CN112889669A

CN112889669A - Culture medium for phoma niveum prothallium and method for rapidly inducing and obtaining sporophyte seedling

Info

Publication number: CN112889669A
Application number: CN202110225385.1A
Authority: CN
Inventors: 冼康华; 唐健民; 苏江; 黄宁珍; 何金祥; 付传明; 刘宝骏
Original assignee: Guangxi Institute of Botany of CAS
Current assignee: Guangxi Institute of Botany of CAS
Priority date: 2021-03-01
Filing date: 2021-03-01
Publication date: 2021-06-04
Anticipated expiration: 2041-03-01
Also published as: CN112889669B

Abstract

The invention provides a culture medium for a phoma niveum prothallium and a method for rapidly inducing and obtaining a sporophyte seedling, belonging to the technical field of incomplete tissue culture. In the invention, spores of Ceratopteris nivea are used as explants to induce prothallium, and after a large amount of robust prothallium is obtained through subculture proliferation, the sporophyte seedlings are directly hardened and transplanted to finally obtain sporophyte seedlings. The method disclosed by the invention is simple to operate, can achieve the effect of rapidly inducing the prothallium to grow the sporophyte, has the advantages of high induction rate and stable induction effect, and can provide technical reference for sustainable development and utilization of the oncophora brightening fern.

Description

Culture medium for phoma niveum prothallium and method for rapidly inducing and obtaining sporophyte seedling

Technical Field

The invention belongs to the technical field of incomplete tissue culture, and particularly relates to a pteridium aquilinum prothallium culture medium and a method for quickly inducing and obtaining a sporophyte seedling.

Background

Bright oncopteris (Phynatodes cubpidatus (D.Don) pic. serm.) is a large epiphytic fern of the oncopteris of the water-dragon orthopaedics, grows under evergreen broad-leaved forest with the altitude of 500-2000 m or on a rock, is commonly found in limestone areas, is fond of warm and humid environment, and is distributed in Yunnan, Tibet, Sichuan and other places of China. The glabrous fern is a traditional Chinese medicinal material in folk, the rhizome of the glabrous fern can be used as a medicine, the medicine is named as the "pig hair fern", and the glabrous fern has the effects of promoting blood circulation, relieving pain, setting a broken bone and eliminating swelling and the like and is mainly used for treating diseases such as traumatic injury, fracture, lumbocrural pain, innominate toxic swelling and the like. The bright pteridium aquilinum leaves are beautiful, the rootstock is thick and strong like fingers, the leaves and the stems can be observed, and the method is a good product for indoor green plant pot culture and garden landscape. In recent years, due to the fact that the application value of the Ceratopteris vittata is more and more concerned by people, wild resources are reduced due to excessive mining and disorderly digging and damage of habitat.

By utilizing the characteristic that the seedlings can be rapidly bred by tissue culture, the development of the research on the tissue culture of the oncopteris shining fern has important significance for protecting, developing and utilizing the oncopteris shining fern. At present, a few reports on tissue culture research of oncopternia lucida exist, the oncopternia lucida belongs to ferns which can be proliferated by gametophytes and cannot be easily induced in bottles, and after prothallium is obtained through spores, the problems of long induction time (11 months), low induction rate, unstable induction and the like of the sporophytes exist, so that the development of the tissue culture technology and industry of the oncopternia lucida is seriously hindered.

Disclosure of Invention

In view of the above, the present invention aims to provide a culture medium for a prothallium of Ceratopteris nivea and a method for rapidly inducing and obtaining a spore seedling, wherein the prothallium culture medium can be used for rapidly inducing the prothallium to grow the sporophyte, and the method of the present invention has the advantages of simple operation, high induction rate and stable induction effect.

In order to achieve the above purpose, the invention provides the following technical scheme:

the invention provides a culture medium of a Ceratopteris niphyllum prothallium, which comprises a primary generation induction culture medium and a secondary generation multiplication culture medium, wherein the formula of the primary generation induction culture medium is as follows: MS + BA 0.2-1.0mg/L + IAA 0.1-0.5mg/L + sucrose 20-30g/L + agar 3-4g/L, pH5.0-5.8; the formula of the subculture multiplication medium is as follows: MS + BA 0.2-1.0mg/L + IAA 0.02-0.1mg/L + ZnSO₄0.2-1.0mg/L + activated carbon 1.0-2.0g/L + sucrose 20-30g/L + agar 3-4g/L, pH 5.0-5.8.

Preferably, the formula of the primary induction medium is as follows: MS + BA 0.8-1.0mg/L + IAA 0.2-0.4mg/L + sucrose 20-30g/L + agar 3-4g/L, pH 5.0-5.8.

Preferably, the formula of the subculture multiplication medium is as follows: MS + BA 0.8-1.0mg/L + IAA 0.08-0.1mg/L + ZnSO₄0.2-0.5mg/L + activated carbon 1.0-1.5g/L + sucrose 20-30g/L + agar 3-4g/L, pH 5.0-5.8.

The invention also provides a method for rapidly inducing and obtaining sporophyte seedlings by using the culture medium, which comprises the following steps: inoculating the oncopteris nivea spores into the primary induction culture medium to obtain oncopteris nivea sterile prothallium; transferring the sterile prothallium into the subculture multiplication culture medium to obtain a prothallium subculture seedling; hardening off the primary leaf subculture seedling, transplanting in the substrate, and culturing to obtain sporophyte seedling.

Preferably, the hardening-seedling time is 5-15 days.

Preferably, the shading degree during seedling exercising is 60-80%.

Preferably, the domesticated successive seedlings of the prothallium are transplanted into a matrix after being soaked in a bactericide.

Preferably, the culture conditions for obtaining sterile prothallium of Ceratopteris nivea and obtaining the prothallium subculture are independently: the temperature is 25-31 ℃, the illumination time is 10-15h/d, and the illumination intensity is 35-45 mu mol.m^-2·s^-1。

Preferably, the substrate comprises two or more of yellow loam, peat soil and fine sand.

Preferably, the substrate is yellow loam and peat soil in a volume ratio of (2-4): 1-2, or fine sand and peat soil in a volume ratio of (2-4): 1-2.

The invention has the beneficial effects that:

according to the invention, mature spores of Ceratopteris nivea are used as explants, and reasonable culture medium is adopted for induction and subculture, so that a large number of robust and uniform prothallium bodies can be obtained, and development of induction of sporophyte is facilitated; in the induction stage of sporophyte, the strong and strong prothallium with yellow rhizoid on the surface is directly transferred to a culture medium for culture, so that juvenile sporophyte can be quickly induced in a short time, and the induction time is far shorter than that of the prior art.

In the invention, the primary induction germination time is 20-30d, and the time required by the successive multiplication of the prothallium is 30-50 d; the time for inducing juvenile sporophyte from prothallium in the culture medium is 30-50d, which greatly shortens the time for obtaining the Ceratopteris nikowii sporophyte by induction.

Drawings

FIG. 1 shows prophyll from example 1 induced by spores;

FIG. 2 shows a plantlet subcultured seedling obtained by subculture propagation of the plantlet of example 1;

FIG. 3 shows the sporophyte seedlings obtained by the induction culture of the tissue culture flask extracellular matrix in example 1.

Detailed Description

The sources of the components in the primary generation induction culture medium and the secondary generation proliferation culture medium are not particularly limited, and the conventional commercial products in the field can be adopted, wherein MS is a conventional MS solid culture medium in the field and comprises agar, macroelements, trace elements, iron salts and organic components.

In the invention, the formula of the primary induction culture medium is preferably MS + BA 0.8-1.0mg/L + IAA 0.2-0.4mg/L + sucrose 20-30g/L + agar 3-4g/L, and the pH is 5.0-5.8, more preferably MS + BA 1.0mg/L + IAA 0.2mg/L + sucrose 30g/L + agar 3.5g/L, and the pH is 5.8. The invention optimizes the culture medium formula used for primary induction, is more beneficial to the germination of spores and the growth of prothallium, has short germination time and higher germination rate of the spores, and leads the prothallium to grow faster and more uniformly, and the primary induction germination time is 20-30 days.

In the present invention, the formulation of the subculture multiplication medium is preferably: MS + BA 0.8-1.0mg/L + IAA 0.08-0.1mg/L + ZnSO₄0.2-0.5mg/L + activated carbon 1.0-1.5g/L + sucrose 20-30g/L + agar 3-4g/L, pH5.0-5.8, more preferably MS + BA 0.8mg/L + IAA 0.08mg/L + ZnSO₄0.2mg/L + activated carbon 1.0g/L + sucrose 30g/L + agar 3-4g/L, pH 5.0-5.8. Exogenous hormones BA, IAA and ZnSO which are properly proportioned and combined are added in the subculture multiplication stage of the prothallium₄The time required for subculture propagation of the prothallium is only 30-50d, and the prothallium has high propagation coefficient, emerald green color, strong growth and yellow rhizoid growing on the surface.

The invention also provides a method for rapidly inducing sporophyte seedlings by using the prothallium obtained by the culture medium, which comprises the following steps: inoculating the spores of the oncophora nivea into the primary induction culture medium to obtain sterile prothallium of the oncophora nivea; transferring the sterile prothallium into the subculture multiplication medium to obtain a prothallium subculture seedling; hardening off the primary leaf subculture seedling, transplanting in the substrate, and culturing to obtain sporophyte seedling.

In the present invention, before inoculating the spores of oncophora nivea to the primary induction medium, it is preferable to sterilize the spores of oncophora nivea, and the sterilization preferably includes the following steps: cutting feather piece with mature sporangia, soaking in 0.2% washing powder water for 5-12min, preferably 10min, brushing and washing feather piece surface with running water, surface sterilizing with 75% alcohol by volume for 30-60s, and 0.1% HgCl₂Sterilizing for 5-8min, rinsing with sterile water, sucking, and removing sporangium from the pinna to obtain sterilized Ceratopteris Nipponica spore. In the present inventionThe brushing is preferably to lightly brush the surface by using a soft brush pen or absorbent cotton, and the sporangia is not scraped; the disinfection process preferably requires constant shaking; the number of the rinsing with the sterile water is preferably 5; the blotting is preferably performed using sterile filter paper. In the present invention, the primary induction medium is preferably inoculated by gently grasping the sporangium with forceps, peeling the sporangium off the pinna, and inoculating the sporocysts into the primary induction medium. In the prior art, most of sterilization technologies for the glabrous fern explant shear sporophylls with mature spores, the mature spores are taken to be pretreated after air drying to remove impurities to be used as the explant, the explant is wrapped by filter paper and then sterilized by the conventional sterilization technology, and the steps are complex, the consumed time is long, the operation is complex, and the loss of the spores is easily caused; the disinfection method is simple and convenient and easy to operate, and greatly improves the experimental efficiency.

In the present invention, after inoculating the spores of Ceratopteris niponensis (Linn.) Kuntze into the primary induction medium, primary culture is performed at a temperature of preferably 25-31 deg.C, more preferably 27-29 deg.C, with a light irradiation time of preferably 10-15h/d, more preferably 12-13h/d, and with a light irradiation intensity of preferably 35-45. mu. mol. m.^-2·s^-1More preferably 38 to 42. mu. mol. m^-2·s^-1The time for the primary culture is preferably 20-30d, more preferably 22-28 d.

According to the invention, the prothallium obtained by spore germination is transferred into a subculture multiplication medium for subculture, so that a large number of robust and uniform prothallium can be obtained, and development of induction of the sporophyte is facilitated. In the present invention, the temperature of the subculture is preferably 25 to 31 ℃ and more preferably 27 to 29 ℃, the illumination time of the subculture is preferably 10 to 15h/d and more preferably 12 to 13h/d, and the illumination intensity of the subculture is preferably 35 to 45. mu. mol. m.^-2·s^-1More preferably 38 to 42. mu. mol. m^-2·s^-1The time for the subculture is preferably 30 to 50 days, more preferably 35 to 45 days.

In the invention, hardening off is carried out after the strong growing prothallium subculture seedling is obtained. The invention has no special limitation on the specific mode of seedling hardening, and can adopt the common oncopteris glabra seedling hardening method in the field. In the invention, the hardening-seedling is preferably carried out in a place with shading degree of 60-80%, the shading degree is more preferably 70%, the place is preferably a hardening-seedling greenhouse, and the hardening-seedling time is preferably 5-15d, more preferably 6-13d, and most preferably 7 d.

In the present invention, after hardening off the primary subculture seedlings, the attached medium is washed, preferably sterilized, and then transplanted into a substrate for culture. The sterilization treatment is preferably soaking in a bactericide, the bactericide can be carbendazim, and the soaking time is preferably 30-60s, and more preferably 40-50 s.

In the invention, the substrate is preferably a substrate which takes two or more of yellow loam, peat soil and fine sand as components, wherein the composition of the transplanting substrate is more preferably yellow loam and peat soil with the volume ratio of (2-4): 1-2 or fine sand and peat soil with the volume ratio of (2-4): 1-2, and is further preferably yellow loam and peat soil with the volume ratio of 3: 1. The culture medium has the advantages of good water retention and air permeability, can shorten the time of inducing juvenile sporophytes in the culture medium by the prothallium to 30-50 days, and has quick induction of the sporophytes and stable effect. In the invention, the primary leaf subculture seedlings after hardening and sterilizing treatment are transplanted in a matrix and then need to be watered regularly, the primary leaf bodies are easy to dry and die due to water shortage, and the watering frequency and the dosage are only needed to keep the primary leaf bodies wet.

In the induction and subculture multiplication stage of sporophyte, the invention directly transfers the strong-growing prothallium with yellow rhizoid on the surface to a culture medium for culture to obtain the sporophyte. The juvenile sporophyte can be obtained by induction in 30-50 days by using the culture medium and the method, so that the induction time of the sporophyte is greatly shortened, and the induction rate of the sporophyte is more stable.

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Example 1

1) Explant sterilizationAnd spore germination: cutting feather pieces with mature sporangia, soaking in 0.2% washing powder water for about 10min, brushing the surface with soft brush or absorbent cotton (taking care not to scrape off sporangia), and washing with running water; transferring the material to a clean bench, surface sterilizing with 75% alcohol for 30-60s, 0.1% HgCl₂Sterilizing for 7min, shaking, rinsing with sterile water for 5 times, and drying with sterile filter paper; gently grasping the sporangia with forceps, peeling the sporangia from the pinna, and then inoculating the pinna with the recipe: MS + BA 1.0mg/L + IAA 0.2mg/L + sucrose 30g/L + agar 3.5g/L, pH 5.8. At 28 deg.C, the illumination time is 10h/d, and the illumination intensity is 40 μmol m^-2·s^-1The spores are germinated to form sterile prothallium after being cultured for 7d under the condition of illumination, and the prothallium is green, heart-shaped, conglobated and well grows as shown in figure 1. And the germination rate is 75.58% at 30 d.

2) Subculturing multiplication of pteridium aquilinum prothallium: the obtained sterile prothallium is transferred into a formula: MS + BA 0.8mg/L + IAA 0.08mg/L + ZnSO₄0.2mg/L + activated carbon 1g/L + sucrose 30g/L + agar 3g/L, pH 5.0. At 28 deg.C, the illumination time is 10h/d, and the illumination intensity is 40 μmol m^-2·s^-1The primary leaf subculture seedlings grow strongly, are emerald green in color and are clustered, and yellow rhizomes grow, as shown in figure 2. The multiplication coefficient can reach 15.6.

3) Selecting strong-growing prothallium subculture seedlings, moving to a seedling hardening greenhouse with shading degree of 70% for hardening seedlings for 7d, lightly clamping prothallium clusters with forceps, cleaning attached culture medium, soaking in carbendazim for 30s, transplanting in a matrix with yellow loam and peat soil in a ratio of 3: 1, placing the transplanted prothallium in a shady and cool ventilated place, preventing direct sun exposure, watering regularly, keeping moisture, inducing for 30d to obtain sporophyte, wherein the survival rate is 90%, and is shown in figure 3.

Example 2

Example 1 was repeated except that:

modifying the formula of the culture medium for subculture multiplication of the metaplasia in the step 2) into that: MS + BA 0.8mg/L + IAA 0.08mg/L + ZnSO₄0.5mg/L + active carbon 1g/L + sucrose 30g/L + agar 3g/L, pH5.0, 50d of prothallium colorEmerald green, medium growth, and a multiplication coefficient of 12.5; the growth of the prothallium is slightly inferior to that of example 1, the proliferation coefficient is lower than that of example 1, and high concentration of ZnSO is illustrated₄The growth of the prophyll body is inhibited;

example 3

Example 1 was repeated except that:

modifying the composition of the culture medium in the step 3) into that: the ratio of fine sand to peat soil is 3: 1, the induction time of sporophyte is 35d, and the survival rate is 82%. Longer induction times than in example 1 increased the mortality of the prothallium, probably due to poor water retention of the sand.

Example 4

Example 1 was repeated except that:

modifying the composition of the culture medium in the step 3) into that: yellow loam, induction time of sporophyte is 38d, and survival rate is 65%. The induction time was longer than that of example 1, and the death rate of the prothallium increased, which may be the cause of poor air permeability of yellow loam.

Example 5

Example 2 was repeated except that:

modifying the formula of the culture medium for subculture multiplication of the metaplasia in the step 2) into that: MS + BA 1.0mg/L + IAA 0.1mg/L + ZnSO₄0.2mg/L + activated carbon 1g/L + sucrose 30g/L + agar 3g/L, pH 5.0; the prototheca bodies cultured for 50d are emerald in color, the individuals are moderate, and the multiplication coefficient is 17.8.

Example 6

Example 1 was repeated except that:

modifying the composition of the culture medium in the step 3) into: fine sand, the induction time of sporophyte is 42 days, and the survival rate is 62 percent. The induction time is longer than that of example 2, and the death rate of the prothallium is increased, which is probably the reason of poor water retention and air permeability of single fine sand.

Example 7

Example 1 was repeated except that:

modifying the composition of the culture medium in the step 3) into that: the induction time of the sporophyte in the peat soil is 50 days, and the survival rate is 60 percent. The induction time was longer than that in example 2, and the death rate of the prothallium increased, which may be a cause of poor water retentivity and air permeability of peat soil.

Example 8

Example 1 was repeated except that:

modifying the formula of the culture medium for subculture multiplication of the metaplasia in the step 2) into that: MS + BA 1.0mg/L + IAA 0.1mg/L + ZnSO₄0.5mg/L + activated carbon 1g/L + sucrose 30g/L + agar 3g/L, pH 5.0; the cultured 50d prototheca bodies are verdure in color, medium in growth and 16.5 in multiplication coefficient.

Modifying the composition of the culture medium in the step 3) into that: the ratio of yellow loam to peat soil is 2: 1, the induction time of sporophyte is 40 days, and the survival rate is 76%.

Example 9

Example 1 was repeated except that:

modifying the formula of the culture medium for subculture multiplication of the metaplasia in the step 2) into that: MS + BA 0.6mg/L + IAA 0.06mg/L + ZnSO₄0.6mg/L + activated carbon 1g/L + sucrose 30g/L + agar 3g/L, pH 5.0; the cultured 50d protosome is verdure in color, medium in growth and 12.5 in proliferation coefficient.

Example 10

Example 1 was repeated except that:

modifying the composition of the culture medium in the step 3) into that: the ratio of yellow loam to peat soil is 4: 1, the induction time of sporophyte is 40d, and the survival rate is 70%.

Example 11

Example 1 was repeated except that:

modifying the formula of the culture medium for subculture multiplication of the metaplasia in the step 2) into that: MS + BA 0.4mg/L + IAA 0.04mg/L + ZnSO₄0.4mg/L + activated carbon 1g/L + sucrose 30g/L + agar 3g/L, pH 5.0; the cultured 50d protosome is verdure in color, medium in growth and has a proliferation coefficient of 10.8.

Example 12

Example 1 was repeated except that:

modifying the composition of the culture medium in the step 3) into that: the ratio of fine sand to peat soil is 4: 1, the induction time of sporophyte is 45d, and the survival rate is 70%.

Comparative example 1

Example 1 was repeated except that:

relaying the frond in the step 2)The formula of the generation multiplication culture medium is modified as follows: MS + BA 8.0mg/L + IAA 0.8mg/L + ZnSO₄2.0mg/L + sucrose 30g/L + agar 3g/L, pH 5.0; the color of the cultured 50d prothallium is light green, part of the prothallium has vitrification phenomenon, the growth is uneven, and the multiplication coefficient is 3.5.

Comparative example 2

Example 1 was repeated except that:

modifying the composition of the culture medium in the step 3) into that: crushing the bricks; culturing for 60 days without induction of sporophyte.

Example 13

1) Explant disinfection: cutting feather pieces with mature sporangia, soaking in 0.2% washing powder water for about 10min, brushing the surface with soft brush or absorbent cotton (taking care not to scrape off sporangia), and washing with running water; transferring the material to a clean bench, surface sterilizing with 75% alcohol for 60s, 0.1% HgCl₂Sterilizing for 5min, shaking, rinsing with sterile water for 5 times, and drying with sterile filter paper; when inoculating, the sporangium is lightly clamped by forceps, and the sporangium is stripped from the pinna, and then the recipe is as follows: MS, BA0.2mg/L, IAA 0.02mg/L, cane sugar 20g/L, agar 4g/L and pH 5.8.

2) Spore germination: at 31 deg.C, the illumination time is 15h/d, and the illumination intensity is 45 μmol m^-2·s^-1The culture is carried out for 7d under illumination, partial treatment of contamination is observed, the later contamination phenomenon occurs along with the increase of the culture time, and the average contamination rate is 50%; and culturing for 30 days, wherein the rest spores germinate to form prothallium, and the prothallium is green, heart-shaped, clustered, well grown and 65% of germination rate.

3) Subculturing multiplication of pteridium aquilinum prothallium: the formula of the obtained sterile prothallium transferred into a culture medium is as follows: MS + BA0.2mg/L + IAA 0.02mg/L + ZnSO₄1.0mg/L, 1g/L of active carbon, 20g/L of sucrose and 4g/L of agar in a subculture multiplication medium with pH of 5.8, at 31 ℃, the illumination time of 15h/d and the illumination intensity of 45 mu mol m^-2·s^-1The prototheca grows strongly, the color is emerald green, the shape is a ball, yellow rhizoid is grown, and the multiplication coefficient can reach 8.6;

4) selecting strong growing prothallium subculture seedlings, moving to a seedling hardening greenhouse with shading degree of 70% for hardening seedlings for one week, lightly clamping prothallium clusters with forceps, cleaning attached culture medium, soaking in carbendazim for 60s, transplanting in a matrix with yellow soil to fine sand ratio of 2: 1, placing the transplanted prothallium in a cool and ventilated place, preventing direct sun exposure, watering regularly, keeping water, and obtaining sporophyte after 42d induction.

Example 14

1) Explant disinfection: cutting feather pieces with mature sporangia, soaking in 0.2% washing powder water for about 10min, brushing the surface with soft brush or absorbent cotton (taking care not to scrape off sporangia), and washing with running water; transferring the material to a clean bench, surface sterilizing with 75% alcohol for 40s, 0.1% HgCl₂Sterilizing for 8min, shaking, rinsing with sterile water for 5 times, and drying with sterile filter paper; when in inoculation, the sporangium is lightly clamped by tweezers, stripped from the pinna, and then inoculated in a culture medium with the following formula: MS + BA 0.4mg/L + IAA 0.04mg/L + sucrose 25g/L + agar 3.5g/L, pH5.2 in the primary induction medium.

2) Spore germination: at 28 deg.C, the illumination time is 12h/d, and the illumination intensity is 40 μmol m^-2·s^-1The spore germinates to form a prothallium after being cultured for 15 days under the condition, the prothallium is green and heart-shaped, forms a cluster and grows well, and the germination rate is 55 percent after 30 days.

3) Subculturing multiplication of pteridium aquilinum prothallium: the formula of the obtained sterile prothallium transferred into a culture medium is as follows: MS + BA 0.4mg/L + IAA 0.04mg/L + ZnSO₄0.8mg/L + activated carbon 1g/+ sucrose 25g/L + agar 3.8g/L, pH5.5, at 29 deg.C with illumination time of 14h/d and illumination intensity of 42 μmol. m^-2·s^-1The prototheca bodies grow strongly, are emerald green in color and are clustered after being cultured for 50 days under illumination, yellow rhizomes are grown, and the multiplication coefficient can reach 9.8.

4) Selecting strong-growing prothallium subculture seedlings, moving to a seedling hardening greenhouse with shading degree of 70% for hardening seedlings for one week, lightly clamping prothallium clusters with forceps, cleaning attached culture medium, soaking in carbendazim for 50s, transplanting in a matrix with a yellow loam to fine sand ratio of 1: 2, placing the transplanted prothallium in a cool and ventilated place, preventing direct sun exposure, watering regularly, keeping moisture, and obtaining sporophyte through induction for 45 d.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. The culture medium of the Ceratopteris niruri prothallium is characterized by comprising a primary induction culture medium and a secondary multiplication culture medium, wherein the primary induction culture medium comprises the following components in percentage by weight: MS + BA 0.2-1.0mg/L + IAA 0.1-0.5mg/L + sucrose 20-30g/L + agar 3-4g/L, pH5.0-5.8; the formula of the subculture multiplication medium is as follows: MS + BA 0.2-1.0mg/L + IAA 0.02-0.1mg/L + ZnSO₄0.2-1.0mg/L + activated carbon 1.0-2.0g/L + sucrose 20-30g/L + agar 3-4g/L, pH 5.0-5.8.

2. The culture medium of claim 1, wherein the primary induction medium is formulated as: MS + BA 0.8-1.0mg/L + IAA 0.2-0.4mg/L + sucrose 20-30g/L + agar 3-4g/L, pH 5.0-5.8.

3. The culture medium according to claim 1, wherein the formulation of the subculture proliferation medium is: MS + BA 0.8-1.0mg/L + IAA 0.08-0.1mg/L + ZnSO₄0.2-0.5mg/L + activated carbon 1.0-1.5g/L + sucrose 20-30g/L + agar 3-4g/L, pH 5.0-5.8.

4. A method for obtaining sporophyte seedlings through rapid induction by using the culture medium of claim 1, which comprises the following steps: inoculating the oncopteris nivea spores into the primary induction culture medium to obtain oncopteris nivea sterile prothallium; transferring the sterile prothallium into the subculture multiplication culture medium to obtain a prothallium subculture seedling; hardening off the primary leaf subculture seedling, transplanting in the substrate, and culturing to obtain sporophyte seedling.

5. The method according to claim 4, wherein the acclimatization time is 5-15 days.

6. The method as claimed in claim 4, wherein the shading degree during seedling exercising is 60-80%.

7. The method as claimed in claim 4, wherein the domesticated successive plantlets of the prothallium are transplanted into a substrate after being soaked in a bactericide.

8. The method of claim 4, wherein the culture conditions for obtaining sterile prothallium of Ceratopteris nivea and obtaining a prothallium subculture are independently: the temperature is 25-31 ℃, the illumination time is 10-15h/d, and the illumination intensity is 35-45 mu mol.m^-2·s^-1。

9. The method of claim 4, wherein the substrate comprises two or more of yellow loam, peat soil and fine sand.

10. The method of claim 9, wherein the substrate is yellow loam and peat soil in a volume ratio of (2-4): 1-2, or fine sand and peat soil in a volume ratio of (2-4): 1-2.