A method for cultivating sporophyte seedlings of Pteris spheniscus

Abstract

The invention discloses a method for cultivating sporophyte seedlings of Pteris spheniscus, comprising the following steps: step 1: spore germination; step 2: prothallus proliferation; step 3: sporophyte induction, moving the prothallus mass into an induction culture medium, and growing the sporophyte in 1-2 months; step 4: rooting and domestication, dividing the prothallus mass growing the sporophyte in step 3, and moving it into a rooting culture medium, and obtaining a mature sporophyte seedling in 1 month; then transplanting and domestication are performed; the induction culture medium and the rooting culture medium both contain soil extract; the soil extract is extracted from peat soil, humus soil and soil in the native habitat of ferns; the method adopts a culture medium containing the soil extract to induce the sporophyte, and compared with a conventional culture medium, the sporophyte induction rate is improved, and the induction rate reaches more than 80%; the culture medium containing the soil extract is used for rooting, and the rooting rate reaches 100%.

Description

Technical Field

[0001] The invention relates to a fern cultivation method, and in particular to a cultivation method for a sporophyte seedling of Pteris sphenanthera. Background Art

[0002] With the rapid growth of the national economy, the development of mineral resources is accelerating, and the heavy metal pollution caused by it is becoming more and more serious. The treatment and restoration of heavy metal contaminated soil has become an environmental problem that needs to be solved urgently. As a friendly restoration technology, phytoremediation can achieve ecological restoration of abandoned mining areas while treating heavy metal pollution. It is one of the mainstream technologies for soil remediation at this stage and in the future.

[0003] The mining ecological type of Pteris spheniscus is a perennial fern found in the typical lead-zinc mining area in western Sichuan, my country. It has a strong lead (Pb) enrichment ability, a certain tolerance to cadmium (Cd), and has the advantages of large biomass, strong vitality, and a well-developed root system. It is one of the pioneer plants for mine ecological restoration. However, the growth process of ferns, such as spore germination, prothallus formation, and fertilization, is affected by many environmental factors, especially the fertilization process is extremely restricted by water, resulting in a low probability of inducing the formation of sporophytes by conventional tissue culture, which limits its large-scale promotion and application. Summary of the invention

[0004] Aiming at the problems existing in the prior art, the invention provides a method for cultivating sporophyte seedlings of Pteris spheniscus.

[0005] The technical solution adopted by the present invention is: a method for cultivating sporophyte seedlings of Pteris spheniscus, comprising the following steps:

[0006] Step 1: Spore germination: inoculate the sterilized spores into a spore germination medium, germinate in 2 to 4 weeks, and produce prothallus;

[0007] Step 2: Prothallus proliferation: 1-2 months after spore germination, the prothallus diameter is not less than 0.5 cm, and the prothallus is transferred to the proliferation medium, and the prothallus group diameter grows to 3-4 cm;

[0008] Step 3: Sporophyte induction: transfer the prothallus mass to the induction medium and grow sporophytes in 1 to 2 months;

[0009] Step 4: Rooting and domestication, the prothallus mass from which the sporophyte grows in step 3 is divided and transferred to a rooting medium, and mature sporophyte seedlings can be obtained in one month; then transplanting and domestication are performed;

[0010] Both the induction medium and the rooting medium contain soil extract; the soil extract is extracted from peat soil, humus soil and soil in the native habitat of ferns.

[0011] Furthermore, the extraction process of the soil extract is as follows:

[0012] The soil was sieved and mixed with deionized water, and then covered with a film and soaked for 24 hours; it was rubbed repeatedly with gauze and filtered, and the filtered liquid was centrifuged to obtain the extract.

[0013] Further, the centrifugation parameters are as follows:

[0014] The centrifugal speed was 4000 r / min and the centrifugation lasted for 10 min.

[0015] Furthermore, the preparation process of the induction medium in step 3 is as follows:

[0016] Select coarse river sand with a particle size of 0.4 to 4 mm, wash it repeatedly until it is clear, dry it and dispense it into culture bottles;

[0017] The product is added with soil extract, sealed after subpackaging, sterilized at high temperature and cooled to obtain the product.

[0018] Furthermore, the induction culture medium and soil extract are above 1 / 3 of the height of the protothalli.

[0019] Furthermore, the preparation process of the rooting medium in step 4 is as follows:

[0020] Select fine river sand with a particle size of 0.1 to 0.25 mm, add soil extract, mix and stir, and then let it stand for 10 minutes; remove the liquid, repeat 3 to 4 times, package it, seal it, sterilize it at high temperature, and cool it to obtain it.

[0021] Furthermore, the soil extract in the rooting medium is in the form of a thin layer of water film on the surface of the fine river sand.

[0022] Furthermore, the spore germination medium is 1 / 4MS+8.0-8.5g / L agar.

[0023] Furthermore, the proliferation culture medium is MS+30g / L sucrose+8.0-8.5g / L agar.

[0024] Furthermore, during the induction process, the temperature was maintained at 23±3°C and the light intensity was 1500-3000 lux.

[0025] The beneficial effects of the present invention are:

[0026] (1) The tissue culture seedlings obtained by the soil extract of the present invention can maintain a stable Cd / Pb enrichment capacity and can be directly transplanted to heavy metal contaminated soil after emergence, thereby providing a large number of seedlings for the application of Pteris spheniscus in the restoration of heavy metal contaminated soil in mining areas and ecological restoration;

[0027] (2) The present invention uses a culture medium containing soil extract to induce sporophytes, and the soil extract is above the sand surface, providing a more sufficient moisture environment, which is conducive to sperm movement; compared with conventional culture media, the sporophyte induction rate is increased, and the induction rate reaches more than 80%;

[0028] (3) The present invention uses a culture medium containing soil extract for rooting, which effectively improves the rooting rate of sporophyte seedlings, making the rooting rate reach 100%, and overcomes the problem that the culture medium is easily contaminated in conventional bottle opening and domestication. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a schematic diagram of the sporangium and spore morphology of Pteris sphenanthera obtained in Example 1 of the present invention. A is sporophylls, B is spore powder, C is sporangium, and D is spores.

[0030] Figure 2 Schematic diagram of the changes in each process of sporophyte induction in Example 1 of the present invention, A is a diagram after 7d of inoculation in step 1, B is a diagram after 20d of inoculation in step 1 (germination), C is a diagram after 60d of inoculation in step 1 (germination), D is a diagram after 30d of proliferation in step 2 transferred to a proliferation medium, F is a diagram after 60d of proliferation in step 2 transferred to a proliferation medium, G is a diagram after 60d of sporophyte induction culture, H is a diagram after 60d of rooting culture, I is a diagram of the growth of the obtained sporophyte seedlings after transplanting, and J is a diagram of the growth of the sporophyte seedlings after transplanting.

[0031] Figure 3 The diagrams are of the prothallus states under different proliferation culture medium culture conditions in Comparative Example 2 of the present invention, A is MS culture medium, B is KNOP'S culture medium, C is hormone culture medium containing NAA and KT, and D is MSMF culture medium.

[0032] Figure 4 This is a growth diagram after 30 days of subculture development in different induction culture media in Comparative Example 3 of the present invention. DETAILED DESCRIPTION

[0033] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0034] A method for cultivating sporophyte seedlings of Pteris spheniscus, comprising the following steps:

[0035] Step 1: Spore germination, inoculate the disinfected spores into a spore germination medium, germinate in 2 to 4 weeks, and produce prothallus; the spore germination medium is 1 / 4MS + 8.0 to 8.5 g / L agar.

[0036] Step 2: Prothallus proliferation. After spore germination for 1 to 2 months, the prothallus diameter is not less than 0.5 cm. The prothallus is transferred to the proliferation medium. The diameter of the prothallus cluster grows to 3 to 4 cm. The proliferation medium is MS + 30 g / L sucrose + 8.0 to 8.5 g / L agar.

[0037] Step 3: Sporophyte induction, the prothallus mass is moved to the induction medium, and the sporophyte grows in 1 to 2 months; before the prothallus mass is moved to the induction medium, the prothallus mass is first soaked in an abscisic acid solution for 30 minutes and then transferred to a bottle.

[0038] The preparation process of induction medium is as follows:

[0039] Select coarse river sand with a particle size of 0.4 to 4 mm, wash it repeatedly until it is clear, dry it and dispense it into culture bottles;

[0040] Add soil extract, seal after subpackaging, sterilize with high-temperature steam at 120°C for 20 to 30 minutes, and then cool to obtain the product.

[0041] In the induction culture medium, the soil extract should cover 1 / 3 of the height of the protothalli.

[0042] Step 4: Rooting, divide the prothallus mass from which the sporophyte grows in step 3 and transfer it to the rooting medium. Mature sporophyte seedlings can be obtained in 1 month; then transplant and acclimate.

[0043] The preparation process of rooting medium is as follows:

[0044] Select fine river sand with a particle size of 0.1 to 0.25 mm, add soil extract, mix and stir, and let it stand for 10 minutes; remove the liquid (generally by absorbing excess liquid on the surface), repeat 3 to 4 times, seal after subpackaging, sterilize with high-temperature steam at 120°C for 20 to 30 minutes, and cool to obtain.

[0045] The soil extract is in the form of a thin water film on the surface of fine river sand.

[0046] The soil extract in the rooting medium appears as a thin layer of water film on the surface of the fine river sand, that is, after absorbing all the excess water on the surface, the sand surface appears to be very saturated with water, but when the culture bottle is tilted, there will not be much water gathering in the low place.

[0047] Both the induction medium and the rooting medium contain soil extract; the soil extract is extracted from peat soil, humus soil and soil in the native habitat of ferns.

[0048] The extraction process of soil extract is as follows:

[0049] The soil is sieved and mixed with deionized water, covered with film and soaked for 24 hours; rubbed repeatedly with gauze and filtered, and the filtered liquid is centrifuged to obtain the extract. The centrifugal speed is 4000r / min, and the centrifugation is 10min. The ratio of deionized water to soil added to the soil is determined according to the actual situation and does not affect the results; the best result is a ratio of water to soil of 2:1, but it is not limited to this ratio.

[0050] During the entire induction process, the temperature was maintained at 23±3°C and the light intensity was 1500-3000 lux.

[0051] Example 1

[0052] A method for cultivating sporophyte seedlings of Pteris spheniscus, comprising the following steps:

[0053] Step 1: Spore germination, take an appropriate amount of Pteris spheniscus spores and wrap them in phosphorus-free filter paper, put them in 75% alcohol for disinfection for 20-30 seconds; then disinfect them with a mixture of 1% sodium hypochlorite and 3-5 drops of Tween 20 for 3 minutes, rinse them with sterile water for 5 times, pour them into spore germination medium (1 / 4MS+8.0g / L agar), and spread them evenly with a sterile glass coating rod.

[0054] Spores begin to germinate around 10 days, and prothallus is formed in around a month.

[0055] Step 2: Prothallus proliferation. Prothallus clusters are formed about 2 months after sporophyte germination. When the prothallus cluster is not less than 0.5 cm, the prothallus is cut into small pieces of about 2 cm and inoculated into a proliferation medium for proliferation culture. The diameter of the prothallus cluster grows to about 3 cm; the proliferation medium is MS + 30g / L sucrose + 8.5g / L agar. After 60 days of proliferation culture, the next step of culture is carried out.

[0056] Before induction, soil extract was prepared.

[0057] Use Yuexi soil (humus soil, organic matter content> 30%, pH 6.13, loose soil, dark color), pass through a 2mm sieve and mix with deionized water in a ratio of 1:2, and soak in the dark for 24 hours after film coating. Use 5 layers of gauze for initial filtration, wrap the soil residue obtained by filtration in gauze and rub it repeatedly to squeeze out water. The filtrate after multiple filtration is placed in a centrifuge tube, centrifuged at 4000r / min for 10 minutes, take the upper clear liquid as the leaching liquid, put it into a container and place it in a 4℃ refrigerator for standby use.

[0058] Step 3: Sporophyte induction

[0059] First prepare the induction medium:

[0060] Select coarse river sand with a particle size of 2 mm, add deionized water and wash repeatedly until the liquid is clear, then drain the river sand, spread it flat on an iron plate and put it into an oven. After complete drying at 110°C, the powder is packed into a culture bottle (2 cm thickness).

[0061] Add soil extract until the liquid is 0.4 cm above the sand surface, sterilize with high-temperature steam at 120°C for 30 minutes and cool to room temperature.

[0062] The protothallus was washed with deionized water and transferred into the induction culture medium in a sand bottle and cultured in a light culture room.

[0063] After about a month, sporophytes will gradually grow and roots will form after 60 days of cultivation.

[0064] Step 4: Rooting

[0065] First prepare the rooting medium:

[0066] Select fine river sand with a particle size of 0.2 mm, add soil extract, mix and stir, let stand for 10 minutes, pour out the liquid, and repeat 4 times. Then, divide the wet fine sand powder into culture bottles, sterilize with high-temperature steam at 120℃ for 30 minutes and cool to room temperature.

[0067] The original leaf mass from which the sporophyte grows is divided into small pieces, taking care not to damage the sporophyte leaves, and is transferred into a sand bottle rooting medium; roots are formed after 1 to 2 months to obtain complete sporophyte seedlings. In this embodiment, the next step of domestication is performed after 60 days of rooting culture.

[0068] After obtaining the sporophyte seedlings, acclimate

[0069] After being exposed to the sun, peat was placed in a rectangular seedling tray and poured with pure water to make the water content of the matrix reach more than 85% and the soil thickness reach 4cm. When the young spore plants were up to 4cm, the cap of the sand bottle was opened and acclimated for 4 days, and then directly transplanted to the seedling tray. The plastic film was kept moisturized, and the film was half-removed after two weeks, and completely removed after four weeks for routine management. The survival rate of young spore seedlings reached 100%.

[0070] Figure 1 The sporangium and spore morphology in Example 1. As can be seen from the figure, the spores of the mining ecotype of the Chinese pteris spheniscus are bilaterally symmetrical, yellow-brown, 30 μm×25 μm in size, with a single crack, the length of the crack is close to 1 / 3 of the equatorial axis, and the equatorial plane view is bean-shaped. The outer layer of the spore wall is thin, the surface is not smooth, and it is scaly.

[0071] Figure 2 The spore germination changes in Example 1. As can be seen from the figure, the spore germination type is centripetal, the gametophyte development is Adiantum type, the antheridia and archegonium are both of the thin capsule type, and the mature prothallus is symmetrical heart-shaped. These common characteristics can be used as the developmental characteristics of the gametophyte of the genus Pteris.

[0072] Example 2

[0073] A method for cultivating sporophyte seedlings of Pteris spheniscus, comprising the following steps:

[0074] Step 1: Spore germination, take an appropriate amount of Pteris spheniscus spores and wrap them in phosphorus-free filter paper, put them in 75% alcohol for disinfection for 20-30 seconds; then disinfect them with a mixture of 1% sodium hypochlorite and 3-5 drops of Tween 20 for 3 minutes, rinse them with sterile water for 5 times, pour them into spore germination medium (1 / 4MS+8.5g / L agar), and spread them evenly with a sterile glass coating rod.

[0075] Spores begin to germinate around 10 days, and prothallus is formed in around a month.

[0076] Step 2: Prothallus proliferation. Prothallus clusters are formed about 2 months after sporophyte germination. When the prothallus cluster is not less than 0.5 cm, the prothallus is cut into small pieces of about 2 cm and inoculated into a proliferation medium for proliferation culture. The diameter of the prothallus cluster grows to about cm; the proliferation medium is MS + 30g / L sucrose + 8.0g / L agar, pH = 5.8. After 60 days of proliferation culture, the next step of culture is carried out.

[0077] Before induction, soil extract was prepared.

[0078] Use peat soil, pass it through a 2mm sieve, and mix it with deionized water in a ratio of 1:2. After covering, soak it in the dark for 24 hours. Use 3 layers of gauze for initial filtration. Wrap the soil residue in the gauze and rub it repeatedly to squeeze out the water. The filtrate after multiple filtrations is placed in a centrifuge tube and centrifuged at 4000r / min for 10 minutes. Take the upper clear liquid as the extract, put it into a container and place it in a 4℃ refrigerator for later use.

[0079] Step 3: Sporophyte induction

[0080] First prepare the induction medium:

[0081] Select coarse river sand with a particle size of 4 mm, add deionized water and wash repeatedly until the liquid is clear, then drain the river sand, spread it flat on an iron plate and put it into an oven. After complete drying at 110°C, the powder is packed into a culture bottle (2 cm thickness).

[0082] Add soil extract so that the liquid is 0.3 cm above the sand surface, sterilize with high-temperature steam at 120°C for 20 minutes and cool to room temperature.

[0083] The protothallus was washed with deionized water and transferred into the induction culture medium in a sand bottle and cultured in a light culture room.

[0084] After about 2 months, sporophytes will gradually grow and roots will form after 60 days of cultivation.

[0085] Step 4: Rooting

[0086] First prepare the rooting medium:

[0087] Select fine river sand with a particle size of 0.25 mm, add soil extract, mix and stir, let stand for 10 minutes, pour out the liquid, and repeat 3 times. Then, divide the wet fine sand powder into culture bottles, sterilize with high-temperature steam at 120℃ for 20 minutes and cool to room temperature.

[0088] The original leaf mass from which the sporophyte grows is divided into small pieces, taking care not to damage the sporophyte leaves, and is transferred into a sand bottle rooting medium; roots are formed after 1 to 2 months to obtain complete sporophyte seedlings. In this embodiment, the next step of domestication is performed after 60 days of rooting culture.

[0089] After obtaining the sporophyte seedlings, acclimate

[0090] After being exposed to the sun, peat was placed in a rectangular seedling tray and poured with pure water to make the water content of the matrix reach more than 85% and the soil thickness reach 3cm. When the young spore plants were up to 5cm, the cap of the sand bottle was opened and acclimated for 3 days, and then directly transplanted to the seedling tray. The plastic film was kept moisturized, and the film was half-opened after two weeks, and completely opened after four weeks for routine management. The survival rate of young spore seedlings reached 100%.

[0091] Example 3

[0092] A method for cultivating sporophyte seedlings of Pteris spheniscus, comprising the following steps:

[0093] Step 1: Spore germination, take an appropriate amount of Pteris spheniscus spores and wrap them in phosphorus-free filter paper, put them in 75% alcohol for disinfection for 20-30 seconds; then disinfect them with a mixture of 1% sodium hypochlorite and 3-5 drops of Tween 20 for 3 minutes, rinse them with sterile water for 5 times, pour them into spore germination medium (1 / 4MS+8.0g / L agar), and spread them evenly with a sterile glass coating rod.

[0094] Spores begin to germinate around 10 days, and prothallus is formed in around a month.

[0095] Step 2: Prothallus proliferation. Prothallus clusters are formed about 2 months after sporophyte germination. When the prothallus cluster is not less than 0.5 cm, the prothallus is cut into small pieces of about 2 cm and inoculated into proliferation culture medium for proliferation culture. The diameter of the prothallus cluster grows to about cm; the proliferation culture medium is MS + 30g / L sucrose + 8.5g / L agar. After 60 days of proliferation culture, the next step of culture is carried out.

[0096] Before induction, soil extract was prepared.

[0097] Use the native soil of the fern, pass it through a 2mm sieve, and mix it with deionized water in a ratio of 1:2. After covering, soak it in the dark for 24 hours. Use 3 layers of gauze for initial filtration. Wrap the soil residue obtained by filtration in gauze and rub it repeatedly to squeeze out the water. The filtrate after multiple filtrations is placed in a centrifuge tube and centrifuged at 4000r / min for 10 minutes. Take the upper clear liquid as the extract, put it into a container and place it in a 4℃ refrigerator for standby use.

[0098] Step 3: Sporophyte induction

[0099] First prepare the induction medium:

[0100] Select coarse river sand with a particle size of 0.4 mm, add deionized water and wash repeatedly until the liquid is clear, then drain the river sand, spread it flat on an iron plate and put it into an oven. After complete drying at 110°C, the powder is packed into a culture bottle (2 cm thickness).

[0101] Add soil extract until the liquid is 0.5 cm above the sand surface, sterilize with high-temperature steam at 120°C for 30 minutes and cool to room temperature.

[0102] The protothallus was washed with deionized water and transferred into the induction culture medium in a sand bottle and cultured in a light culture room.

[0103] After about a month, sporophytes will gradually grow and roots will form after 60 days of cultivation.

[0104] Step 4: Rooting

[0105] First prepare the rooting medium:

[0106] Select fine river sand with a particle size of 0.1 mm, add soil extract, mix and stir, let stand for 10 minutes, pour out the liquid, and repeat 4 times. Then, divide the wet fine sand powder into culture bottles, sterilize with high-temperature steam at 120℃ for 30 minutes and cool to room temperature.

[0107] The original leaf mass from which the sporophyte grows is divided into small pieces, taking care not to damage the sporophyte leaves, and is transferred into a sand bottle rooting medium; roots are formed after 1 to 2 months to obtain complete sporophyte seedlings. In this embodiment, the next step of domestication is performed after 60 days of rooting culture.

[0108] After obtaining the sporophyte seedlings, acclimate

[0109] After being exposed to the sun, peat was placed in a rectangular seedling tray and pure water was poured in to make the water content of the matrix reach more than 85% and the soil thickness reach 3cm. When the young spore plants were up to 3cm, the cap of the sand bottle was opened and acclimated for 5 days, and then directly transplanted to the seedling tray. The plastic film was kept moisturized, and the film was half-opened after two weeks, and completely opened after four weeks for routine management. The survival rate of young spore seedlings reached 100%.

[0110] Comparative Example 1

[0111] A method for cultivating sporophyte seedlings of Pteris spheniscus

[0112] The other steps are the same as those in Example 1, except that the germination medium in step 1 is different. In this comparative example, 1 / 2MS+8.0g / L agar and 1 / 4MS+30g / L sucrose+8.0g / L agar are respectively selected to repeat the experiment according to the steps of Example 1. The results of spore germination and prothallus proliferation culture are shown in Table 1.

[0113] Table 1. Results of spore germination and prothallus proliferation culture on different germination media

[0114]

[0115] From the above statistical results, it can be seen that the selected germination medium has the best results and the germination rate has been significantly improved.

[0116] Comparative Example 2

[0117] A method for cultivating sporophyte seedlings of Pteris spheniscus

[0118] The other steps are the same as those in Example 1, except that the proliferation medium in step 2 is different. In this comparative example, 1 / 2MS+30g / L sucrose+8.5g / L agar and MS+8.5g / L agar are respectively selected to repeat the experiment according to the steps of Example 1. The results of the proliferation and differentiation of the prothallus are shown in Table 2.

[0119] Table 2. Proliferation and differentiation results of prothallus in different proliferation medium

[0120]

[0121]

[0122] From the above statistical results, it can be seen that the selected proliferation culture medium has the best results and the proliferation multiples have been significantly improved.

[0123] The results of prothallus status obtained from each culture medium are as follows Figure 3 As shown in the figure, after inoculation into MS medium, the leaves of the prothallus are stretched, the color is emerald green, and the growth and proliferation are normal as shown in A. Inoculation into KNOP'S medium. This medium has simple ingredients, and it can be seen that the leaves of the prothallus group are seriously degraded, as shown in Figure B. Inoculation into hormone medium containing NAA and KT, it can be seen that the leaves become very dense, which is not conducive to the subsequent transformation into sporophyte, as shown in Figure C. Inoculation into MSMF medium, the medium has a high concentration of inorganic salts, which causes the leaves of the prothallus group to become thicker, which is also not conducive to the subsequent transformation into sporophyte, as shown in Figure D.

[0124] Comparative Example 3

[0125] A method for cultivating sporophyte seedlings of Pteris spheniscus

[0126] The other steps are the same as those in Example 1, except that the induction medium in step 3 is different. This comparative example selects the existing solid culture medium and liquid culture medium to compare with the sand bottle culture method in Example 1. The induction liquid in each culture medium is selected from soil extract, clean water, and 0.1% KH2PO4. The results are shown in Table 3. The induction liquid in the sand bottle culture in the table is soil extract, which is the result of Example 1. It can be seen from the following statistical results that the induction medium used in the present invention has a significant effect on the induction of sporophytes.

[0127] Table 3. Sporophyte induction culture results

[0128]

[0129] Note: +++ represents the induction of a large number of sporophytes, ++ represents the induction of more sporophytes, + represents the induction of a small number of sporophytes, and - represents no or very little sporophyte formation.

[0130] As can be seen from Table 3, when only soil extract and 0.1% KH2PO4 were used as induction solutions in the liquid culture medium, a small amount of sporophytes appeared and the number of induced sporophytes decreased significantly.

[0131] The growth of sporophytes Figure 4 As shown in the figure, it can be seen that soil extract + sand bottle has induced the formation of more spores.

[0132] Comparative Example 4

[0133] A method for cultivating sporophyte seedlings of Pteris spheniscus

[0134] The other steps are the same as those in Example 1, except that the rooting medium in step 4 is different. In this comparative example, 1 / 2MS+15g / L sucrose+IBA (0.2-0.5mg / L) and 1 / 2MS+15g / L sucrose+2,4-D (0.2-0.5mg / L) are respectively selected to repeat the experiment according to the steps of Example 1. The rooting results are shown in Table 4 after statistics.

[0135] Table 4. Rooting culture effect results

[0136]

[0137] As can be seen from Table 4, the rooting rate of other existing culture media is low and the root hairs are thin. However, the rooting efficiency is significantly improved and the root hairs are thicker when the rooting culture medium of the present invention is used, which shows that the culture medium of the present invention has a significant effect on rooting.

[0138] The present invention adopts a unique induction and rooting culture medium and method to ensure sufficient water to induce fertilization, so that young sporophytes can grow in 1-2 months, the sporophyte induction rate reaches 80%, and the rooting effect is excellent, with a rooting rate of about 100%. The rooting method adopted by the present invention effectively improves the rooting rate of sporophyte seedlings, so that the rooting reaches 100%; it overcomes the problem that the culture medium is easily contaminated in the conventional bottle opening and domestication, so that the survival rate of seedling domestication and transplantation reaches more than 90%. The tissue culture method provided does not require any additional addition of any hormones from spore germination to rooting, and the operation is simple. A set of simple, feasible and complete Sinensis rapid propagation system is provided, and the tissue culture seedlings obtained by soil immersion can maintain a stable Cd / Pb enrichment ability, and can be directly transplanted to heavy metal contaminated soil after germination, providing a large number of seedlings for the application of Sinensis in the restoration of heavy metal contaminated soil and ecological restoration in mining areas.

Claims

1. A method for cultivating sporophyte seedlings of Pteris spheniscus, characterized in that: The following steps are involved: Step 1: Spore germination, inoculate the sterilized spores into a spore germination medium, germinate in 2 to 4 weeks, and produce prothallus; the spore germination medium is 1 / 4MS + 8.0 to 8.5 g / L agar; Step 2: Prothallus proliferation: 1-2 months after spore germination, the prothallus diameter is not less than 0.5 cm, and the prothallus is transferred to the proliferation medium, and the prothallus group diameter grows to 3-4 cm; the proliferation medium is MS + 30g / L sucrose + 8.0-8.5g / L agar; Step 3: Sporophyte induction: Move the prothallus mass to the induction medium and grow sporophytes in 1 to 2 months. The preparation process of the induction medium is as follows: Select coarse river sand with a particle size of 0.4 to 4 mm, wash it repeatedly until it is clear, dry it and dispense it into culture bottles; Add soil extract, seal after subpackaging, sterilize at high temperature, and cool to obtain; Step 4: Rooting, the prothallus mass from which the sporophyte grows in step 3 is split and transferred to a rooting medium. Mature sporophyte seedlings can be obtained in one month, and then transplanted and domesticated; Both the induction medium and the rooting medium contain soil extract; the soil extract is extracted from peat soil, humus soil and soil in the native habitat of ferns; the preparation process of the rooting medium is as follows: Select fine river sand with a particle size of 0.1 to 0.25 mm, add soil extract, mix and stir, and then let it stand for 10 minutes; remove the liquid, repeat 3 to 4 times, package it, seal it, sterilize it at high temperature, and cool it to obtain it.

2. The method for cultivating sporophyte seedlings of Pteris spheniscus according to claim 1, characterized in that: The extraction process of the soil extract is as follows: The soil was sieved and mixed with deionized water, and then covered with a film and soaked for 24 hours; it was rubbed repeatedly with gauze and filtered, and the filtered liquid was centrifuged to obtain the extract.

3. The method for cultivating sporophyte seedlings of Pteris spheniscus according to claim 2, characterized in that: The centrifugation parameters are as follows: The centrifugal speed was 4000 r / min and the centrifugation lasted for 10 min.

4. The method for cultivating sporophyte seedlings of Pteris spheniscus according to claim 1, characterized in that: The induction culture medium and soil extract solution cover 1 / 3 of the height of the protothalli.

5. The method for cultivating sporophyte seedlings of Pteris spheniscus according to claim 1, characterized in that: The soil extract in the rooting medium is in the form of a thin layer of water film on the surface of the fine river sand.

6. The method for cultivating sporophyte seedlings of Pteris spheniscus according to claim 1, characterized in that: During the induction process, the temperature was maintained at 23±3°C and the light intensity was 1500-3000 lux.

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