Method for simulating high-efficiently breeding unique rare Adiantum nelumboides in wild environment

Abstract

A method for simulating high-efficiently breeding of Adiantum nelumboides in a wild environment comprises collecting spores from the right and left sides of a bottom and top of a leaf wherein the midd

Description

The present invention relates to the technical field of breeding, and in particular relates to a method for simulating high-efficiently breeding unique rare Adiantum nelumboides in a wild environment. BACKGROUND OF THE INVENTION Adiantum nelumboides, belonging to Adiantum capillus-veneris Genus, Pteridaceae Family, as a variant of Adiantum erythrochlamys Diels, is a first level national key protected wild plant, as wells as a sole single-leaved pteridophyte growing in Asia, only distributed in low-altitude areas of Chongqing City such as Wanzhou District, Fuling and Shizhu Country, so it is an endemic species of China, a relict plant of the Quaternary Period, known as the "treasure of China". Adiantum nelumboides faces a series of challenges in the natural environment, such as poor adaptability and weak natural renewal ability, so how to breed and protect it through technology is a worldwide problem. Establishing a system of "ex situ conservation-scientific research and breeding-return and re-protection" for Adiantum nelumboides and enabling it to grow normally under natural conditions so as to alleviate the endangered status of this species in the wild, has become an urgent problem to be solved. For this, how to obtain a large number of high-quality seedlings suitable for field environments and return requirements has become the key to solving the problem. At present, most research institutions, scholars and researchers mainly focus on traditional breeding and tissue culture for Adiantum nelumboides. Adiantum nelumboides cannot achieve mass reproduction or occupy new areas, due to severe germination conditions and low survival rate of its spores; in addition, the growth cycle of Adiantum nelumboides is long, taking nearly 1 year for spores to form young sporophytes from germination, and changes in various environmental factors during this process will affect the germination and growth of spores(Zeng Jianhong et al., Biological Characteristics and Protection Countermeasures of the Rare and Endangered Adiantum nelumboides in the Three Gorges Reservoir Area, 2002). (Huang Fang et al. Tissue Culture and Rapid Propagation of Adiantum nelumboides, 2008) By ways of researching tissue culture and rapid propagation of Adiantum nelumboides, adopting technology about aseptic seeding, tissue culture and rapid propagation, and using prothallia for proliferation, about 20%~30% of gametophytes convert to young sporophytes, so the conversion rate is low. (Zhang Guorong, Investigation and Analysis on the Endangered Causes of Endemic Medicinal and Ornamental Adiantum nelumboides in the Three Gorges Reservoir Area, 2010) The highest conversion rate of converting a gametophyte to a sporophyte is only 46.6%, and the study shows that increasing the conversion rate from a gametophyte to a young sporophyte is of great significance for artificial breeding of Adiantum nelumboides. (Yang Qinghua et al., 2021) A method for seeding and propagating spores of Adiantum nelumboides is disclosed in "A Spore Propagation Method for Rare and Endangered Adiantum nelumboides ", using two or more isochoric mixtures of grass charcoal, humus soil, red jade soil, black soil and vermiculite as a cultivation substrate, and it takes about 105-147 days from seeding to the emergence of sporophyte seedlings, but this method is cumbersome and the emergence time is long. (Chen Huiyuan et al., 2021) In "a Method for the Propagation of Adiantum nelumboides Ramets ", two substrates, namely river sand and peat(peat: perlite: river sand = 3:1:1) are used to treat propagules before transplanting and manage them after transplanting, it takes 60 days to have bred potted Adiantum nelumboides with ornamental value, this method has distinct seasonal characteristics and obvious disadvantages of higher requirements for the time of ramets and the growth state of maternal plants, as well as in terms of reproductive speed and proliferation coefficient compared with tissue culture. SUMMARY OF THE INVENTION In order to solve the above problems, the present invention provides a method for simulating high-efficiently breeding unique rare Adiantum nelumboides in a wild environment, optimizing the breeding technology system for Adiantum nelumboides, establishing a seedlings’ growth and acclimatization method that adapts to the environmental conditions in the wild from the initial stage, and obtaining a large number of high-quality seedlings that are completely suitable for return to the wild. The present invention is of great ecological, social and practical significance for carrying out research on the breeding and application of Adiantum nelumboides, stabilizing the population of rare and endangered species, improving the stability and resilience of ecosystems, effectively avoiding species extinction, protecting plant diversity, and playing a major role in the protection of the Yangtze River, so as to build a beautiful China with harmonious symbiosis of human and nature. The present invention adopts the following technical solutions: A method for simulating high-efficiently breeding unique rare Adiantum nelumboides in a wild environment, comprising the steps of: SI. collecting and pretreating spores, and inducing and propagating prothallia: collecting Adiantum nelumboides' spores, the collection beginning with the occurrence that the front sides of leaves become darker, their waviness become larger, and the sorus cover on the back of leaves change from green to gray-brown without cracking, after collection, placing them horizontally in a vibrator’s glass container, after sterilization, inoculating the spores on a induction medium, so that the prothallia grow out on a proliferation medium; and S2. simulating inducting and acclimatizing sporophytes in wild environments: directly taking the prothallia out of the culture vessel, dividing them into blocks according to aunit area of 0.8-1.2cmx0.8~l. 2cm, placing them on the substrate of an open seedling box, directly spraying mixed solution on the surface of the substrate, until the bottom water accumulates for 0.5cm~l ,0cm, then putting a transparent cover on it, in 28-35 days beginning to germinate sporophytes, in 60 days obtaining a large number of seedlings in verdancy and vigorous growth and suitable for return to the wild. Preferably, in SI, the spores are clockwise vibrated for 30~45min with a vibrating frequency of 250~300rpm and a vibrating amplitude of 20~26mm. Preferably, in SI, during collecting the Adiantum nelumboides' spores, selecting a round sorus in the middle of its leaf, the number of sorus is decided by regarding the middle of each leaf as the axis of symmetry and then respectively taking 5-6 spores on the left and right of the top of the leaf, the spores’ collection number in the whole leaf is decided by regarding the middle of each leaf as the axis of symmetry and then respectively taking 3-4 spores on the left and right of the bottom of the leaf and 2 spores on the left and right of the top of the leaf. Preferably, in SI, the induction medium includes MS+0.05-0.1mg / L KT-0.001-0.003mg / L TDZ+0.5~0.8mg / L NAA+3.0% of sucrose +0.7% of agar with a pH of 5.5-6.0; the proliferation medium includes MS+0.6-0.8mg / L 6-BA+0.1~0.3mg / L NAA+3.0% of sucrose +0.7% of agar, and its pH is 5.5-6.0. Preferably, in S2, the seedling box has a dimension of length x width x height = (41~45)cm x (30~36)cm x (25~30)cm and ventholes with a diameter of 0.8-1.2cm, the transparent upper cover made from a PET material is 16~20cm in height, the upper cover is provided with two rotary buttons, each of which controls 2-4 ventholes. Preferably, in S2, the seedling box has a dimension of length x width x height = 43cm x 33cm x 28cm and ventholes with a diameter of 1.0cm, the transparent upper cover made from a PET material is 18cm in height, the upper cover is provided with two rotary buttons, each of which controls 4 ventholes. Preferably, in S2, in the first week, the rotary buttons are adjusted to open one hole and close three holes; in the second week, the rotary buttons are adjusted to open two holes and close two holes; in the third week, the rotary buttons are adjusted to open three holes and close one hole; in the fourth week, the rotary buttons are adjusted to close all the holes. Preferably, in S2, the substrate components of the seedling box are formed by weathering and eroding rock, and its main component is gravel of silica, the diameter of the gravel is 0.06mm~5.0mm, the thickness of the substrate is 5cm, divided into upper, middle and lower layers, the upper layer is mixed with gravel having diameters of 3mm~5.0mm, the middle layer is mixed gravel having diameters of 0.06mm~l .0mm, and the lower layer is mixed with gravel having diameters of lmm~3.0mm. The organic combination of measures such as mixture with 3 layers of gravel with different particle sizes and environmental adjustment of the seeding box can not only ensure the moisture required by the prothallia in the process of producing sporophyte seedlings, but also adjust air humidity and temperature, enabling the plants to breathe fresh oxygen and providing a place and environment for the smooth germination of Adiantum nelumboides’ prothallia. Its most importance is to realize the germination of sporophytes in the simulated original ecological state of the wild field and the integration and intensification of seedling’s growth and plant’s acclimatization, simplify experimental technical processes and save time and effort. At the time of return, the seedlings are watered regularly in the first half of the month after planting, and the plant’s preservation rate reaches 91.1% after half a year. Preferably, in S2, the mass concentration of perilla leaf juice in the mixed solution is 45-95%, the concentration of e-polylysine is 0.01~0.05g / L, the concentration of GA3 is 0.0l~0.03g / L, the concentration of IAA is 0.1~0.5g / L, the concentration of NAA is 0.3~0.6mg / L, the concentration of KH2PO4 is 0.5~1.0g / L, the concentration of (NH4)2SO4is 1.0~2.0g / L. Further preferably, in S2, the production method of perilla leaf juice is as follows: taking 100g of perilla leave, washing them, adding 0-100ml of purified water to crush them, then filtering out the leaf residue with a multi-mesh filter to obtain perilla leaf juice. The present invention has the following beneficial effects: 1 .The method can simulate a wild environment, efficiently breed the unique rare Adiantum nelumboides, and breed, induce and acclimate seedlings from a primary stage and source, so as to realize perfectly integrating of the breeding of seedlings and their adaptive acclimatization of the wild environment. 2. A short-term regular vibrate to the spores is beneficial to stimulate their internal vitality and raise induction and induction speed of prothallia. 3.The combination of grow seedlings box + gravel substrate precisely controls the environmental factors such as temperature, humidity, moisture and oxygen required for the induction of sporophyte seedlings, while achieving synchronizing substrates in natural growth. 4.The treatment by mixed solution can greatly promote growth quantity and growth potential of Adiantum nelumboides' seedlings, and the seedlings can be directly used for natural return of rare plants in the wild, so as to realize the germination of sporophytes in the simulated original ecological state of the wild field and the integration and intensification of seedling’s growth and plant’s acclimatization, simplify experimental technical processes and save time and effort. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows collecting Adiantum nelumboides’ spores, where a stands for a collection process, b stands for a collection detail map. FIG.2 shows proliferation and expansion of Adiantum nelumboides’ prothallia. FIG. 3 shows growth comparison of Adiantum nelumboides’ seedlings between a seedling box and a culture vessel, where a stands for a collection process, b stands for a collection detail map, there are significant differences in seedling growth rates, plant sizes and growth potential. FIG.4 shows growth of sporophyte seedlings in 10 days. FIG.5 shows growth of sporophyte seedlings in 20 days. FIG.6 shows growth of sporophyte seedlings in 30 days. FIG.7 shows batch reproduction and vigorous growth of Adiantum nelumboides' seedlings. FIG.8 shows simulation of the return of Adiantum nelumboides efficiently bred in the wild. FIG.9 shows observations of Adiantum nelumboides returned in the wild. DETAILED DESCRIPTION OF SOME EMBODIMENTS The present invention is further described in combination with the examples as follows, but the protection scope claimed by the present invention is not limited to the described scope of the examples. EXAMPLE 1 Collecting and pretreating spores, inducing and propagating prothallia, inducting and acclimatizing sporophytes in wild environments. The spore’s collection complies with the principle of advantage and superior species, that is, during collecting the Adiantum nelumboides' spores, selecting a round sorus in the middle of its leaf. The number of sorus is decided by regarding the middle of each leaf as the axis of symmetry and then respectively taking 6 spores on the left and right of the top of the leaf, in this way, the total number of spores in the middle is 6*2=12. The spores’ collection number in the whole leaf is decided by regarding the middle of each leaf as the axis of symmetry and then respectively taking 4 spores on the left and right of the bottom of the leaf and 2 spores on the left and right of the top of the leaf, in this way, the total number of whole-leaf spores is the number taken at the bottom plus the number taken in the middle, that is 12. This method can ensure that the number of two samples is equal, avoiding collecting short linear sorus of the lower portion of plants and the surrounding leaves, as well as ensure spores’ quality. The collection begins with the occurrence that the front sides of leaves become darker, their waviness become larger, and the sorus cover on the back of leaves change from green to graybrown without cracking. After collection, they are placed horizontally in a vibrator’s glass container, and clockwise vibrated for 35min with a vibrating frequency of260rpm and vibrating amplitude of 24mm, and the short-term regular vibration conduces to stimulating the internal vitality of the spores and raising the induction and induction speed of prothallia. After sterilization, the spore is inoculated on a culture medium including MS+0.08mg / L KT+0.002mg / L TDZ+0.6mg / L NAA+3.0% of sucrose + 0.7% of agar with a pH of 5.5. In 20-25 days, the prothallium grows out, the proliferation medium includes MS+0.7mg / L 6-BA+0.2mg / L NAA+3.0% of sucrose + 0.7% of agar, and its pH is 5.5. EXAMPLE 1-1 Based on Example 1, the difference is that the spores stimulated by vibration are the whol e-leaf spores. CONTROL1 Based on Example 1, the difference is that the spores are the middle-leaf spores and not vibrated, and the others conditions are the same as Example 1. CONTROL 2 Based on Example 1, the difference is that the spores are the whole-leaf spores and not vibrated, and the others conditions are the same as Example 1. The induction state of prothallia through different treatments of Adiantum nelumboides’ spores is shown in Table 1. Table 1 The induction state of prothallia through different treatments of Adiantum nelumboides’ spores No. Stimulation by vibration (rpm / min) spore number prothalliu m number inductio n rate (%) prolifer ation rate(%) proliferati on coefficient 1 Example 1 (middle-leaf spore 90 82 91.1 98.5 6.9 +stimulated by vibration) 2 Examplel-1 (whole-leaf spore +stimulated by vibration) 90 81 91.2 98.4 6.8 3 Control group 1 (middle-leaf spore) 90 35 34.4 32.3 1.5 4 Control group 2 (whole -leaf spore) 90 21 23.3 22,4 1.1 It can be seen from Table 1 that short-term regular vibration is beneficial to stimulate the internal vitality of the spores and raise the induction and induction speed of prothallia. Moreover, the induction rate and proliferation rate of the prothallia using whole-leaf spores can reach the level of middle-leaf spores after vibration and stimulation. Although according to the central effect of plants, that is, nutrients first converge to the central portion and the central spores grow more vigorously, so that the middle-leaf spores are normally better, but after appropriate vibration and stimulation, the vitality of the whole-leaf spores including inactive spores can be stimulated, promoting the induction and proliferation in the later stage, so that the vitality of inactive spores can be stimulated and more adaptable to changes in the environment. Moreover, the whole leaf spores are used for treatment, omitting a step for screening spores and cutting down on procedures. EXAMPLE 2 Simulating the induction and acclimatization of sporophyte seedlings in wild environments. The prothallia obtained by the method for collecting and pretreating spores and inducing and proliferating prothallia in Example 1 are used for the experiment in this example. Processing Number A: The primary goal of plant protection is to realize ex situ conservation and batch reproduction for rare and endangered plants, on this basis, the ultimate goal for rare plants is to allow rare and endangered plants to set foot on the road of return to the wild, achieve survival in the wild, rebuild the population and maintain ecological balance. For those, the ability to obtain a large number of Adiantum nelumboides’ seedlings adapted to the complex and changeable wild environment is the key core factor. After researching the induction and acclimatization of sporophytes by means of different combinations such as indoor sterile culture vessels, outdoor square plug trays and seedling boxes, it is found that the seedling box is the best choice. The prothallia are directly taken out of the culture vessel, divided into blocks according to a unit area of 1cm x 1cm, and placed on the substrate of the open seedling box having a dimension of length x width x height = 430mm x 330mm x 280mm and ventholes with a diameter of 1.0cm. Mixed solution (100ml of perilla leaf juice + 0.03g / L c-polylysine +0.02g / L GA3 +0.3g / L IAA +0.5mg / L NAA +0.8g / L KH2PO4 +1.5g / L (NH4)2SO4) is directly sprayed on the surface of the substrate, until the bottom water accumulates for 0 ,8cm, then a transparent cover of PET material is covered, in 28-35 days it begins to germinate sporophytes, in 60 days a large number of seedlings in verdancy and vigorous growth and suitable for return to the wild can be obtained. Creating and controlling microenvironments is important for the sporophyte seedlings generated through induction of Adiantum nelumboides’ prothallia. The control of a rotary ventilation switch near both sides on the top of the transparent upper cover of the seedling box can play a key role in adjusting air temperature, humidity and oxygen content in the box. In the first week, the rotary ventilation switch is adjusted to open one hole and close three holes; in the second week, the rotary ventilation switch is adjusted to open two holes and close two holes; in the third week, the rotary ventilation switch is adjusted to open three holes and close one hole; in the fourth week, the rotary ventilation switch is adjusted to close all the holes. The substrate components of the seedling box are formed by weathering and eroding rock, and its main component is ordinary gravel of silica, which can be collected at the foot of the nearby granite mountain, and from which clean gravel is selected without weeds, lichen and moss on its surface, so that it can be used directly during laying at the bottom of the seedling box, without sterilization and spraying herbicides. The diameter of the gravel is 0.06mm - 5.0mm, the thickness of the gravel is 50mm, divided into upper, middle and lower layers, the upper layer is mixed with gravel 1 (the gravel having a particle size of 3mm is mixed with the gravel having a particle size of 5mm according to the mass ratio of 1:1), the thickness of the upper layer is 10mm, the middle mixed gravel 2 (the gravel having a particle size of 0.08mm is mixed with the gravel having a particle size of 1mm according to the mass ratio of 1:1), the thickness is 20mm, and the lower layer is mixed with gravel 3 (the gravel having a particle size of 1mm is mixed with and the gravel having a particle size of 2mm according to the mass ratio of 1:1), the thickness of the lower layer is 20mm. The organic combination of measures such as mixture with 3 layers of gravel with different particle sizes and environmental adjustment of the seeding box can not only ensure the moisture required by the prothallia in the process of producing sporophyte seedlings, but also adjust air humidity and temperature, enabling the plants to breathe fresh oxygen and providing a place and environment for the smooth germination of Adiantum nelumboides’ prothallia. Its most importance is to realize the germination of sporophytes in the simulated original ecological state of the wild field and the integration and intensification of seedling’s growth and plant’s acclimatization, simplify experimental technical processes and save time and effort. At the time of return, the seedlings are watered regularly in the first half of the month after planting, and the plant’s preservation rate reaches 91.1% after half a year. Processing Number B: The prothallia are placed in a culture vessel, the culture medium is a mixture of gravel and peat (1:1), the peat is HAWITA peat, its volume-weight is 0.25-0.5, its total porosity is 50%, the culture medium is sterilized for 20min under the sterilization 5 conditions of temperature of 121 °C and pressure of O.IMPa in a sterilizing autoclave, and the others are same with Processing Number A. Processing Number C: The prothallia are placed in a square plug tray, the dimension of the square tray is 380mmx300mmx56mm, its top is not covered, and the others are same with Processing Number A. 10 Processing Number D: compared with the specific treatment method that the prothallia induced by the spores early sterilized and cultivated in the sporangia continue to be on MS inorganic solid plant tissue culture medium without any changes to the culture medium and container. Table 2 Comparison table about the induction of Adiantum nelumboides’ sporophyte 15 seedlings by different ways No. Induction combination of seedings Prothall ium number Seeding number Inducti on rate (%) Seedling growth in 60 days Survival rate after return A Seeding box +mixed solution 90 88 97.8 The seedlings are numerous in verdancy and vigorous growth 91.1% B Culture vessel + mixed solution 90 73 81.1 The seedlings are less numerous, gradually become yellow and weak in growth 67.2% C Square plug tray + mixed solution 90 15 16.7 Few seedlings occur, wither and die 59.8% D Control 90 0 0.0 No seedlings occur in inorganic medium 0 It can be seen from Table 2 that the order of Treatment A (seedling box + mixed solution), Treatment B (culture vessel + mixed solution), Treatment C (square tray + mixed solution) and Treatment D (control) in the aspect of the induction number and induction rate of sporophyte seedlings is Treatment A> Treatment B> Treatment C> Treatment D. The number of the 20 sporophyte seedlings induced by prothallia in the combination of Treatment A is the largest, reaching 88, which is 15, 73 and 88 higher than those of Treatment B, Treatment C and Treatment D, respectively; the induction rate of sporophyte seedlings is the highest, 97.8%, which increases by 16.7%, 81.1% and 97.8% compared with Treatment B, Treatment C and Treatment D, respectively. The number of induced sporophyte seedlings in Treatment B is 58 and 73 higher than those of Treatment C and Treatment D, respectively, and the induction rate increases by 64.4%, respectively. It was proved that there is a significant difference in the type of container + mixed solution in the aspect of the induction number and induction rate of Adiantum nelumboides’ sporophyte. Treatment A (seedling box + mixed solution) can accurately control the environmental factors such as temperature, humidity, moisture and oxygen required for the induction of sporophyte seedlings on the mixed gravel substrate, and maximize the growth quantity and growth potential of Adiantum nelumboides’ seedlings, and the seedlings can be directly used for natural return of rare plants in the wild. EXAMPLE 3 Based on Example 2, the composition of the mixed solution is changed, and the others are same with Treatment A of Example 2. Treatment A of Example 2: 100ml of perilla leaf juice + 0.03g / L s-polylysine +0.02g / L GA3 +0.3g / L IAA +0.5mg / L NAA +0.8g / L KH2PO4 +1 5g / L (NH4)2SO4. EXAMPLE 3-1: 0.03g / L c-polylysine +0.02g / L GA3 +0.3g / LIAA+0.5mg / LNAA+0.8g / L KH2PO4 +1.5g / L (NH4)2SO4 EXAMPLE 3-2: 100ml of perilla leaf juice + 0.02g / L GA3 +0.3g / L IAA +0.5mg / L NAA +0.8g / L KH2PO4 +L5g / L (NH4)2SO4. EXAMPLE 3-3: 100ml of perilla leaf juice + 0.03g / L s-polylysine +0.3g / L IAA+0.5mg / L NAA +0.8g / L KH2PO4 +1 5g / L (NH4)2SO4. EXAMPLE 3-4: 100ml of perilla leaf juice + 0.03g / L e-polylysine +0.02g / L GA3 +0.5mg / L NAA +0.8g / L KH2PO4 +L5g / L (NH4)2SO4 EXAMPLE 3-5: 100ml of perilla leaf juice + 0.03g / L s-polylysine +0.02g / L GA3 +0.3mg / LNAA+0.8g / L KH2PO4 +1.5g / L (NH4)2SO4. EXAMPLE 3-6: 100ml of perilla leaf juice + 0.03g / L s-polylysine +0.02g / L GA3 +0.3mg / LNAA+1.5g / L (NH4)2SO4. Table 3 Comparison table about the induction of Adiantum nelumboides’ sporophyte seedlings by different mixed solutions No. Combmation(g-L-1) Prothallium number Inducted seeding number Induction rate(%) Example2-1 Pretreatment A with mixed solution 90 88 97.8 Examples-1 Pretreatment B with mixed solution 90 70 77.8 Examples-2 Pretreatment C with mixed solution 90 80 88.9 Examples-3 Pretreatment D with mixed solution 90 71 78.9 Example3-4 Pretreatment E with mixed solution 90 81 90.0 Examples-5 Pretreatment F with mixed solution 90 83 92.2 Examples-6 Pretreatment G with mixed solution 90 85 94.4 No control group with mixed solution 90 0 0 It can be seen from Table 3 that the induction number of sporophyte seedlings under Pretreatment A with mixed solution is at most 88, and its induction rate is 97.8%; the induction number of sporophyte seedlings under Pretreatment B with mixed solution is 70, and its induction rate is 77.8%, compared with Treatment A, the number decreases by 18 and the rate decreases by 20.0%; the induction number of sporophyte seedlings under Pretreatment C with mixed solution is 80, and its induction rate is 88.9%, compared with Treatment A, the number decreases by 8 and the rate decreases by 8.9%; the induction number of sporophyte seedlings under Pretreatment D with mixed solution is 71, and its induction rate is 78.9%, compared with Treatment A, the number decreases by 17 and the rate decreases by 18.9%; the induction number of sporophyte seedlings under Pretreatment E with mixed solution is 81, and its induction rate is 90.0%, compared with Treatment A, the number decreases by 7 and the rate decreases by 7.8%; the induction number of sporophyte seedlings under Pretreatment F with mixed solution is 83, and its induction rate is 92.2%, compared with Treatment A, the number decreases by 5 and the rate decreases by 5.6%; the induction number of sporophyte seedlings under Pretreatment G with mixed solution is 85, and its induction rate is 94.4%, compared with Treatment A, the number decreases by 3 and the rate decreases by 3.4%. Among the combinations of pretreatment with mixed solution, there is an extremely significant difference between Pretreatment B and Pretreatment A, while the differences between Pretreatment C, D, E, F and G and Pretreatment A are small, indicating that perilla leaf juice has a significant promoting effect on the induction of Adiantum nelumboides’ sporophyte seedlings. However, the induction number of Adiantum nelumboides’ sporophyte in the control group (not under the pretreatment with mixed solution) is 0, and the induction rate was 0.0%, compared with the control group, the number increases by 88 and the rate increases by 97.8%; compared with the control group, the induction number of Pretreatment A as an optimal combination of the pretreatment with mixed solution increases by 88 and its induction rate increases by 97.8%, the result shows that the mixed solution of perilla leaf juice (100ml of perilla leaf juice + 0.03g / L s-polylysine +0.02g / L GA3 +0.3g / L IAA +0.5mg / L NAA +0.8g / L KH2PO4 +1.5g / L (NH4)2SO4)can greatly increase the induction number of sporophyte seedlings in the aspect of the induction of Adiantum nelumboides’ sporophyte seedlings, and significantly increase the induction rate of sporophyte seedlings. EXAMPLE 4 Based on Example 2, the composition of the substrate components is changed, and the others are same with Treatment A of Example 2. Treatment A of Example 2: the upper layer is mixed with gravel 1 (the gravel having a particle size of 3mm is mixed with the gravel having a particle size of 5mm according to the mass ratio of 1:1), the thickness of the upper layer is 10mm, the middle mixed gravel 2 (the gravel having a particle size of 0.08mm is mixed with the gravel having a particle size of 1mm according to the mass ratio of 1:1), the thickness is 20mm, and the lower layer is mixed with gravel 3 (the gravel having a particle size of 1mm is mixed with and the gravel having a particle size of 2mm according to the mass ratio of 1:1), the thickness of the lower layer is 20mm. EXAMPLE 4-1: the particle sizes of the upper, middle and lower layers are same with each other, all 3.0mm. EXAMPLE 4-2: the particle sizes of the upper, middle and lower layers are same with each other, all 10mm. Table 4 Comparison table about the induction of Adiantum nelumboides’ sporophyte seedlings by different substrate components No. Combin ation(g • L_ 1) Prothallium number Inducted seeding number Induction rate(%) Example 2-1 Treatment A with mixed gravel 90 88 97.8 Example 4-1 Treatment B with gravel having a size of 3.0mm 90 75 83.3 Example 4-2 Treatment C with gravel having a size of 1.0mm 90 62 68.9 It can be seen from Table 4 that the induction number of sporophyte seedlings under Treatment A with mixed gravel is at most 88, and its induction rate is 97.8%; the induction number of sporophyte seedlings under Treatment B with mixed gravel is 75, and its induction rate is 83.3%, compared with Treatment A, the number decreases by 13 and the rate decreases by 14.5%; the induction number of sporophyte seedlings under Treatment C with mixed gravel is 62, and its induction rate is 68.9%, compared with Treatment A, the number decreases by 26 and the rate decreases by 28.9%; among the combinations of mixed gravel, Treatment B and C differ significantly from Treatment A, indicating that deferent combinations of mixed gravel have a significant promoting effect on the induction of Adiantum nelumboides’ sporophyte seedlings. Although the present invention has been disclosed as above in preferred examples, it is not intended to limit the present invention, any person skilled in the art can make various changes 12 and modifications without departing from the spirit and scope of the present invention, so the protection scope of the present invention should subject to the claims. The above embodiments are only preferred technical solutions of the present invention, and should not be regarded as limiting the present invention. The embodiments in the present application and the features in 5 the embodiments can be arbitrarily combined with each other without conflict. The scope of protection of the present invention shall be the technical solutions recorded in the claims, including the equivalent alternatives of the technical features in the technical solutions recorded in the claims. Equivalent substitutions and improvements in the scope are also included in the scope of protection of the present invention.

Claims

What is claimed is:

1. A method for simulating high-efficiently breeding unique rare Adiantum nelumboidesin a wild environment, comprising the steps of:SI. collecting and pretreating sorus, and inducing and propagating prothallia: collecting Adiantum nelumboides' sorus, selecting round sorus in the middle of its leaf, the number of sorus is decided by regarding the middle of each leaf as the axis of symmetry and then respectively taking 5-6 sorus on the left and right of the top of the leaf, or the sorus’ collection number in the whole leaf is decided by regarding the middle of each leaf as the axis of symmetry and then respectively taking 3~4 sorus on the left and right of the bottom of the leaf and 2 sorus on the left and right of the top of the leaf;the collection beginning with the occurrence that the front sides of leaves become darker, their waviness become larger, and the sori cover on the back of leaves change from green to graybrown without cracking,after collection, placing them horizontally in a vibrator’s glass container, the sorus are clockwise vibrated for 30~45min with a vibrating frequency of 250~300rpm and a vibrating amplitude of 20~26mm:after sterilization, inoculating the sorus on an induction medium, so that prothallia grow out on a proliferation medium; andS2. simulating inducting and acclimatizing sporophytes in wild environments:directly taking the prothallia out of the culture vessel, dividing them into blocks according to a unit area of 0.8-1.2cm*0.8-1.2cm, placing them on a substrate of an open seedling box, directly spraying a mixed solution on the surface of the substrate, until the bottom water accumulates for 0.5cm-1.0cm, then putting a transparent cover on the open seedling box, in 28-35 days the prothallia beginning to germinate into sporophytes, in 60 days obtaining a large number of seedlings in verdancy and vigorous growth and suitable for return to the wild; in SI, the induction medium includes MS+0.05~0.1mg / L KT+0.001~0.003mg / L TDZ+0.5~0.8mg / L NAA+3.0% of sucrose +0.7% of agar with a pH of 5.5-6.0; the proliferation medium includes MS+0.6-0.8mg / L 6-BA+O.l~0.3mg / L NAA+3.0% of sucrose +0.7% of agar, and its pH is 5.5-6.0.in S2, in the mixed solution, a mass percent of a perilla leaf juice is 45-95%, theconcentration of £-polylysine is 0.03g / L, the concentration of GA3 is 0.02g / L, the concentration of IAA is 0.3g / L, the concentration of NAA is 0.5mg / L, the concentration of KH2PO4 is 0.8g / L, the concentration of (NH4)2SO4 is 1.5g / L;m ,the seedling box has a dimension of length x width x height = (41~45)cm x (30-36)cm x (25~30)cm and ventholes with a diameter of 0.8-1.2cm, the transparent upper cover made from a PET material is 16~20cm in height, the upper cover is provided with two rotary buttons, each of which controls 4 ventholes;in S2,in the first week, the rotary buttons are adjusted to open one hole and close three holes; in the second week, the rotary buttons are adjusted to open two holes and close two holes; in the third week, the rotary buttons are adjusted to open three holes and close one hole; in the fourth week, the rotary buttons are adjusted to close all the holes.

2. The method for simulating high-efficiently breeding unique rare Adiantum nelumboides in a wild environment, according to claim 1. wherein in S2,the substrate components of the seedling box are formed by weathering and eroding rock, and its main component is gravel of silica, the diameter of the gravel is 0.06mm-5.0mm, the thickness of the substrate is 5cm, divided into upper, middle and lower layers, the upper layer is mixed with gravel having diameters of 3mm~5.0mm, the middle layer is mixed gravel having diameters of 0.06mm~l .0mm, and the lower layer is mixed with gravel having diameters of lmm~3.0mm.

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