Method for rapidly propagating spores of cyatheridae rare and valuable fern type pteridium aquilinum

Abstract

The invention discloses a rapid spore propagation method of a cyatheridae rare fern type pteridium aquilinum var. Spinulosa var. Spinulosa. Comprising the following steps: (1) collecting and storing spores; (2) selecting and preparing a spore culture medium; (3) spore treatment and sowing; (4) culturing spores; (5) observing and maintaining gametophytes; (6) transplanting sporophyte young plants; (7) seedling management and protection; (8) transplanting seedlings. When seedlings in the seedling raising box grow to about 5-8 cm high, the seedling raising box is moved into a non-imitation cloth seedling raising bag in an outdoor greenhouse nursery. Compared with tissue culture, the method has the advantages of high success rate, simplicity and high efficiency. A manual climatic box is not needed in a spore-gametophyte cultivation stage, but on a light-cared cultivation frame, so that the operation is simple and easy, and the limitation of space is avoided.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant breeding, and particularly relates to a method for rapid spore propagation of Sphaeropteris lepifera, a rare fern of the Cyatheaceae family. Background Art

[0002] The reproduction of lycophytes and ferns is different from that of seed plants, showing an obvious alternation of generations, namely the sporophyte generation and the gametophyte generation, and both their gametophytes and sporophytes can live independently.

[0003] The main reproduction methods of ferns include spore reproduction, division propagation, vegetative propagation, and tissue culture. Among them, spore reproduction is the main way of fern reproduction, but spore reproduction has high requirements for the environment, which restricts the reproduction of ferns to a certain extent. Ornamental ferns with structures such as creeping rhizomes, bulbils, thickened auricles, or tubers can be propagated vegetatively or by division. If ferns cannot be effectively propagated by spore reproduction, tissue culture methods can be adopted. At present, only a very small part has been successfully tissue-cultured, mainly concentrated in a few genera and species such as Nephrolepis cordifolia, Platycerium bifurcatum, Platycerium wandae, Matteuccia struthiopteris, Pteris vittata, Neottopteris nidus, etc., and some of them have entered commercial operation. Although some genera and species have been successfully tissue-cultured, there are problems such as long cycle and low proliferation rate (Wu Fanhua, Zhang Chao, Zhou Yadong, etc. Species, Reproduction Methods and Applications in Gardens of Chinese Ornamental Ferns. Acta Tropica Sinica, 2012, 3(4): 387-392).

[0004] Rare ferns are protected by the state or local governments. There are few existing plants, and they have high requirements for the living environment. In the wild, due to their low reproductive ability, they have difficulties in breeding, low population renewal ability, and are at risk of endangerment or extinction, which is not conducive to the protection and sustainable utilization of their species. Therefore, carrying out artificial spore breeding or tissue culture breeding is an effective measure to promote the expansion of their populations.

[0005] At present, the research on fern reproduction mainly focuses on tissue culture and rapid propagation technology. However, this method is costly and requires strict aseptic conditions, with high requirements for equipment. While conventional soil cultivation has the characteristics of relatively low cost, simple management, large output, and being suitable for the spore reproduction of various ferns, it has more development space in actual production.

[0006] The Cyatheaceae is one of the few fern families with an above-ground trunk among extant ferns. It is usually tree-like and is also known as tree fern. It is widely distributed in humid regions from the tropics to the subtropics around the world. There are about 18 species of Cyatheaceae in China, belonging to 3 genera, including Sphaeropteris, Alsophia, and Gymnosphaera. With its ancient and unique morphology, tree fern is not only suitable for the production of handicrafts and garden ornamentation, but also can be used in traditional Chinese medicine treatment. It has important ornamental, horticultural, and medicinal values, and has received extensive social attention. It is listed as a first-class protected plant in many countries. Globally, the species of Cyatheaceae are continuously decreasing due to natural and human reasons. At present, they have been listed in Appendix II of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES), and most species of Cyatheaceae have also been listed in the Red List of the International Union for Conservation of Nature (IUCN). In China, except for Gymnosphaera dentitulata and G. metteniana, the plants of Cyatheaceae are all listed in the List of National Key Protected Wild Plants in 2021.

[0007] Sphaeropteris lepifera, a relic plant known as a "living fossil", belongs to the genus Sphaeropteris of the Cyatheaceae family. Currently, there are only a small number of remnants in southern China and some countries in Southeast Asia, and the number of wild plants is scarce. Sphaeropteris lepifera has high requirements for the living environment and likes a shady, humid, warm and hot environment. Due to its own medicinal value, ornamental value, and rarity, it is very easy to be discovered and dug up illegally. Scholars have conducted relatively in-depth research on the morphological development, systematic classification, and germplasm preservation of Sphaeropteris lepifera. In terms of its reproduction, there are relatively many studies on tissue culture. For example, Ma Hongna et al. used 1 / 2MS medium, with the macro and micro elements being 1 / 2 of the MS medium, and the content of iron salts and organic substances being the same as that of the MS medium, to carry out the experimental study on the spore inoculation of Sphaeropteris lepifera (Ma Hongna, Li Yang, Tan Longyan, etc. Spore Propagation and Rejuvenation of Sphaeropteris lepifera. Acta Horticulturae Sinica, 2010, 37(10): 1679-1684).

[0008] CN111096173A discloses an artificial breeding method for Sphaeropteris lepifera. Based on the technical problems such as the difficulty of spore reproduction of Sphaeropteris lepifera, and the harsh requirements of spores for the water retention, air permeability, and nutrient supply of the substrate, the invention preliminarily constructs a spore breeding system for Sphaeropteris lepifera by regulating factors such as different harvesting periods, sowing methods, transplanting times, and substrate types. However, the spore reproduction in this method is relatively difficult, and the time for growing into gametophytes is relatively long.

[0009] CN110786243A discloses a method for tissue culture and propagation of Alsophila spinulosa, specifically inducing and transferring the curled leaves of Alsophila spinulosa to the GGB medium, and then obtaining Alsophila spinulosa seedlings through proliferation culture and differentiation culture. After a long time of tissue culture, the explants are easily contaminated during disinfection, which affects the vitality of Alsophila spinulosa. The survival rate of this culture method is relatively low, and a large-scale propagation system cannot be formed.

[0010] CN113349058B discloses a propagation technology and a method for field reintroduction of Alsophila spinulosa. Based on the technical problems that it is difficult to propagate the spores of Alsophila spinulosa, and the spores have strict requirements for the water retention, air permeability and nutrient supply of the substrate, the invention inoculates the spores into the germination medium, optimizes the disinfection scheme, changes the culture substrate at the same time, explores placing sterile filter paper in the culture dish, and regulates the growth conditions of the spores of Alsophila spinulosa to achieve the breeding effect of accelerating the spore to gametophyte stage.

[0011] CN110786243B discloses a propagation method for tissue culture of Alsophila spinulosa. Using the young and tender leaves of Alsophila spinulosa as explants, culturing to form GGB, transferring and culturing in the primary medium to form prothallus clusters, then transferring and culturing in the prothallus proliferation medium to proliferate a large number of prothallus clusters, and then transferring to the differentiation medium for induction culture of clustered buds. Some of the clustered buds form rootless young sporophytes, and the rooted seedlings are continuously cultured, and the clustered buds quickly grow into Alsophila spinulosa seedlings with roots.

[0012] At present, the disclosed technologies are mainly tissue culture and spore sowing propagation methods. The defect of using the MS medium is that the experimental cost is high, and the medium is easily contaminated, which easily leads to experimental failure. When using spore soil sowing for propagation, the spores have defects such as short lifespan and long propagation cycle. When the spores of Alsophila spinulosa germinate, they have high requirements for the environment and substrate. Unlike the spores of conventional fern plants, good germination rate can be achieved by controlling temperature, humidity and light. It has high requirements for humidity and substrate. Often, when the inappropriate substrate is used, the germination rate drops significantly.

[0013] The growth of fern plants is divided into gametophyte stage and sporophyte stage. From the germination of very tiny spores to gametophytes and then growing new spore leaves, and then through multiple sub-planting and transplanting to become high-quality seedlings that can be directly planted in the ground or potted is a very complex process. During the spore germination and gametophyte stage, plants have high requirements for environmental factors such as light, temperature and water. Therefore, it is necessary to find a method for spore soil sowing germination and seedling raising of the endangered plant Alsophila spinulosa that is lower in cost, higher in efficiency, easy to operate, convenient for sampling and observation at any time, and has a high seedling emergence rate in scientific experiments and seedling raising practices. Summary of the Invention

[0014] The object of the present invention is to provide a method for rapid propagation of spores of the rare fern Sphaeropteris lepifera in the Cyatheaceae family, which is a simple and efficient general method with remarkable advantages such as low cost, easy operation, easy management, convenient sampling and observation, fast growth rate, high emergence rate, and strong universality.

[0015] The method for rapid propagation of spores of the rare fern Sphaeropteris lepifera in the Cyatheaceae family according to the present invention includes the following steps:

[0016] (1) Spore collection and preservation

[0017] Collect the leaves of Sphaeropteris lepifera in which the spores are mature and the spore powder has not dispersed between May and August every year, place them in a cool and dry place, remove impurities from the spores after drying, collect the naturally fallen spores, and store the collected spores at low temperature for standby or directly sow them.

[0018] (2) Selection and preparation of spore culture substrate

[0019] Substrate treatment: Use the Norwegian Jiffy seedling block as the seedling culture substrate, remove the non-woven fabric wrapped around the seedling block, sterilize it with a high-pressure steam sterilizer, dry it, and cool it for standby. Sterilize pure water or ultrapure water in the same way for standby;

[0020] Add pure water to the seedling culture substrate, expand and crush it, then spread it flat in a transparent seedling tray. After sowing, cover it with a lid and seal it. Sow as evenly as possible, pour pure water to keep it in a moist state, and place it in an incubator for cultivation;

[0021] (3) Spore treatment and sowing

[0022] After taking out the spores and sterilizing them, make a spore suspension with water, shake it well, suck out the spore suspension, and spray it evenly on the seedling culture substrate. Cover the box lid, and keep the air holes on the box lid closed;

[0023] (4) Spore culture

[0024] Place the seedling box sown with spores on a light culture rack, and install fluorescent lamps on the culture rack. The culture conditions are set as follows: temperature 21–26°C, with fluorescent lamp illumination both during the day and at night, and the light intensity is 1100–1600 lux;

[0025] (5) Gametophyte observation and maintenance

[0026] The spores will form a preliminary heart-shaped sheet in 3–5 weeks after sowing. When tiny green sheets appear on the surface of the substrate in the seedling box and gradually cover the surface of the substrate like a layer of moss, spray water once every 2–3 days to keep it moist. When the gametophytes grow densely and many sporophytes appear, prepare for dividing and transplanting. After transplanting, water it to keep it moist and promote fertilization;

[0027] (6) Transplanting of young sporophyte plants

[0028] When the newly grown sporophytes reach about 1 cm in height, transplant them by changing the tray. The transplanting substrate is a mixture of sterilized peat soil, vermiculite, and perlite. When the sporophytes reach 1.5 - 2 cm in height, use sterilized forceps to pick up a cluster of sporophytes for transplanting into a seedling-raising tray.

[0029] (7) Seedling management

[0030] After the seedlings are transplanted, keep the ventilation holes of the seedling-raising tray open. Spray water once every 1 - 2 weeks. If mold appears in the seedling box, remove the mold manually in time, and spray and sterilize with 0.5% - 1% baking soda solution, spraying 1 - 2 times of the baking soda solution.

[0031] (8) Seedling transplanting

[0032] When the seedlings in the seedling box grow to about 5 - 8 cm in height, transfer the seedling box to a non-woven fabric seedling bag in an outdoor greenhouse nursery.

[0033] Preferably, the spores of the Sphaeropteris lepifera are mature, and the spores are brown.

[0034] Preferably, in step (1), the collected leaves are sealed in a clean sealed bag to prevent the mixing of different plant germplasms. Then, cut the fresh plant leaves into small pieces and place them in a sulfur paper bag, and place them in a cool and dry place. After about one week, remove impurities from the spores, collect the naturally shed spores, and avoid artificial interference and peeling the sporangia on the sporophylls. Wear gloves for operation and change gloves for each sample to avoid spore mixing. The collected spores are put into a centrifuge tube and placed in a -80°C low-temperature refrigerator for standby.

[0035] Preferably, in step (2), pay attention to making the substrate as flat as possible before sowing, sowing as evenly as possible, watering with a small sprayer, wearing a mask and gloves throughout the process, using sterile water to avoid contamination by miscellaneous bacteria. After sowing, water to keep the substrate in a moist state. Observe once every 2 days and supplement water appropriately.

[0036] Preferably, for the sterilization in step (3), take out the centrifuge tube containing the spores, add 1 ml of 5% NaClO aqueous solution, shake and disinfect continuously for 10 minutes, then centrifuge at 10000 r / min for 2 minutes, wash and centrifuge with sterile water and repeat 3 times. After pouring out the waste liquid, make a spore suspension according to a volume ratio of 1:100.

[0037] Preferably, for the light cultivation in step (4), the light cultivation rack should be placed in a separate room that has been sterilized, and no mosquitoes or other insects can enter or exit.

[0038] Preferably, for the transplanting in step (5), a cluster is about the size of a soybean, and the spacing is 1.2 - 1.5 cm.

[0039] Preferably, the peat soil: vermiculite: perlite are mixed in a mass ratio of 8:1:1.

[0040] Preferably, the substrate of the non-woven fabric seedling bag is: peat soil, pine bark, vermiculite, perlite mixture = 6:2:1:1, by mass ratio.

[0041] Preferably, the peat soil is NOVARBO peat.

[0042] Preferably, the collection date of the spores of Sphaeropteris lepifera is preferably in June.

[0043] Preferably, steps (4)-(7) of the rapid propagation method are carried out in a greenhouse equipped with facilities such as a blower, a sunshade net, and a plastic film, ensuring that the high temperature in summer does not exceed 30 °C and the low temperature in winter is not lower than 10 °C.

[0044] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0045] 1. Excellent substrate: In the stage of cultivating the sporophyte into the gametophyte, the selected substrate is the Jiffy seedling block from Norway, which is generally used for the growth after seed sowing and the cultivation substrate for small seedlings. Currently, it is not used for the spore germination of ferns. The Jiffy seedling block is made of coconut coir, peat, and nutrients, with good water permeability and air permeability, sufficient nutrition, meeting the requirements for spore germination, and having a high spore germination rate. The NOVARBO peat used in the stage of transplanting the sporophyte has the characteristics of light weight, strong water retention ability, balanced nutrition, and does not contain harmful substances, pathogens, and weed seeds.

[0046] 2. The selected seedling box is a thickened pp resin seedling tray with multiple rows of small holes at the bottom of the box. A tray is placed under the seedling box, and a transparent plastic lid is placed on it. There are 3 ventilation holes on the top of the lid. This combination of seedling boxes can not only prevent bacteria and impurities from entering, but also can be directly watered on the tray, which is easy to water and maintain, and effectively reduces the contact between the gametophyte and the outside world.

[0047] 3. The propagation process is simple and efficient, and industrial production can be realized: Compared with tissue culture, this method has a high success rate, is simple and efficient. In the stage of cultivating spores to gametophytes, an artificial climate chamber is not required, but on a light and material culture rack, the operation is simple, avoiding the limitation of space.

[0048] 4. Low cost: All materials of the present invention are conventional materials for seedling experiments, with low cost. Description of the Drawings

[0049] Figure 1 is the mature sporangiophore of Sphaeropteris lepifera;

[0050] Figure 2 is the seedling block;

[0051] Figure 3It is the sporangiophyll of the spore of the seedling-raising box and the divided Sphaeropteris lepifera;

[0052] Figure 4 It is the Sphaeropteris lepifera plant transplanted into the experimental greenhouse;

[0053] Figure 5 It is the seedling-raising box, which is respectively the tray 1, the seedling-raising tray 2, the box cover 3 and the whole 4. Specific implementation mode

[0054] The following embodiments are further descriptions of the present invention, rather than limitations on the present invention.

[0055] The seedling-raising box used in the following embodiments ( Figure 5 ) Specification: It is a thickened pp resin seedling-raising tray, rectangular, 8 cm high, with an inner diameter of 30 cm * 23 cm in length and width, and an outer diameter of 34.5 cm * 27.5 cm. There are multiple rows of small holes at the bottom of the box. A tray is placed under the seedling-raising box, and a transparent plastic lid is placed on it. There are 4 ventilation holes on the top of the lid, which are divided into two groups.

[0056] Flowerpot tray: rectangular, with an inner diameter of 31.5 cm * 23.5 cm in length and width, and 2 cm high.

[0057] The box cover is a transparent plastic cover, 15.5 cm high, with a top diameter of 30.5 * 23 cm in length and width. There are two circular ventilation holes on it, and there are rotatable black plastic knobs on the ventilation holes, which can cover the ventilation holes after rotation.

[0058] Example 1:

[0059] Collect mature spores on the wild Sphaeropteris lepifera in Houhuayuan Village, Nan'ao County, Shantou City. Obtain spore resources by collecting spore leaves in June. At this time, the spores on the back of the mature Sphaeropteris lepifera leaves are brown. Select the leaves where the spores are mature and the spore powder has not dispersed ( Figure 1 ).

[0060] The materials collected in the wild are sealed with a clean envelope and sealed bag. Cut the fresh plant leaves into small pieces and place them in a sulfur paper bag, and place them in a cool and dry place. After about a week, remove impurities from the spores, collect the naturally fallen spores, and be careful not to peel the sporangium groups on the sporangiophyll. Wear gloves and change gloves for each sample processed to avoid spore mixing.

[0061] The collected spores are put into a centrifuge tube and placed in a -80 °C low-temperature refrigerator for standby.

[0062] Use the Norwegian Jefe seedling-raising block (Norwegian Jefe diameter 30 mm, Weifang Jinnong Biotechnology Co., Ltd.) as the seedling-raising substrate ( Figure 2 ), remove the non-woven fabric wrapped around the seedling-raising block, sterilize it with a high-pressure steam sterilizer (120 °C), dry it for 12 minutes, and cool it for standby; sterilize pure water or ultrapure water in the same way for standby.

[0063] Add an appropriate amount of pure water to the seedling-raising substrate at a ratio of 1:200. After swelling and crushing, spread it evenly in the pp resin seedling-raising tray in the seedling-raising box. After sowing, cover it with a transparent lid and seal it. There are 3 ventilation holes on the box lid. Then place the seedling-raising tray on the flower pot tray. Sow as evenly as possible, pour pure water to keep it in a moist state, and place it in an incubator for cultivation.

[0064] Before sowing, the substrate should be as flat as possible, and sowing should be as even as possible. Use a small sprayer for watering. Wear a mask and gloves throughout the process and use sterile water. After sowing, water to keep the substrate in a moist state. Observe it every 2 days and supplement water appropriately.

[0065] Take out the centrifuge tube containing spores, add 1 ml of 5% NaClO aqueous solution by mass fraction, shake it continuously for disinfection for 10 minutes, then centrifuge at 10000 r / min for 2 min, wash and centrifuge with sterile water and repeat 3 times. After pouring out the waste liquid, make a spore distilled water suspension according to a volume ratio of 1:100.

[0066] Shake the spores well, suck the spore suspension with a pipette, and spray it evenly on the substrate. Cover the box lid, and keep the ventilation holes on the box lid closed. Place the seedling-raising box sown with spores on the light culture rack. There are fluorescent lamps installed on the culture rack, and the culture conditions are set as follows: temperature 25 °C, with fluorescent lamp illumination both during the day and at night, and the light intensity is 1200 lux.

[0067] The spores will form heart-shaped flakes in about 5 weeks after sowing. When tiny green flakes appear on the surface of the substrate in the seedling-raising box ( Figure 3 ), and gradually cover the surface of the substrate like a layer of moss, spray and water once every 2 days to keep it moist. After 6 weeks, the gametophytes grow densely and many sporophytes appear. The spore germination rate is 69%, and it is ready for ramet transplantation. When transplanting, a cluster the size of a soybean is taken as one group, and the spacing is 1.5 cm. After transplantation, water frequently to keep it moist.

[0068] After 9 weeks, when the newly grown sporophytes grow to about 1 cm high, transplant them to a new seedling-raising tray. The transplanting substrate is a mixture of sterilized peat soil (PH = 5.6), vermiculite, and perlite (peat soil: vermiculite: perlite = 8:1.5:1.5 by mass ratio). The substrate is fully supplied with water, and the spacing of the transplanted seedlings is 2 cm. Use sterilized forceps to pick up a cluster of sporophytes for transplantation. When the sporophytes grow to 1.5 - 2 cm high (1 - 2 months), use sterilized forceps to pick up a cluster of sporophytes for tray-changing transplantation.

[0069] After the seedlings are transplanted, keep the ventilation holes of the seedling-raising box open, and spray and water once every 1 - 2 weeks.

[0070] When the seedlings in the seedling-raising box grow to about 5 - 8 cm high, move the seedling-raising box to a non-woven fabric seedling bag in the outdoor greenhouse nursery (Figure 4 ) Among them, the nursery bag has a specification of 15 cm in diameter and 10 cm in height. The matrix ratio is: peat soil: pine bark: vermiculite: perlite = 6:2:1:1 by mass ratio. The transplanting survival rate reaches 93%.

[0071] Example 2:

[0072] Collect mature spores on the tree of the fern collection penholder in the South China Botanical Garden, Guangzhou. Obtain spore resources by collecting spore leaves in July. At this time, the spores on the back of the mature penholder tree leaves are brown. Select the leaves with mature spores and the spore powder not scattered.

[0073] Wrap the materials collected in the wild with clean newspapers. Cut the fresh plant leaves into small pieces and place them in a sulfur paper bag. Place it in a cool and dry place. After 8 days, remove impurities from the spores and collect the naturally fallen spores.

[0074] Put the collected spores into a centrifuge tube, sow them in time or place them in a -80 °C low-temperature refrigerator for standby.

[0075] Preparation of nursery substrate: Use the Norwegian Jefe nursery block (Norwegian Jefe with a diameter of 30 mm, Weifang Jinnong Biotechnology Co., Ltd.) as the nursery substrate. Remove the non-woven fabric wrapped around the nursery block, sterilize it with a high-pressure steam sterilizer (120 °C) for 10 minutes, dry it, and cool it for standby; sterilize pure water in the same way for standby.

[0076] Add an appropriate amount of pure water to the nursery substrate at a ratio of 1:200. After swelling and crushing, spread it evenly in the pp resin nursery tray in the nursery box. After sowing, cover it with a transparent lid and seal it. Then place the nursery tray on the flower pot tray. Sow as evenly as possible, pour pure water to keep it in a moist state, and place it in an incubator for cultivation.

[0077] Before sowing, the substrate should be as flat as possible, sowing should be as even as possible, and a small sprayer should be used for watering. Wear a mask and gloves throughout the process and use sterile water. After sowing, water to keep the substrate in a moist state. Observe once every 2 days and supplement water appropriately.

[0078] Take out the centrifuge tube containing spores, add 1 ml of 5% NaClO aqueous solution by mass fraction, shake and disinfect continuously for 10 minutes, then centrifuge at 10000 r / min for 3 min, wash and centrifuge with sterile water and repeat 2 times. After pouring out the waste liquid, make a spore distilled water suspension according to a volume ratio of 1:100.

[0079] Shake the spores well, suck the spore suspension with a pipette, and spray it evenly on the substrate. Cover the plastic box lid, and keep the ventilation holes on the box lid closed. Place the nursery box sown with spores on the light culture rack. There are fluorescent lamps installed on the culture rack, and the culture conditions are set as: temperature 23 °C, with fluorescent lamp illumination during both day and night, and the illumination intensity is 1200 lux.

[0080] The spores will form a heart-shaped thallus about 5 weeks after sowing. When tiny green thalli appear on the surface of the substrate in the seedling-raising box and gradually cover the surface of the substrate like a layer of moss, spray water once every 3 days to keep it moist. After 6 weeks, the gametophytes grow densely and many sporophytes appear. The spore germination rate is 62%-70%, and it is ready for ramet transplantation. When transplanting, a cluster the size of a soybean is used, with a spacing of 2 cm. After transplantation, water regularly to keep it moist and open the lid of the seedling-raising box.

[0081] After 9 weeks, when the newly grown sporophytes reach about 1 cm in height, transplant them to a new seedling-raising tray. The transplanting substrate is a mixture of sterilized peat soil (PH = 5.6), vermiculite, and perlite (peat soil: vermiculite: perlite = 8:2:1.5 by mass ratio). The substrate is fully wetted, and the spacing between transplanted seedlings is 2 cm. Use sterilized forceps to pick up a cluster of sporophytes for transplantation. When the sporophytes reach 1.5 - 2 cm in height (2 months), use sterilized forceps to pick up a cluster of sporophytes for tray-changing transplantation.

[0082] After the seedlings are transplanted, keep the ventilation holes of the seedling-raising box open and spray water once every 1 - 2 weeks.

[0083] When the seedlings in the seedling-raising box grow to about 5 - 8 cm in height, transfer the seedling-raising box to a non-woven fabric seedling bag in the outdoor greenhouse nursery. The specifications of the seedling bag are a diameter of 15 cm and a height of 10 cm. The substrate ratio is: peat soil: pine bark: vermiculite: perlite = 6:2:1.5:1 by mass ratio. The transplanting survival rate reaches 90%.

Claims

1. A method for rapid propagation of spores of Sphaeropteris lepifera, a rare fern of the Cyatheaceae family, characterized in that, It includes the following steps: (1) Spore collection and preservation Collect the leaves of Sphaeropteris lepifera when the spores are mature and the spore powder has not dispersed between May and August every year. Place them in a cool and dry place. After drying, remove impurities from the spores, collect the naturally shed spores, and store the collected spores in a low-temperature storage for later use or directly sow them; (2) Selection and preparation of spore culture medium Substrate treatment: Use Norwegian Jiffy seedling blocks as the seedling culture substrate. Remove the non-woven fabric wrapped around the seedling blocks, sterilize them with a high-pressure steam sterilizer, dry them, and cool them for later use. Sterilize pure water or ultrapure water in the same way for later use; Add pure water to the seedling culture substrate, expand and crush it, then spread it evenly in a transparent seedling tray. After sowing, cover it with a lid and seal it. Sow as evenly as possible, water with pure water to keep it in a moist state, and place it in an incubator for cultivation; (3) Spore treatment and sowing After taking out the spores and sterilizing them, make a spore suspension with water, shake it well, suck out the spore suspension, and spray it evenly on the seedling culture substrate. Cover the box lid, and keep the ventilation holes on the box lid closed; (4) Spore cultivation Place the seedling box sown with spores on a light cultivation rack. Fluorescent lamps are installed on the cultivation rack. The cultivation conditions are set as follows: temperature 21–26°C, with fluorescent lamp illumination both during the day and at night, and the light intensity is 1100–1600 lux; (5) Gametophyte observation and maintenance Spores will form a preliminary heart-shaped flake in 3–5 weeks after sowing. When tiny green flakes appear on the surface of the substrate in the seedling box and gradually cover the substrate surface like a layer of moss, spray water once every 2–3 days to keep it moist. When the gametophytes grow densely and many sporophytes appear, prepare for dividing and transplanting. After transplanting, water to keep it moist to promote fertilization; (6) Transplanting of young sporophyte plants When the newly grown sporophytes reach about 1 cm in height, transplant them to a new tray. The transplanting substrate is a mixture of sterilized peat soil, vermiculite, and perlite; when the sporophytes reach 1.5 - 2 cm in height, use sterilized forceps to pick up the sporophytes for transplanting to a new seedling tray; (7) Seedling management and protection After transplanting the seedlings, keep the ventilation holes of the seedling tray open, spray water once every 1–2 weeks. If mold appears in the seedling box, remove the mold manually in time, and spray with a 0.5% - 1% baking soda solution for disinfection, spraying 1 - 2 times; (8) Seedling transplanting When the seedlings in the seedling box grow to about 5 - 8 cm in height, move the seedling box to a non-woven fabric seedling bag in an outdoor greenhouse nursery.

2. The spore rapid propagation method according to claim 1, wherein The spores of Sphaeropteris lepifera are mature, and the spores are brown.

3. The spore rapid propagation method according to claim 1, characterized in that In step (1), the collected leaves are sealed with a clean sealed bag to prevent the mixing of different plant sources. As soon as possible, cut the fresh plant leaves into small pieces and place them in a sulfur paper bag, and place them in a cool and dry place. After about one week, remove impurities from the spores, collect the naturally shed spores, and avoid artificial interference and peeling the sporangium groups on the spore leaves; operate while wearing gloves and change gloves for each sample processed to avoid spore mixing. The collected spores are put into a centrifuge tube and stored in a -80°C low-temperature refrigerator for later use.

4. The spore rapid propagation method according to claim 1, wherein For the described step (2), note that the substrate should be as flat as possible before sowing, sowing should be as uniform as possible, a small sprayer should be used for watering, masks and gloves should be worn throughout the process, sterile water should be used to avoid contamination by miscellaneous bacteria. After sowing, water the substrate to keep it in a moist state. Observe it every 2 days and supplement water appropriately.

5. The spore rapid propagation method according to claim 1, wherein The sterilization in the described step (3) is to take out the centrifuge tube containing spores, add 1 ml of 5% NaClO aqueous solution, continuously oscillate and disinfect for 10 minutes, then centrifuge at 10000 r / min for 2 minutes, wash and centrifuge with sterile water and repeat 3 times. After pouring out the waste liquid, make a spore suspension according to a volume ratio of 1:

100.

6. The spore rapid propagation method according to claim 1, characterized in that, For the light cultivation in the described step (4), the light cultivation rack should be placed in a separate room that has been sterilized and disinfected, and no mosquitoes or other insects are allowed to enter or exit.

7. The spore rapid propagation method according to claim 1, characterized in that, For the transplanting in the described step (5), when transplanting, a cluster should be the size of a soybean, and the spacing should be 1.2 - 1.5 cm.

8. The spore rapid propagation method according to claim 1, wherein, The described peat soil: vermiculite: perlite are mixed according to a mass ratio of 8:1:

1.

9. The spore rapid propagation method according to claim 1, characterized in that, The substrate of the described non-woven fabric seedling bag is: peat soil, pine bark, vermiculite, perlite mixture = 6:2:1:1, by mass ratio.

10. The spore rapid propagation method according to claim 1, characterized in that, The steps (4) - (7) of the described rapid propagation method are carried out in a greenhouse. The greenhouse is equipped with facilities such as a blower, a sunshade net, and a plastic film to ensure that the high temperature in summer does not exceed 30 °C and the low temperature in winter is not lower than 10 °C.

Images