Live seedling raising method of dryopteris filix-mas

Abstract

The application provides a live sowing and seedling raising method for Dryopteris filix-mas spores and belongs to the technical field of spore propagation of medicinal ferns. The live sowing and seedling raising method for Dryopteris filix-mas spores comprises the following steps: collecting Dryopteris filix-mas spores, sowing on the surface of a culture medium, spraying nutrient solution according to different growth periods, and growing into spore bodies; the culture medium is composed of humus soil, peat soil and limestone at a volume ratio of 3-5:1-3:1-2; during sowing and germination of prothallus, 1 / 8MS+0.01-0.05mg / L GA3+0.3-0.7mg / L 6-BA nutrient solution is sprayed; during the period from prothallus to spore body growth, 1 / 2MS+0.3-0.7mg / L KT+0.3-0.7mg / L 6-BA+1.5-2.5mg / L IBA nutrient solution is sprayed. The application can significantly improve the emergence speed and propagation efficiency of Dryopteris filix-mas by using a specific culture medium and supplementing suitable nutrient solution, and has important significance for realizing artificial planting and resource protection of live sowing and seedling raising of Dryopteris filix-mas.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicinal fern spore propagation, and particularly relates to a Drynaria roosii Nakaike spore direct-seeding seedling raising method. BACKGROUND

[0002] Drynaria roosii Nakaike, also known as Drynaria, is a plant of Drynariaceae Drynaria. There are 8 genera and about 100 species in Drynariaceae. Most of the Drynariaceae plants have high ornamental value due to their elegant posture and beautiful shape. The rhizome of Drynaria is used as medicine and is rich in flavonoids, alkaloids, phenols and other effective components, and has the effects of removing blood stasis, relieving pain, setting bones and tendons, treating toothache, backache and chronic diarrhea. In recent years, due to blind excavation, natural resources are gradually exhausted, and the ecological environment is seriously damaged. In 2002, Drynaria was recommended to be included in the national endangered plant list.

[0003] In the natural environment, most of the Drynariaceae plants are epiphytic on rocks or tall tree trunks, and have high requirements for environmental conditions, such as temperature range, humidity range and soil pH range. It is difficult to survive when introduced and cultivated, which brings great difficulties to germplasm preservation and utilization. As for the artificial propagation of Drynaria, CN109691372A (publication date: April 30, 2019) discloses a Drynaria artificial propagation method, which uses rhizome as explant for tissue culture to propagate Drynaria, so as to improve the survival rate and the rate of leaving the nursery. However, the cost of tissue culture is high, the culture conditions are harsh, and it is not suitable for large-scale promotion. In addition, the collection of explants will cause damage to the original plant. CN100358414C (publication date: January 2, 2008) and CN1788549A (publication date: June 21, 2006) both disclose the use of spore propagation to propagate Drynaria. The former is mainly to improve the seeding efficiency of Drynaria spores, and the latter is to culture spores on a sterile medium to improve the seedling rate. However, how to accelerate the propagation efficiency of Drynaria spores and improve the seedling speed of Drynaria spore propagation is still a blank in the existing research. SUMMARY

[0004] Therefore, the purpose of the present application is to provide a Drynaria spore direct-seeding seedling raising method, which uses suitable culture medium for planting and sprays suitable nutrient solution according to different growth periods of Drynaria, so as to improve the seedling speed of Drynaria, improve the propagation efficiency and avoid damage to the original plant caused by other propagation methods.

[0005] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions:

[0006] The method comprises the following steps: collecting the spores of Drynaria fortunei, sowing on the surface of culture medium, spraying nutrient solution according to different growth periods until the sporophytes are grown; the culture medium is composed of humus soil, peat soil and limestone in a volume ratio of 3-5:1-3:1-2; during the sowing and germination of the prothallus, 1 / 8MS+0.01-0.05mg / L GA3+0.3-0.7mg / L 6-BA nutrient solution is sprayed; during the growth of the prothallus into the sporophytes, 1 / 2MS+0.3-0.7mg / L KT+0.3-0.7mg / L 6-BA+1.5-2.5mg / L IBA nutrient solution is sprayed.

[0007] Preferably, the spores of Drynaria fortunei are fresh and mature spores; more preferably, the collection method of the spores of Drynaria fortunei comprises the following steps: collecting leaf blades with fresh and mature soralia on the back, scraping off the soralia with a sterilized scalpel and then storing the soralia in a sterile dry EP tube.

[0008] Preferably, the spores of Drynaria fortunei are sterilized before sowing; more preferably, the sterilization process comprises the following steps: placing the spores of Drynaria fortunei in a 40mu cell filter, adding 75% alcohol for sterilization for 5-10s, washing with sterile water for 5-10 times, adding 0.1% HgCl2 for sterilization for 10-15min, and washing with sterile water for 5-10 times.

[0009] Preferably, the nutrient solution is sprayed once every 5-10 days.

[0010] Preferably, during the sowing and germination of the prothallus, the daytime temperature is controlled to be 22-25 DEG C, the night temperature is controlled to be 18-20 DEG C, the humidity is controlled to be 70%-90%, the light time is controlled to be 10-12h / d, the light intensity is controlled to be 1000-1500lx, and the culture medium is kept moist.

[0011] Preferably, during the growth of the prothallus into the sporophytes, the daytime temperature is controlled to be 22-25 DEG C, the night temperature is controlled to be 18-20 DEG C, the humidity is controlled to be 70%-90%, the light time is controlled to be 10-12h / d, the light intensity is controlled to be 1500-2000lx, and the culture medium is kept moist.

[0012] Compared with the prior art, the method has the following beneficial effects:

[0013] The method provided by the application improves the germination speed and the propagation efficiency of Drynaria fortunei by adjusting the culture medium and supplementing suitable nutrient solution and exogenous hormones. 2And avoid other breeding methods to destroy the original plant. In addition, the direct seeding method of the application is simple and easy to operate, low in cost, and suitable for large-scale popularization and application. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 Fresh and mature sporangia of Drynaria fortunei;

[0015] Figure 2 A sporophyte of Drynaria fortunei obtained by the direct seeding method of the application under a stereomicroscope. DETAILED DESCRIPTION

[0016] The application provides a direct seeding method for Drynaria fortunei, which improves the germination speed and reproduction efficiency of Drynaria fortunei, and comprises the following steps: collecting Drynaria fortunei spores, sowing on the surface of a culture medium, spraying nutrient solution according to different growth periods until sporophytes grow; the culture medium is composed of humus soil, peat soil and limestone at a volume ratio of 3-5:1-3:1-2; during sowing to the emergence of prothallus, 1 / 8MS+0.01-0.05mg / L GA3+0.3-0.7mg / L 6-BA nutrient solution is sprayed; during the prothallus to the growth of sporophytes, 1 / 2MS+0.3-0.7mg / L KT+0.3-0.7mg / L 6-BA+1.5-2.5mg / L IBA nutrient solution is sprayed.

[0017] The application preferably uses fresh and mature Drynaria fortunei spores, and the fresh and mature spores have higher activity and higher germination rate than mature spores stored for a period of time. Further preferably, the collection method of Drynaria fortunei spores comprises: collecting leaf blades covered with fresh and mature sporangia, scraping off the sporangia from the leaf blades with a sterilized scalpel, and then placing the sporangia in a sterile dry EP tube for storage.

[0018] The application preferably performs disinfection treatment on the Drynaria fortunei spores before sowing, and the disinfection treatment further preferably comprises: placing the Drynaria fortunei spores in a 40μ cell filter, adding 75% alcohol for disinfection for 5-10s, more preferably for 7-8s, washing with sterile water for 5-10 times, more preferably for 7-8 times, adding 0.1% HgCl2 for disinfection for 10-15min, more preferably for 12-13min, and washing with sterile water for 5-10 times, more preferably for 7-8 times.

[0019] The culture medium of the application preferably comprises humus soil, peat soil and limestone in a volume ratio of 4:2:1.5. The humus soil and the peat soil are loose and breathable after being mixed, and provide nutrients for the germination and growth of the sporophytes of the Cyatheasporita; the limestone is the environment on which the Cyatheasporita grows in nature. As an implementation manner, the humus soil is commercially available general-purpose humus soil, which is balanced in terms of acidity and alkalinity and is sterilized; the peat soil is imported peat soil from Denmark, which has a size of 5.0-20.0 mm and is rich in natural organic matter; and the limestone is limestone collected from the karst landform in Guizhou, which has a size of 5-10 mm.

[0020] During the period from sowing to the germination of the prothallus, the culture medium is sprayed with a 1 / 8MS+0.01mg / L GA3+0.5mg / L 6-BA nutrient solution; and during the period from the prothallus to the growth of the sporophytes, the culture medium is sprayed with a 1 / 2MS+0.5mg / L KT+0.5mg / L 6-BA+2.0mg / L IBA nutrient solution.

[0021] The nutrient solution is preferably sprayed once every 5-10 days, and more preferably once every 7 days.

[0022] During the cultivation, the daytime temperature is controlled to be 22-25°C, and more preferably 23.5°C, the nighttime temperature is controlled to be 18-20°C, and more preferably 19°C, the humidity is controlled to be 70%-90%, and more preferably 80%, the light time is controlled to be 10-12h / d, and more preferably 11h / d, and the culture medium is kept moist. As an implementation manner, distilled water is sprayed before 9am in the morning and before 6pm in the afternoon every day to keep the culture medium moist.

[0023] The light intensity during the period from sowing to the germination of the prothallus is 1000-1500lx, and the prothallus is germinated after about 25 days; the light intensity during the period from the prothallus to the growth of the sporophytes is 1500-2000lx, and the prothallus develops into sporophytes after about 50 days; and more preferably, the light intensity during the period from sowing to the germination of the prothallus is 1250lx, and the light intensity during the period from the prothallus to the growth of the sporophytes is 1750lx.

[0024] The technical solutions of the application will be described in detail below with reference to the examples, but they should not be understood as limiting the scope of protection of the application.

[0025] Example 1

[0026] A live seedling cultivation method of Cyatheasporita is provided, and the steps are as follows:

[0027] (1) Collect fresh and mature sporangia of Cyatheasporita: collect leaf blades covered with fresh and mature sporangia on the back of the leaf blades, scrape off the sporangia with a sterilized scalpel, and then store them in a sterile and dry EP tube;

[0028] (2) Disinfection treatment: the Cyatheaspores were placed in a 40μ cell filter, 75% alcohol was added for disinfection for 8s, sterile water was washed for 8 times, 0.1% HgCl2 was added for disinfection for 13min, and sterile water was washed for 8 times;

[0029] (3) Sowing: humus soil, peat soil and limestone were mixed in a volume ratio of 4:2:1.5 to prepare the culture medium, which was placed in the seedling tray, and the spores treated by disinfection were uniformly scattered on the surface of the culture medium;

[0030] (4) Management during the prothallus germination period: the sowing seedling tray was placed in the artificial climate box for culture, the artificial climate box culture temperature was 23℃ in the daytime and 19℃ at night, the humidity was 80%, the light intensity was 1250lx, the light time was 11h per day, and distilled water was sprayed before 9am in the morning and before 6pm in the afternoon every day to keep the culture medium moist; the nutrient solution was sprayed every 7 days, the nutrient solution formula was 1 / 8MS+0.01mg / L GA3+0.5mg / L 6-BA, until the prothallus germinated;

[0031] (5) Management during the sporophyte formation period: the light intensity of the artificial climate box was adjusted to 1750lx, the nutrient solution was sprayed every 7 days, the nutrient solution formula was 1 / 2MS+0.5mg / L KT+0.5mg / L 6-BA+2.0mg / L IB A, other conditions were unchanged, until the sporophyte was formed.

[0032] Example 2

[0033] A Cyatheaspore direct sowing seedling method, the steps are as follows:

[0034] (1) Collect fresh and mature Cyatheaspores: collect leaf blades covered with fresh and mature sporangia, scrape off the sporangia with a sterilized scalpel and place them in a sterile dry EP tube for storage;

[0035] (2) Disinfection treatment: the Cyatheaspores were placed in a 40μ cell filter, 75% alcohol was added for disinfection for 8s, sterile water was washed for 8 times, 0.1% HgCl2 was added for disinfection for 13min, and sterile water was washed for 8 times;

[0036] (3) Sowing: humus soil, peat soil and limestone were mixed in a volume ratio of 4:2:1.5 to prepare the culture medium, which was placed in the seedling tray, and the spores treated by disinfection were uniformly scattered on the surface of the culture medium;

[0037] (4) Prothallial stage management: the seedling tray after sowing was placed in the artificial climate box for culture, the culture temperature of the artificial climate box was 22°C in the daytime and 18°C at night, the humidity was 90%, the light intensity was 1000 lx, the light time was 12 hours per day, and distilled water was sprayed before 9 am in the morning and before 6 pm in the afternoon every day to keep the culture medium moist; the nutrient solution was sprayed every 10 days, the formula of the nutrient solution was 1 / 8MS+0.05 mg / L GA3+0.3 mg / L 6-BA, until the prothallial stage was germinated;

[0038] (5) Sporophyte formation management: the light intensity of the artificial climate box was adjusted to 1500 lx, the nutrient solution was sprayed every 10 days, the formula of the nutrient solution was 1 / 2MS+0.3 mg / L KT+0.3 mg / L 6-BA+1.5 mg / L IBA, other conditions were unchanged, until the sporophyte was formed.

[0039] Example 3

[0040] A live sowing seedling method of Cyatheasporita, the steps are as follows:

[0041] (1) Collect fresh and mature Cyatheasporita spores: collect leaf blades covered with fresh and mature sporangia, scrape off the sporangia with a sterilized scalpel and then place them in a sterile dry EP tube for storage;

[0042] (2) Disinfection treatment: place the Cyatheasporita spores in a 40 μ cell filter, add 75% alcohol for disinfection for 10 s, wash with sterile water for 10 times, then add 0.1% HgCl2 for disinfection for 10 min, and wash with sterile water for 5 times;

[0043] (3) Sowing: mix humus soil, peat soil and limestone at a volume ratio of 5:3:2 to prepare a culture medium, place the culture medium in a seedling tray, and uniformly spread the disinfected spores on the surface of the culture medium;

[0044] (4) Prothallial stage management: the seedling tray after sowing was placed in the artificial climate box for culture, the culture temperature of the artificial climate box was 22°C in the daytime and 18°C at night, the humidity was 90%, the light intensity was 1000 lx, the light time was 12 hours per day, and distilled water was sprayed before 9 am in the morning and before 6 pm in the afternoon every day to keep the culture medium moist; the nutrient solution was sprayed every 10 days, the formula of the nutrient solution was 1 / 8MS+0.05 mg / L GA3+0.3 mg / L 6-BA, until the prothallial stage was germinated;

[0045] (5) During the management of sporophyte formation, the light intensity of the artificial climate chamber was adjusted to 2000 lx, and the nutrient solution was sprayed every 5 days. The formula of the nutrient solution was 1 / 2MS + 0.7 mg / L KT + 0.7 mg / L 6-BA + 2.5 mg / L IBA, and other conditions remained unchanged until the sporophyte was formed.

[0046] Example 4

[0047] Effect of spores from different sources on the production of prothallus

[0048] The leaves of wild fresh Dryopteris eburnea with sporangia were collected and put into a kraft paper bag. The spores on the back of the leaves were carefully scraped off with a sterilized scalpel, and the fresh and mature spores obtained by this treatment were used as the treatment group. The spores of Dryopteris eburnea stored in a refrigerator at 4°C for one year were used as the control group. The culture was carried out according to the method of Example 1, and the spore germination and the production of prothallus were observed and counted.

[0049] The S-type five-point sampling method was used to sample on the seedling tray, and the number of prothallus produced in the two groups was counted. The results showed that the prothallus production rate of the fresh spore treatment group was 35% higher than that of the one-year stored spore control group, indicating that the germination rate of Dryopteris eburnea was higher when fresh spores were used for reproduction.

[0050] Example 5

[0051] Effect of different culture substrates on the production of prothallus of Dryopteris eburnea

[0052] Example 1 was used as the treatment group, and control groups A and B were set up. The culture substrate of control group A was pure humus soil, and the culture substrate of control group B was washed fine river sand (particle size 1-3 mm). Other conditions were the same, and the spore germination and the production of prothallus were observed until the spores of Dryopteris eburnea germinated into prothallus.

[0053] The growth and development of prothallus in the three groups were observed on the seedling tray. The difference in spore germination rate between the treatment group and the control groups A and B was not large, and the germination rate of the treatment group was slightly higher than that of the control groups A and B. However, the size of prothallus in the three groups was significantly different, with the size of prothallus being treatment group > control group A > control group B, indicating that the mixed culture substrate of humus soil, peat soil and limestone was more conducive to the reproduction of spores of Dryopteris eburnea.

[0054] Example 6

[0055] Effect of different nutrient solutions on the growth of prothallus of Dryopteris eburnea

[0056] Example 1 was used as the treatment group, and a control group was set up. The control group was sprayed with 1 / 8MS during the period from sowing to germination into prothallus, i.e. no exogenous hormones were added to the nutrient solution. Other conditions were the same, and the spore germination and the production of prothallus were observed until the spores of Dryopteris eburnea germinated into prothallus.

[0057] The growth and development of the two groups of prothallus are observed on the seedling tray, and the spore germination rate of the treatment group and the control group of Drynaria fortunei is shown as treatment group > control group, and the size of the prothallus is shown as treatment group > control group, which indicates that adding exogenous hormones in the nutrient solution is beneficial to the spore germination of Drynaria fortunei and the formation of prothallus.

[0058] Example 7

[0059] Effect of different nutrient solutions on the growth of spore of Drynaria fortunei

[0060] Example 1 is taken as the treatment group, and the control group is set, and the control group sprays 1 / 2MS during the period from prothallus to the growth of spore, that is, no exogenous hormones are added in the nutrient solution. Other conditions are the same, until the prothallus of Drynaria fortunei develops into spore.

[0061] The growth and development of the two groups of spore of Drynaria fortunei are observed and counted on the seedling tray, and the S-type five-point sampling method is used for counting, and the results show that the emergence rate of the treatment group of prothallus developing into spore is 40%, and the average is 240 plants / dm 2 The emergence rate of the control group of prothallus developing into spore is less than 10%, and the number of seedlings is small. It is shown that adding exogenous hormones in the nutrient solution can effectively promote the development of prothallus of Drynaria fortunei and form spore. And the spore of Drynaria fortunei in the treatment group germinates into prothallus in 25 days, and the prothallus develops into spore in 50 days, which indicates that the method can effectively improve the emergence speed and the propagation efficiency of Drynaria fortunei.

[0062] The above only describes the preferred embodiments of the present application, and it should be noted that for ordinary skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should be considered as the protection scope of the present application.

Claims

1. A method for direct seeding of Cyatheas spores, characterized in that, The method comprises the following steps: Collecting the spores of Cyatheas spicata, sowing on the surface of culture medium, spraying nutrient solution according to different growth periods until the sporophytes grow; The culture medium is composed of humus soil, peat soil and limestone in a volume ratio of 3-5:1-3:1-2; During the sowing and germination of the prothallus, 1 / 8MS+0.01-0.05mg / L GA3+0.3-0.7mg / L 6-BA nutrient solution is sprayed; during the growth of the prothallus into sporophytes, 1 / 2MS+0.3-0.7mg / L KT+0.3-0.7mg / L 6-BA+1.5-2.5mg / L IBA nutrient solution is sprayed; The spores of Cyatheas spicata are fresh and mature spores. The nutrient solution is sprayed once every 5-10 days.

2. The method of claim 1, wherein the method is characterized by, The collection method of the spores of Cyatheas spicata comprises the following steps: collecting the leaf blades with fresh and mature sporangia, scraping off the sporangia with a sterilized scalpel and then storing them in a sterile dry EP tube.

3. The method of claim 1, wherein the method is characterized by, Before sowing, the spores of Cyatheas spicata are subjected to sterilization treatment.

4. The method of claim 3, wherein the method is characterized by, The sterilization treatment comprises the following steps: placing the spores of Cyatheas spicata in a 40μ cell filter, adding 75% alcohol for sterilization for 5-10s, washing with sterile water for 5-10 times, adding 0.1% HgCl2 for sterilization for 10-15min, and washing with sterile water for 5-10 times.

5. The method according to claim 1, wherein the method is characterized by, During the sowing and germination of the prothallus, the temperature is controlled to be 22-25℃ during the day and 18-20℃ at night, the humidity is controlled to be 70%-90%, the light time is controlled to be 10-12h / d, the light intensity is controlled to be 1000-1500lx, and the culture medium is kept moist.

6. The method of claim 1, wherein the method is characterized by, During the growth of the prothallus into sporophytes, the temperature is controlled to be 22-25℃ during the day and 18-20℃ at night, the humidity is controlled to be 70%-90%, the light time is controlled to be 10-12h / d, the light intensity is controlled to be 1500-2000lx, and the culture medium is kept moist.

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