.
In the invention, the MS culture medium comprises 30g/L of white granulated sugar and 4g/L of agar, and the pH value of the culture medium is 5.8-6.2; 1/2MS is half of macroelement and sucrose based on MS, 1/4MS is 1/4 for the macroelement and sucrose, and 1/8MS is 1/8 for the macroelement and sucrose.
The invention has the following beneficial effects:
1. the tissue culture rapid propagation method of the Dryopteris fragrans is successfully constructed for the first time, the artificial efficient rapid propagation of the Dryopteris fragrans is realized, and the protection, development and application of the Dryopteris fragrans are facilitated.
2. The transplanting and seedling hardening of the Dryopteris fragrans adopts rootless test-tube seedlings, and the process of rooting in a test tube is omitted, so that the seedling cultivation time is greatly shortened, and the production cost of tissue culture seedlings is reduced.
3. The rapid propagation mode of the invention adopts a basic 1/2MS culture medium, realizes the rapid propagation and seedling strengthening of the Dryopteris fragrans by controlling the culture temperature of the culture room, and has simple operation and low cost.
4. When the method is used for transplanting the rootless seedlings, the sphagnum is adopted, and the rootless seedlings can root and survive through specific temperature and humidity control, so that the management cost is low, and the mass production is easy.
5. Through strong seedling culture, the basal part of the Dryopteris fragrans is made into a cluster rhizome, and the characteristics that the rhizome has the tenacious vitality and has the function of absorbing nutrients and water are utilized, so that the transplanting and seedling hardening are directly carried out, and the process flow is reduced.
6. By removing 40-50% of the leaves above the base, on one hand, the water loss of the plants can be effectively reduced, on the other hand, the air permeability of the rhizomes clustered at the base is increased, and the breeding of hyphae can be effectively reduced, so that the survival rate of the acclimatized seedlings is better ensured.
Drawings
FIG. 1 is a photograph showing germination of Dryopteris fragrans spores at different temperatures;
FIG. 2 is a photograph showing germination of Dryopteris fragrans spores in different germination media;
FIG. 3 is a photograph showing the proliferation of Dryopteris fragrans seedlings in 1/2MS medium;
FIG. 4 is a photograph showing the strong seedlings of Dryopteris fragrans in 1/2MS medium;
FIG. 5 is a photograph of the formation of tufted rhizomes at the base of Dryopteris fragrans;
FIG. 6 is a photograph of transplanted Paulownia fern tissue culture seedlings.
Detailed Description
The following further describes embodiments of the present invention with reference to the drawings. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
Example 1
A tissue culture method of Dryopteris fragrans comprises the following steps:
(1) germination of Dryopteris fragrans spores; sterilizing mature Dryopteris fragrans spores with 0.1% mercury bichloride solution by mass concentration for 5 minutes, washing with sterile water for 3-5 times, and inoculating the sterilized Dryopteris fragrans spores in 1/8MS culture medium for culture and germination to obtain clumpy gametophytes; the temperature for culturing and germinating is 20 ℃, the illumination intensity for culturing and germinating is 1500-2000 Lx, and the illumination time for culturing and germinating is 12 hours/day.
(2) Inducing to grow seedlings; dividing the gametophyte (cultured for 90 days) of the Dryopteris fragrans obtained in the step (1) into the sizes of 0.5-1.0cm in length and width, inoculating the gametophyte into 1/2MS culture medium for culturing for about 66 days to obtain sporophytes, wherein the induction rate is more than 95%.
(3) Proliferation; the sporophyte mass cultured for about 45 days is cut into pieces with length and width of 0.5-1.0cm, and inoculated into 1/2MS culture medium for proliferation, and cultured for about 60 days, and the proliferation multiple is above 10 (see figure 3).
(4) Strengthening seedlings; transferring the propagated seedling with the height of 2cm or more to 1/2MS culture medium for strong seedling culture. After 60 days of strong seedling culture, the height of the cluster seedlings is more than 3cm, cluster rootstocks are formed at the base parts of the seedlings, the diameter is more than 0.2cm (figure 4 and figure 5), and the important thing that the large and small Dryopteris fragrans have the rootstocks is very important. The diameter of the rhizome is more than 0.2cm, the adaptability of the Dryopteris fragrans to the outside is greatly enhanced, and meanwhile, certain nutrition is stored, so that the Dryopteris fragrans can root after transplanting of rootless seedlings, and the success of seedling hardening is greatly influenced.
(5) Transplanting and hardening seedlings; transplanting and hardening the rootless seedlings of the Dryopteris fragrans which have the height of more than 3cm, form rootstocks at the base parts and have the diameter of more than 0.2 cm. Before transplanting, placing the tissue culture seedlings in an indoor natural light, closing bottles, hardening the seedlings for 14 days, and then transplanting; during transplanting, the culture medium at the base part of the seedling is cleaned, and 40% -50% of leaves above the base part are removed, so that on one hand, the water loss of the plant can be effectively reduced, on the other hand, the pollution probability is greatly reduced, and the survival rate of the acclimatized seedling can be better ensured. If not removed, the survival rate of the seedlings is greatly reduced. Then, one in a thousand of carbendazim is fully sprayed for disinfection, and finally, the carbendazim is aired for 20 minutes and then transplanted into a disinfected substrate; wherein the matrix proportion used for transplanting is sphagnum: the river sand is 2:1, the water permeability of the matrix is increased, and the ventilation property of the plant base is more favorable than that of sphagnum used alone; during the period, the temperature and the humidity are controlled, the temperature is kept between 20 ℃ and 30 ℃, the humidity is kept above 80%, and the survival rate can reach above 90% (figure 6).
Example 2
A tissue culture method of Dryopteris fragrans comprises the following steps:
(1) germination of Dryopteris fragrans spores; sterilizing mature Dryopteris fragrans spores for 6 minutes by using 0.1% mercuric chloride solution, washing the sterilized water for 3-5 times, and inoculating the sterilized water into 1/8MS culture medium for culture and germination to obtain clumpy gametophytes; the temperature for culturing and germinating is 15 ℃, the illumination intensity for culturing and germinating is 1500-2000 Lx, and the illumination time for culturing and germinating is 12 hours/day.
(2) Inducing to grow seedlings; dividing the gametophyte (cultured for 90 days) of the Dryopteris fragrans obtained in the step (1) into the sizes of 0.5-1.0cm in length and width, inoculating the gametophyte into 1/2MS culture medium for culturing for about 66 days to obtain sporophytes, wherein the induction rate is more than 95%.
(3) Proliferation; cutting the cultured sporophyte blocks of about 45 days into pieces with length and width of 0.5-1.0cm, inoculating into 1/2MS culture medium, and culturing for about 60 days with proliferation multiple of above 10.
(4) Strengthening seedlings; transferring the propagated seedling with the height of 2cm or more to 1/2MS culture medium for strong seedling culture. After 60 days of strong seedling culture, the height of the cluster seedlings is more than 3cm, cluster rootstocks are formed at the base parts of the seedlings, the diameter is more than 0.2cm, and the important thing is that the large and small Dryopteris fragrans have the rootstocks. The diameter of the rhizome is more than 0.2cm, the adaptability of the Dryopteris fragrans to the outside is greatly enhanced, and meanwhile, certain nutrition is stored, so that the Dryopteris fragrans can root after transplanting of rootless seedlings, and the success of seedling hardening is greatly influenced.
(5) Transplanting and hardening seedlings; transplanting and hardening the rootless seedlings of the Dryopteris fragrans which have the height of more than 3cm, form rootstocks at the base parts and have the diameter of more than 0.2 cm. Before transplanting, placing the tissue culture seedlings in an indoor natural light, closing bottles, hardening the seedlings for 14 days, and then transplanting; during transplanting, the culture medium at the base part of the seedling is cleaned, and 40% -50% of leaves above the base part are removed, so that on one hand, the water loss of the plant can be effectively reduced, on the other hand, the pollution probability is greatly reduced, and the survival rate of the acclimatized seedling can be better ensured. If not removed, the survival rate of the seedlings is greatly reduced. Then, one in a thousand of carbendazim is fully sprayed for disinfection, and finally, the carbendazim is aired for 20 minutes and then transplanted into a disinfected substrate; wherein the matrix proportion used for transplanting is sphagnum: the river sand is 2:1, the water permeability of the matrix is increased, and the ventilation property of the plant base is more favorable than that of sphagnum used alone; during the period, the temperature and the humidity are controlled, the temperature is kept between 20 ℃ and 30 ℃, the humidity is kept above 80 percent, and the survival rate can reach above 90 percent.
Example 3
A tissue culture method of Dryopteris fragrans comprises the following steps:
(1) germination of Dryopteris fragrans spores; sterilizing mature Dryopteris fragrans spores with 0.1% mercury bichloride solution by mass concentration for 5 minutes, washing with sterile water for 3-5 times, and inoculating the sterilized Dryopteris fragrans spores in 1/8MS culture medium for culture and germination to obtain clumpy gametophytes; the temperature for culturing and germinating is 18 ℃, the illumination intensity for culturing and germinating is 1500-2000 Lx, and the illumination time for culturing and germinating is 12 hours/day.
(2) Inducing to grow seedlings; dividing the gametophyte (cultured for 90 days) of the Dryopteris fragrans obtained in the step (1) into the sizes of 0.5-1.0cm in length and width, inoculating the gametophyte into 1/2MS culture medium for culturing for about 66 days to obtain sporophytes, wherein the induction rate is more than 95%.
(3) Proliferation; cutting the cultured sporophyte blocks of about 45 days into pieces with length and width of 0.5-1.0cm, inoculating into 1/2MS culture medium, and culturing for about 60 days with proliferation multiple of above 10.
(4) Strengthening seedlings; transferring the propagated seedling with the height of 2cm or more to 1/2MS culture medium for strong seedling culture. After 60 days of strong seedling culture, the height of the cluster seedlings is more than 3cm, cluster rootstocks are formed at the base parts of the seedlings, the diameter is more than 0.2cm, and the important thing is that the large and small Dryopteris fragrans have the rootstocks. The diameter of the rhizome is more than 0.2cm, the adaptability of the Dryopteris fragrans to the outside is greatly enhanced, and meanwhile, certain nutrition is stored, so that the Dryopteris fragrans can root after transplanting of rootless seedlings, and the success of seedling hardening is greatly influenced.
(5) Transplanting and hardening seedlings; transplanting and hardening the rootless seedlings of the Dryopteris fragrans which have the height of more than 3cm, form rootstocks at the base parts and have the diameter of more than 0.2 cm. Before transplanting, placing the tissue culture seedlings in an indoor natural light, closing bottles, hardening the seedlings for 14 days, and then transplanting; during transplanting, the culture medium at the base part of the seedling is cleaned, and 40% -50% of leaves above the base part are removed, so that on one hand, the water loss of the plant can be effectively reduced, on the other hand, the pollution probability is greatly reduced, and the survival rate of the acclimatized seedling can be better ensured. If not removed, the survival rate of the seedlings is greatly reduced. Then, one in a thousand of carbendazim is fully sprayed for disinfection, and finally, the carbendazim is aired for 20 minutes and then transplanted into a disinfected substrate; wherein the matrix proportion used for transplanting is sphagnum: the river sand is 2:1, the water permeability of the matrix is increased, and the ventilation property of the plant base is more favorable than that of sphagnum used alone; during the period, the temperature and the humidity are controlled, the temperature is kept between 20 ℃ and 30 ℃, the humidity is kept above 80 percent, and the survival rate can reach above 90 percent.
In order to verify the scientific reasonability of the technical scheme of the invention, the following tests are specially carried out:
(1) comparison of contamination at different Disinfection times
Taking mature Dryopteris fragrans spores, respectively sterilizing with mercuric chloride with the mass concentration of 0.1% for 4min, 5min, 6min and 7min, washing with sterile water for 3-5 times, and inoculating to 1/2MS culture medium. The culture medium comprises 15g/L of white granulated sugar and 4g/L of agar, and the pH value of the culture medium is 5.8-6.2. The light intensity of the culture conditions is 1500-2000 Lx, the illumination time is 12 hours/day, and the temperature is about 20 ℃. The results (table 1) show that: the preferred disinfection time is 5-6 min.
The spore of the Dryopteris fragrans is disinfected in different disinfection time, and the pollution rate and the germination time are evaluated. The results are shown in Table 1.
TABLE 1 evaluation of different disinfection time results
| Disinfection time (min) | Contamination ratio (%) | Germination time (Tian) |
| 4 | 13.89 | 14 |
| 5 | 5.56 | 16 |
| 6 | 5.56 | 16 |
| 7 | 2.78 | 20 |
(2) Comparison of spore germination of Dryopteris fragrans at different culture temperatures
Taking mature Dryopteris fragrans spores, sterilizing the spores for 5min by using mercuric chloride with the mass concentration of 0.1%, washing the spores for 3-5 times by using sterile water, inoculating the spores to 1/2MS culture medium, and respectively culturing the spores in a constant temperature box with the temperature of 5 ℃, 10 ℃, 15 ℃, 20 ℃ and 25 ℃. 1/2MS culture medium comprises 15g/L white granulated sugar and 4g/L agar, and the pH value of the culture medium is 5.8-6.2. The light intensity of the culture conditions is 1500-2000 Lx, the illumination time is 12 hours/day, and the temperature is about 20 ℃. The results (table 2) show that: the germination of the Dryopteris fragrans spores is facilitated at the temperature of 15-20 ℃ (figure 1).
TABLE 2 Germination of Dryopteris fragrans spores at different temperatures
| Temperature of germination | Germination Rate (%) | Growth conditions |
| 5℃ | 0 | - |
| 10℃ | 13-17 | Diapause after germination |
| 15℃ | 54-57 | Health gametophyte |
| 20℃ | 56-58 | Health gametophyte |
| 25℃ | 4-6 | Diapause after germination |
(3) Comparing the spore germination effects of different basic culture mediums
Taking mature Dryopteris fragrans spores, sterilizing the spores for 5min by mercuric chloride with the mass concentration of 0.1%, washing the spores for 3-5 times by using sterile water, and respectively inoculating the spores into MS, 1/2MS, 1/4MS and 1/8MS culture media. The MS culture medium comprises 30g/L of white granulated sugar and 4g/L of agar, the pH value of the culture medium is 5.8-6.2, 1/2MS is obtained by halving macroelements and cane sugar on the basis of MS, 1/4MS means that the dosage of the macroelements and cane sugar is 1/4, and 1/8MS means that the dosage of the macroelements and cane sugar is 1/8. The culture conditions are the same as in step (1). The results (table 3) show that: the Dryopteris fragrans spores can germinate in the 4 basic culture media. The germination speed of the Dryopteris fragrans spores in the 1/8MS culture medium is the fastest, only about 13d is needed, but the 1/8MS culture medium is not beneficial to the growth and development of the prothallium; the growth and development conditions in 1/2MS culture medium are good, but the required germination time is about 15 days longer than that in 1/8MS culture medium; to sum up: since the difference in the time required for the spores of Dryopteris fragrans to germinate in the 1/2MS and 1/8MS media was small, the preferred germination medium for Dryopteris fragrans spores was considered to be 1/2MS (FIG. 2). In addition, the germination rate of the Dryopteris fragrans spores in 1/2MS medium is about 57.38%.
TABLE 3 Observation of spore germination of Dryopteris fragrans in different media
The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, and the scope of protection is still within the scope of the invention.
Claims (10)
1. A tissue culture method of Dryopteris fragrans is characterized by comprising the following steps: the method comprises the following steps:
(1) germination of Dryopteris fragrans spores; inoculating Dryopteris fragrans spores into a germination culture medium for culturing and germinating to obtain clumpy gametophytes;
(2) inducing to grow seedlings; inoculating the gametophyte into an induction culture medium for culturing to obtain a sporophyte;
(3) proliferation; inoculating the sporophyte into a subculture medium for culturing to obtain a cluster seedling;
(4) strengthening seedlings; inoculating the cluster seedlings into 1/2MS culture medium for culture to obtain rootless seedlings;
(5) transplanting and hardening seedlings; transplanting and hardening the rootless seedlings to obtain the seedling-hardening agent.
2. The tissue culture method of Dryopteris fragrans as claimed in claim 1, wherein: the Dryopteris fragrans spore in the step (1) also comprises a step of disinfection before inoculation; the disinfection is carried out by adopting 0.1% mercury bichloride solution by mass concentration, and the disinfection time is 4-7 minutes.
3. The tissue culture method of Dryopteris fragrans as claimed in claim 2, wherein: the disinfection time is 5-6 minutes.
4. The tissue culture method of Dryopteris fragrans as claimed in claim 1, wherein: the temperature for culturing and germinating in the step (1) is 5-25 ℃, the illumination intensity for culturing and germinating is 1500-2000 Lx, and the illumination time for culturing and germinating is 8-14 hours/day.
5. The tissue culture method of Dryopteris fragrans as claimed in claim 4, wherein: the temperature for culturing and germinating is 15-20 ℃.
6. The tissue culture method of Dryopteris fragrans as claimed in claim 1, wherein: the germination culture medium in the step (1) adopts one of an MS culture medium, an 1/2MS culture medium, a 1/4MS culture medium or a 1/8MS culture medium.
7. The tissue culture method of Dryopteris fragrans as claimed in claim 1, wherein: the induction medium in the step (2) is 1/2MS medium.
8. The tissue culture method of Dryopteris fragrans as claimed in claim 1, wherein: the subculture medium in the step (3) is 1/2MS medium.
9. The tissue culture method of Dryopteris fragrans as claimed in claim 1, wherein: before transplanting, the rootless seedlings are firstly placed in an indoor natural light condition and then sealed and hardened; cleaning the culture medium of the root-free seedling base part during transplanting, removing 40-50% of leaves above the base part, and sterilizing with carbendazim; transplanting is carried out by adopting a substrate comprising sphagnum and river sand.
10. The tissue culture method of Dryopteris fragrans as claimed in claim 9, wherein: the time for closing the bottle and hardening the seedlings is 10-20 days; the carbendazim with the mass percentage concentration of one thousandth is adopted for disinfection, and then the sterilized carbendazim is cooled for 15-30 minutes; the matrix is prepared from sphagnum and river sand, and the mass ratio of the sphagnum to the river sand is 2: 1; the temperature during transplanting is kept between 20 ℃ and 30 ℃, and the humidity is kept above 80%.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101133704A (en) * | 2007-09-28 | 2008-03-05 | 中国科学院华南植物园 | Sterilized spore germination of platycerium wallichii and test tube seedling cultivating method |
| CN101297635A (en) * | 2008-06-30 | 2008-11-05 | 中国科学院华南植物园 | Method for breeding spore of Dryopteris varia |
| CN106718878A (en) * | 2016-11-22 | 2017-05-31 | 云南省农业科学院花卉研究所 | A kind of fan fern quick breeding by group culture method with young sporangiorus as explant |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101133704A (en) * | 2007-09-28 | 2008-03-05 | 中国科学院华南植物园 | Sterilized spore germination of platycerium wallichii and test tube seedling cultivating method |
| CN101297635A (en) * | 2008-06-30 | 2008-11-05 | 中国科学院华南植物园 | Method for breeding spore of Dryopteris varia |
| CN106718878A (en) * | 2016-11-22 | 2017-05-31 | 云南省农业科学院花卉研究所 | A kind of fan fern quick breeding by group culture method with young sporangiorus as explant |
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