Method for improving proliferation rate of tissue culture seedlings of cibotium barometz and promoting sporophyte propagation of cibotium barometz

Abstract

The invention relates to the technical field of medicinal plant propagation, in particular to a method for increasing the proliferation rate of cibotium barometz tissue culture seedlings and promoting sporophyte propagation of the cibotium barometz tissue culture seedlings. The method comprises the following steps: preparing a propagation culture medium; and culturing the golden dog. According to the method provided by the invention, the proliferation multiple of the tissue culture seedlings of the cibotium barometz can be remarkably increased, the propagation of sporophytes of the cibotium barometz can be remarkably promoted, the method has important significance in relieving the problem of resource shortage of the cibotium barometz and improving the resource utilization rate of the cibotium barometz, and meanwhile, a technical support is provided for resource protection and artificial cultivation of the cibotium barometz.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medicinal plant propagation, in particular to a method for improving the propagation multiple of Cibotium barometz (L.) J. Sm. tissue culture seedlings and promoting the sporophyte propagation. BACKGROUND

[0002] Cibotium barometz (L.) J. Sm., also known as guogu in the Dai nationality, has other names such as Baizhi, Dog Green, Fugai, Dog Ridge, Gold Silk Hair and Gold Hair Monkey, is a perennial large tree-like terrestrial fern of the Dicksoniaceae family and Cibotium Kaulf. and is an important medicinal plant. Cibotium barometz (L.) J. Sm. is mainly used for medicinal purposes with roots and stems, which can be collected throughout the year, but the quality of the medicinal materials collected in late autumn to winter is the best. It has the effects of tonifying liver and kidney, strengthening waist and knees and relieving rheumatism and is mainly used for treating rheumatic bone pain, lumbago and weakness, hemiplegia and other diseases. The golden yellow hair on the surface of the root and stem is a special medicine for treating external injuries and bleeding. The chemical components isolated from the aboveground parts and rhizomes of Cibotium barometz (L.) J. Sm. mainly include fernin, terpenes, flavonoids, aromatic compounds, steroids and pyrones, which have the effects of hemostasis, bacteriostasis, prevention and treatment of osteoporosis, anticancer and liver protection.

[0003] Based on the good medicinal and ornamental values of Cibotium barometz (L.) J. Sm., the development and utilization of its wild resources have attracted much attention in recent years. However, the collection of Cibotium barometz (L.) J. Sm. is in disorder without considering its growth and propagation and distribution rules, which, combined with the internal factors causing the species to be endangered, leads to the continuous decrease of the wild resources of Cibotium barometz (L.) J. Sm. and the species has been listed in the Convention on International Trade in Endangered Species of Wild Fauna and Flora (Appendix II) (CITES 2016) and the National List of Key Protected Wild Plants (First Batch) as a national second-class protected plant. Therefore, exploring a method for improving the tissue culture efficiency of Cibotium barometz (L.) J. Sm. has important significance for alleviating the shortage of Cibotium barometz (L.) J. Sm. resources and improving the utilization rate of Cibotium barometz (L.) J. Sm. resources.

[0004] Therefore, the present application provides a method for improving the propagation multiple of Cibotium barometz (L.) J. Sm. tissue culture seedlings and promoting the sporophyte propagation by using macroelement nitrogen, aiming to solve the above problems and provide technical support for the resource protection and artificial cultivation of Cibotium barometz (L.) J. Sm. SUMMARY

[0005] The present application aims to provide a method for improving the propagation multiple of Cibotium barometz (L.) J. Sm. tissue culture seedlings and promoting the sporophyte propagation.

[0006] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows: The method for improving the propagation multiple of Cibotium barometz (L.) J. Sm. tissue culture seedlings and promoting the sporophyte propagation provided by the present application comprises the following steps: S1, preparing a propagation culture medium: Take NH4NO3, KH2PO4, KNO3, CaCl2 and MgSO4 to prepare nitrogen treatment solution; take agar, trace elements, sucrose, organic matter, iron salt and the above prepared nitrogen treatment solution to prepare MS culture medium; add 6-benzyladenine, naphthalene acetic acid and activated carbon to the above prepared MS culture medium to prepare propagation culture medium; Wherein, the trace elements are prepared by dissolving 22.3mg MnSO4·4H2O, 8.6mg ZnSO4·7H2O, 6.2mg H3BO3, 0.83mg KI, 0.025mg CuSO4·5H2O, 0.025mg CoCl2·6H2O and 0.25mg Na2MoO4·2H2O in 1L water; the organic matter is prepared by dissolving 2mg glycine, 0.1mg VB1, 0.5mg VB6, 0.5mg VB5 and 100mg myo-inositol in 1L water; the iron salt is prepared by dissolving 37.3mg Na2·EDTA and 27.8mg FeSO4·7H2O in 1L water; S2, gold dog culture: Take the gold dog tissue culture seedling to the propagation culture medium prepared in step S1 for subculture; take the subculture obtained gold dog tissue culture seedling to continue to culture in the propagation culture medium, and the method is completed.

[0007] Preferably, in the method for improving the propagation multiple of gold dog tissue culture seedling and promoting the sporophyte propagation, the concentration of NH4NO3 in the nitrogen treatment solution in step S1 is 0.825-6.6g / L; the concentration of KH2PO4 is 3.4g / L; the concentration of KNO3 is 38g / L; the concentration of CaCl2 is 8.8g / L; and the concentration of MgSO4 is 3.61g / L.

[0008] Further preferably, in the method for improving the propagation multiple of gold dog tissue culture seedling and promoting the sporophyte propagation, the concentration of NH4NO3 in the nitrogen treatment solution in step S1 is 0.825g / L.

[0009] Preferably, in the method for improving the propagation multiple of gold dog tissue culture seedling and promoting the sporophyte propagation, the concentration of agar in the MS culture medium in step S1 is 4.5g / L; the concentration of trace elements is 1mL / L; the concentration of sucrose is 20g / L; the concentration of organic matter is 10mL / L; the concentration of nitrogen treatment solution is 50mL / L; and the concentration of iron salt is 10mL / L.

[0010] Preferably, in the method for improving the propagation multiple of gold dog tissue culture seedling and promoting the sporophyte propagation, the concentration of 6-benzyladenine in the propagation culture medium in step S1 is 1.5mg / L; the concentration of naphthalene acetic acid is 0.5mg / L; and the concentration of activated carbon is 1mg / L.

[0011] Preferably, in the method for increasing the propagation multiple of the in vitro cultured seedlings of Cinnamomum wilsonii and promoting the sporophyte propagation of the application, the in vitro cultured seedlings in step S2 are obtained by the following method: 10 mg of Cinnamomum wilsonii mature spore powder is weighed in a 1.5 mL centrifuge tube, soaked with distilled water for 1 h, the supernatant is removed by centrifugation, the lower layer of the precipitate is sterilized with 70% alcohol for 30 s, centrifuged and the supernatant is removed, the lower layer of the precipitate is sterilized with 4% NaClO solution for 15 min, centrifuged and the supernatant is removed, the lower layer of the precipitate is washed with sterile water for 4-6 times, after centrifugation, 1.5 mL of sterile water is added to the obtained precipitate and mixed, thereby obtaining a spore suspension; the spore suspension is inoculated into 1 / 2MS culture medium, 1 mL of sterile spore suspension is inoculated into each bottle, and after 60-90 days of culture, prothalli are formed, and when the prothalli grow into heart-shaped, they are transferred to new culture medium for continuous culture, thereby obtaining the in vitro cultured seedlings of Cinnamomum wilsonii.

[0012] Preferably, in the method for increasing the propagation multiple of the in vitro cultured seedlings of Cinnamomum wilsonii and promoting the sporophyte propagation of the application, the subculture in step S2 is specifically as follows: the well-grown sterile emerald gametophyte clusters are selected on the clean bench, cut into small prothallus clusters with a size of 1 cm×1 cm, inoculated into the Cinnamomum wilsonii propagation culture medium, 5 or 6 prothallus clusters are inoculated into each bottle, and after inoculation, constant temperature culture is carried out. The propagation culture medium is composed of MS+agar or carrageenan+ sucrose+IAA+6-BA+activated carbon without agar and sucrose.

[0013] Further preferably, in the method for increasing the propagation multiple of the in vitro cultured seedlings of Cinnamomum wilsonii and promoting the sporophyte propagation of the application, the culture conditions of step S2 are as follows: the temperature is 25±2℃; the humidity is 50%; the light intensity is 1000 Lx; and the light time is 12 h / d.

[0014] Preferably, in the method for increasing the propagation multiple of the in vitro cultured seedlings of Cinnamomum wilsonii and promoting the sporophyte propagation of the application, the continuous culture time in step S2 is 60-90 d.

[0015] Further preferably, in the method for increasing the propagation multiple of the in vitro cultured seedlings of Cinnamomum wilsonii and promoting the sporophyte propagation of the application, the continuous culture time in step S2 is 60 d.

[0016] The beneficial effects of the application are as follows: The application provides a method for increasing the propagation multiple of the tissue culture seedlings of golden thread and promoting the propagation of the sporophytes of the tissue culture seedlings by using nitrogen, and the optimal concentration of NH4NO3 in the nitrogen treatment solution is 0.825 g / L by comparing the effects of different concentrations of nitrogen treatment on the agronomic characters of the tissue culture seedlings of golden thread. Under the optimal concentration of NH4NO3: the propagation multiple of the tissue culture seedlings of golden thread is increased by about 2 times after 60 days of culture compared with the group without adding NH4NO3; and the number of sporophytes of the tissue culture seedlings of golden thread is increased by about 3 times per bottle after 90 days of culture compared with the group without adding NH4NO3. The above proves that the method provided by the application can significantly increase the propagation multiple of the tissue culture seedlings of golden thread and promote the propagation of the sporophytes of the tissue culture seedlings of golden thread, and has important significance for relieving the shortage of the resources of golden thread, improving the utilization rate of the resources of golden thread, and providing technical support for the resource protection and artificial cultivation of golden thread. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 The agronomic character statistical results of the tissue culture seedlings of golden thread treated by different concentrations of nitrogen. DETAILED DESCRIPTION

[0018] The technical solutions of the application are described in detail below in combination with specific embodiments. The following embodiments are only used for explanation and illustration, and do not constitute a limitation on the technical solutions of the application.

[0019] Embodiment 1 The method for increasing the propagation multiple of the tissue culture seedlings of golden thread and promoting the propagation of the sporophytes of the tissue culture seedlings of golden thread comprises the following steps: (1) Preparation of propagation culture medium NH4NO3, KH2PO4, KNO3, CaCl2 and MgSO4 are taken to prepare a nitrogen treatment solution, and the concentrations of the components are as follows: the concentration of NH4NO3 is 0.825 g / L; the concentration of KH2PO4 is 3.4 g / L; the concentration of KNO3 is 38 g / L; the concentration of CaCl2 is 8.8 g / L; and the concentration of MgSO4 is 3.61 g / L.

[0020] MS medium was prepared by taking agar, trace elements (prepared by dissolving 22.3 mg of MnSO4·4H2O, 8.6 mg of ZnSO4·7H2O, 6.2 mg of H3BO3, 0.83 mg of KI, 0.025 mg of CuSO4·5H2O, 0.025 mg of CoCl2·6H2O, and 0.25 mg of Na2MoO4·2H2O in 1 L of water), sucrose, organic matter (prepared by dissolving 2 mg of glycine, 0.1 mg of VB1, 0.5 mg of VB6, 0.5 mg of VB5, and 100 mg of inositol in 1 L of water), iron salt (prepared by dissolving 37.3 mg of Na2·EDTA and 27.8 mg of FeSO4·7H2O in 1 L of water), and the nitrogen treatment solution prepared above, with the concentrations of the components being: the concentration of agar was 4.5 g / L; the concentration of trace elements was 1 mL / L; the concentration of sucrose was 20 g / L; the concentration of organic matter was 10 mL / L; the concentration of the nitrogen treatment solution was 50 mL / L; and the concentration of iron salt was 10 mL / L.

[0021] A propagation medium was prepared by adding 6-benzyladenine, naphthaleneacetic acid, and activated carbon to the MS medium prepared above, with the concentrations of the components being: the concentration of 6-benzyladenine was 1.5 mg / L; the concentration of naphthaleneacetic acid was 0.5 mg / L; and the concentration of activated carbon was 1 mg / L.

[0022] (2) Culturing of C. fortunei C. fortunei tissue culture seedlings were obtained by taking C. fortunei tissue culture seedlings (10 mg of C. fortunei mature spore powder was taken in a 1.5 mL centrifuge tube, soaked in distilled water for 1 h, the supernatant was removed by centrifugation, the lower layer of the precipitate was sterilized with 70% alcohol for 30 s, the supernatant was removed by centrifugation, the lower layer of the precipitate was sterilized with 4% NaClO solution for 15 min, the supernatant was removed by centrifugation, the lower layer of the precipitate was washed with sterile water for 4-6 times, the obtained precipitate was centrifuged again, 1.5 mL of sterile water was added to the precipitate, and the mixture was mixed to obtain a spore suspension; the spore suspension was inoculated into 1 / 2 MS medium, 1 mL of sterile spore suspension was inoculated into each bottle, and protocom-like bodies were formed after 60-90 days of culture; when the protocom-like bodies grew into heart-shaped, they were transferred to new medium of the same type for continuous culture, and C. fortunei tissue culture seedlings were obtained), which were placed in the propagation medium prepared in step S1 for subculture (well-grown sterile and emerald gametophyte clusters were selected on a clean bench, cut into small protocom-like body clusters with a size of 1 cm x 1 cm, inoculated into C. fortunei propagation medium, and the propagation medium was composed of MS+agar or carrageenan+sucrose+IAA+6-BA+activated carbon without agar and sucrose, 5 or 6 protocom-like body clusters were inoculated into each bottle, and the inoculated bottles were incubated at a constant temperature); the C. fortunei tissue culture seedlings obtained by subculture were placed in the propagation medium for continuous culture for 90 days. The culture conditions were: the temperature was 25°C; the humidity was 50%; the light intensity was 1000 Lx; and the light time was 12 h / d.

[0023] Example 2 The method for increasing the propagation multiple of the tissue culture seedlings of the golden dog and promoting the sporophyte propagation is as follows: (1) Preparation of the propagation culture medium Take NH4NO3, KH2PO4, KNO3, CaCl2 and MgSO4 to prepare the nitrogen treatment solution, and the concentration of each component is as follows: the concentration of NH4NO3 is 1.65 g / L; the concentration of KH2PO4 is 3.4 g / L; the concentration of KNO3 is 38 g / L; the concentration of CaCl2 is 8.8 g / L; and the concentration of MgSO4 is 3.61 g / L.

[0024] Take agar, trace elements (prepared by dissolving 22.3 mg of MnSO4·4H2O, 8.6 mg of ZnSO4·7H2O, 6.2 mg of H3BO3, 0.83 mg of KI, 0.025 mg of CuSO4·5H2O, 0.025 mg of CoCl2·6H2O and 0.25 mg of Na2MoO4·2H2O in 1 L of water), sucrose, organic matter (prepared by dissolving 2 mg of glycine, 0.1 mg of VB1, 0.5 mg of VB6, 0.5 mg of VB5 and 100 mg of inositol in 1 L of water), iron salt (prepared by dissolving 37.3 mg of Na2·EDTA and 27.8 mg of FeSO4·7H2O in 1 L of water) and the above prepared nitrogen treatment solution to prepare the MS culture medium, and the concentration of each component is as follows: the concentration of agar is 4.5 g / L; the concentration of trace elements is 1 mL / L; the concentration of sucrose is 20 g / L; the concentration of organic matter is 10 mL / L; the concentration of the nitrogen treatment solution is 50 mL / L; and the concentration of iron salt is 10 mL / L.

[0025] Add 6-benzyladenine, naphthalene acetic acid and activated carbon to the above prepared MS culture medium to prepare the propagation culture medium, and the concentration of each component is as follows: the concentration of 6-benzyladenine is 1.5 mg / L; the concentration of naphthalene acetic acid is 0.5 mg / L; and the concentration of activated carbon is 1 mg / L.

[0026] (2) Cultivation of the golden dog The gold dog tissue culture seedlings (referred to as the mature spore powder of gold dog spine 10 mg in a 1.5 mL centrifuge tube, soaked with distilled water for 1 h, centrifuged to remove the supernatant, the lower layer of the precipitate was sterilized with 70% alcohol for 30 s, centrifuged and the supernatant was removed, the lower layer of the precipitate was sterilized with 4% NaClO solution for 15 min, centrifuged and the supernatant was removed, the lower layer of the precipitate was washed with sterile water for 4-6 times, centrifuged again, 1.5 mL of sterile water was added to the obtained precipitate, mixed well, and the spore suspension was obtained; the spore suspension was inoculated into 1 / 2 MS medium, 1 mL of sterile spore suspension was inoculated into each bottle, and protoplasts were formed after 60-90 days of culture, and the protoplasts were transferred to new identical culture medium for continuous culture when the protoplasts grew to heart-shaped, and the gold dog tissue culture seedlings were obtained) were placed in the propagation medium prepared in step S1 for subculture (the sterile and green gametophyte clusters growing well were selected on the clean bench, cut into 1 cm x 1 cm small protoplast clusters, inoculated into the gold dog propagation medium, and the propagation medium was composed of MS without agar and sucrose + agar or carrageenan + sucrose + IAA + 6-BA + activated carbon, 5 or 6 protoplast clusters were inoculated into each bottle, and the constant temperature culture was carried out after inoculation) ; the gold dog tissue culture seedlings obtained by subculture were placed in the propagation medium for continuous culture for 90 days. The culture conditions are as follows: temperature is 25°C; humidity is 50%; light intensity is 1000 Lx; and light time is 12 h / d.

[0027] Example 3 The method for improving the propagation multiple of gold dog tissue culture seedlings and promoting the propagation of spore bodies is as follows: (1) Preparation of propagation medium NH4NO3, KH2PO4, KNO3, CaCl2 and MgSO4 were taken to prepare a nitrogen treatment solution, and the concentrations of the components were as follows: the concentration of NH4NO3 was 3.3 g / L; the concentration of KH2PO4 was 3.4 g / L; the concentration of KNO3 was 38 g / L; the concentration of CaCl2 was 8.8 g / L; and the concentration of MgSO4 was 3.61 g / L.

[0028] MS medium was prepared by taking agar, trace elements (prepared by dissolving 22.3 mg of MnSO4·4H2O, 8.6 mg of ZnSO4·7H2O, 6.2 mg of H3BO3, 0.83 mg of KI, 0.025 mg of CuSO4·5H2O, 0.025 mg of CoCl2·6H2O, and 0.25 mg of Na2MoO4·2H2O in 1 L of water), sucrose, organic matter (prepared by dissolving 2 mg of glycine, 0.1 mg of VB1, 0.5 mg of VB6, 0.5 mg of VB5, and 100 mg of inositol in 1 L of water), iron salt (prepared by dissolving 37.3 mg of Na2·EDTA and 27.8 mg of FeSO4·7H2O in 1 L of water), and the nitrogen treatment solution prepared above, with the concentrations of the components being: the concentration of agar was 4.5 g / L; the concentration of trace elements was 1 mL / L; the concentration of sucrose was 20 g / L; the concentration of organic matter was 10 mL / L; the concentration of the nitrogen treatment solution was 50 mL / L; and the concentration of iron salt was 10 mL / L.

[0029] A propagation medium was prepared by adding 6-benzyladenine, naphthaleneacetic acid, and activated carbon to the MS medium prepared above, with the concentrations of the components being: the concentration of 6-benzyladenine was 1.5 mg / L; the concentration of naphthaleneacetic acid was 0.5 mg / L; and the concentration of activated carbon was 1 mg / L.

[0030] (2) Culturing of C. fortunei C. fortunei tissue culture seedlings were obtained by taking C. fortunei tissue culture seedlings (10 mg of C. fortunei mature spore powder was taken in a 1.5 mL centrifuge tube, soaked in distilled water for 1 h, the supernatant was removed by centrifugation, the lower layer of the precipitate was sterilized with 70% alcohol for 30 s, the supernatant was removed by centrifugation, the lower layer of the precipitate was sterilized with 4% NaClO solution for 15 min, the supernatant was removed by centrifugation, the lower layer of the precipitate was washed with sterile water for 4-6 times, the obtained precipitate was centrifuged again, 1.5 mL of sterile water was added to the precipitate, and the mixture was mixed to obtain a spore suspension; the spore suspension was inoculated into 1 / 2 MS medium, 1 mL of sterile spore suspension was inoculated into each bottle, and protocom-like bodies were formed after 60-90 days of culture; when the protocom-like bodies grew into heart-shaped, they were transferred to new medium of the same type for continuous culture, and C. fortunei tissue culture seedlings were obtained), which were placed in the propagation medium prepared in step S1 for subculture (well-grown sterile and emerald gametophyte clusters were selected on a clean bench, cut into small protocom-like body clusters with a size of 1 cm x 1 cm, inoculated into C. fortunei propagation medium, and the propagation medium was composed of MS+agar or carrageenan+sucrose+IAA+6-BA+activated carbon without agar and sucrose, 5 or 6 protocom-like body clusters were inoculated into each bottle, and the inoculated bottles were incubated at a constant temperature); the C. fortunei tissue culture seedlings obtained by subculture were placed in the propagation medium for continuous culture for 90 days. The culture conditions were: the temperature was 25°C; the humidity was 50%; the light intensity was 1000 Lx; and the light time was 12 h / d.

[0031] Example 4 The method for increasing the propagation multiple of the tissue culture seedlings of the golden dog and promoting the sporophyte propagation is as follows: (1) Preparation of the propagation culture medium Take NH4NO3, KH2PO4, KNO3, CaCl2 and MgSO4 to prepare the nitrogen treatment solution, and the concentration of each component is as follows: the concentration of NH4NO3 is 4.95 g / L; the concentration of KH2PO4 is 3.4 g / L; the concentration of KNO3 is 38 g / L; the concentration of CaCl2 is 8.8 g / L; and the concentration of MgSO4 is 3.61 g / L.

[0032] Take agar, trace elements (prepared by dissolving 22.3 mg of MnSO4·4H2O, 8.6 mg of ZnSO4·7H2O, 6.2 mg of H3BO3, 0.83 mg of KI, 0.025 mg of CuSO4·5H2O, 0.025 mg of CoCl2·6H2O and 0.25 mg of Na2MoO4·2H2O in 1 L of water), sucrose, organic matter (prepared by dissolving 2 mg of glycine, 0.1 mg of VB1, 0.5 mg of VB6, 0.5 mg of VB5 and 100 mg of inositol in 1 L of water), iron salt (prepared by dissolving 37.3 mg of Na2·EDTA and 27.8 mg of FeSO4·7H2O in 1 L of water) and the above prepared nitrogen treatment solution to prepare the MS culture medium, and the concentration of each component is as follows: the concentration of agar is 4.5 g / L; the concentration of trace elements is 1 mL / L; the concentration of sucrose is 20 g / L; the concentration of organic matter is 10 mL / L; the concentration of the nitrogen treatment solution is 50 mL / L; and the concentration of iron salt is 10 mL / L.

[0033] Add 6-benzyladenine, naphthalene acetic acid and activated carbon to the above prepared MS culture medium to prepare the propagation culture medium, and the concentration of each component is as follows: the concentration of 6-benzyladenine is 1.5 mg / L; the concentration of naphthalene acetic acid is 0.5 mg / L; and the concentration of activated carbon is 1 mg / L.

[0034] (2) Cultivation of the golden dog The gold dog tissue culture seedlings (referred to as the mature spore powder of gold dog spine 10 mg in a 1.5 mL centrifuge tube, soaked with distilled water for 1 h, centrifuged to remove the supernatant, the lower layer of the precipitate was sterilized with 70% alcohol for 30 s, centrifuged and the supernatant was removed, the lower layer of the precipitate was sterilized with 4% NaClO solution for 15 min, centrifuged and the supernatant was removed, the lower layer of the precipitate was washed with sterile water for 4-6 times, centrifuged again, 1.5 mL of sterile water was added to the obtained precipitate, mixed well, and the spore suspension was obtained; the spore suspension was inoculated into 1 / 2 MS medium, 1 mL of sterile spore suspension was inoculated into each bottle, and protoplasts were formed after 60-90 days of culture, and the protoplasts were transferred to new identical culture medium for continuous culture when the protoplasts grew to heart-shaped, and the gold dog tissue culture seedlings were obtained) were placed in the propagation medium prepared in step S1 for subculture (the sterile and green gametophyte clusters growing well were selected on the clean bench, cut into 1 cm x 1 cm small protoplast clusters, inoculated into the gold dog propagation medium, and the propagation medium was composed of MS without agar and sucrose + agar or carrageenan + sucrose + IAA + 6-BA + activated carbon, 5 or 6 protoplast clusters were inoculated into each bottle, and the constant temperature culture was carried out after inoculation) ; the gold dog tissue culture seedlings obtained by subculture were placed in the propagation medium for continuous culture for 90 days. The culture conditions are as follows: temperature is 25°C; humidity is 50%; light intensity is 1000 Lx; and light time is 12 h / d.

[0035] Example 5 The method for improving the propagation multiple of gold dog tissue culture seedlings and promoting the propagation of spore bodies is as follows: (1) Preparation of propagation medium NH4NO3, KH2PO4, KNO3, CaCl2 and MgSO4 were used to prepare a nitrogen treatment solution, and the concentrations of the components were as follows: the concentration of NH4NO3 was 6.6 g / L; the concentration of KH2PO4 was 3.4 g / L; the concentration of KNO3 was 38 g / L; the concentration of CaCl2 was 8.8 g / L; and the concentration of MgSO4 was 3.61 g / L.

[0036] MS medium was prepared by taking agar, trace elements (prepared by dissolving 22.3 mg of MnSO4·4H2O, 8.6 mg of ZnSO4·7H2O, 6.2 mg of H3BO3, 0.83 mg of KI, 0.025 mg of CuSO4·5H2O, 0.025 mg of CoCl2·6H2O, and 0.25 mg of Na2MoO4·2H2O in 1 L of water), sucrose, organic matter (prepared by dissolving 2 mg of glycine, 0.1 mg of VB1, 0.5 mg of VB6, 0.5 mg of VB5, and 100 mg of inositol in 1 L of water), iron salt (prepared by dissolving 37.3 mg of Na2·EDTA and 27.8 mg of FeSO4·7H2O in 1 L of water), and the nitrogen treatment solution prepared above, with the concentrations of the components being: the concentration of agar was 4.5 g / L; the concentration of trace elements was 1 mL / L; the concentration of sucrose was 20 g / L; the concentration of organic matter was 10 mL / L; the concentration of the nitrogen treatment solution was 50 mL / L; and the concentration of iron salt was 10 mL / L.

[0037] A propagation medium was prepared by adding 6-benzyladenine, naphthaleneacetic acid, and activated carbon to the MS medium prepared above, with the concentrations of the components being: the concentration of 6-benzyladenine was 1.5 mg / L; the concentration of naphthaleneacetic acid was 0.5 mg / L; and the concentration of activated carbon was 1 mg / L.

[0038] (2) Culturing of C. fortunei C. fortunei tissue culture seedlings were obtained by taking C. fortunei tissue culture seedlings (10 mg of C. fortunei mature spore powder was taken in a 1.5 mL centrifuge tube, soaked in distilled water for 1 h, the supernatant was removed by centrifugation, the lower layer of the precipitate was sterilized with 70% alcohol for 30 s, the supernatant was removed by centrifugation, the lower layer of the precipitate was sterilized with 4% NaClO solution for 15 min, the supernatant was removed by centrifugation, the lower layer of the precipitate was washed with sterile water for 4-6 times, the obtained precipitate was centrifuged again, 1.5 mL of sterile water was added to the precipitate, and the mixture was mixed to obtain a spore suspension; the spore suspension was inoculated into 1 / 2 MS medium, 1 mL of sterile spore suspension was inoculated into each bottle, and protocom-like bodies were formed after 60-90 days of culture; when the protocom-like bodies grew into heart-shaped, they were transferred to new medium of the same type for continuous culture, and C. fortunei tissue culture seedlings were obtained), which were placed in the propagation medium prepared in step S1 for subculture (well-grown sterile and emerald gametophyte clusters were selected on a clean bench, cut into small protocom-like body clusters with a size of 1 cm x 1 cm, inoculated into C. fortunei propagation medium, and the propagation medium was composed of MS+agar or carrageenan+sucrose+IAA+6-BA+activated carbon without agar and sucrose, 5 or 6 protocom-like body clusters were inoculated into each bottle, and the inoculated bottles were incubated at a constant temperature); the C. fortunei tissue culture seedlings obtained by subculture were placed in the propagation medium for continuous culture for 90 days. The culture conditions were: the temperature was 25°C; the humidity was 50%; the light intensity was 1000 Lx; and the light time was 12 h / d.

[0039] In order to further verify the reliability of the application and screen out the best scheme, the research team respectively used the method of examples 1-5 to cultivate the gold dog tissue culture seedlings, and used the group without adding NH4NO3 as a comparative example, each group was set 10 times in parallel, the agronomic shape of the gold dog tissue culture seedlings treated with different concentrations of nitrogen was measured and counted, and the results are shown in Table 1 and Figure 1 N0 corresponds to the comparative example, N1-N5 correspond to examples 1-5 respectively.

[0040] ; From the results, it can be seen that different concentrations of exogenous nitrogen elements have a certain influence on the agronomic traits (proliferation multiple, induced spore and growth status) of the gold dog tissue culture seedlings. After 60 days of culture, the proliferation multiple of the gold dog tissue culture seedlings in order is: N1 (0.825 g / L) > N2 (1.65 g / L) > N0 (0) > N3 (3.3 g / L) > N4 (4.95 g / L) > N5 (6.6 g / L). Among them, the average proliferation multiple of the gold dog tissue culture seedlings under N1 (0.825 g / L) treatment is the largest, which is 9.74; under N5 (6.6 g / L) treatment, the proliferation multiple is the smallest, which is 2.89; the largest group (N1 group) is 3.37 times of the smallest group (N5 group), and 2.1 times of the group without adding NH4NO3 (N0 group).

[0041] After 90 days of culture, the total number of spores induced by the gold dog tissue culture seedlings in order is: N1 (0.825 g / L) > N2 (1.65 g / L) > N3 (3.3 g / L) > N0 (0) > N4 (4.95 g / L) > N5 (6.6 g / L). Among them, the spores induced by the gold dog tissue culture seedlings under N1 (0.825 g / L) treatment are the most, with an average of 28.8 strains per bottle; under N5 (6.6 g / L) treatment, the induced spores are the least, with an average of 3 strains per bottle; the largest group (N1 group) is 9.6 times of the smallest group (N5 group), and 3 times of the group without adding NH4NO3 (N0 group).

[0042] Therefore, appropriate amount of nitrogen element can promote plant growth and is beneficial to the reproduction of induced spores. However, when the nitrogen concentration is too high, the growth of the plant and the induced spores will be inhibited.

[0043] Although the application has been described in detail in the foregoing general description, specific embodiments and experiments, some modifications or improvements can be made on the basis of the application, which is obvious to those skilled in the art. Therefore, these modifications or improvements made on the basis of not deviating from the spirit of the application, all belong to the scope of protection claimed by the application.

Claims

1. A method for increasing the multiplication rate of tissue culture seedlings of Cynodon dactylon and promoting their sporophyte propagation, characterized in that, It comprises the following steps: S1, preparing a propagation medium: Take NH4NO3, KH2PO4, KNO3, CaCl2 and MgSO4 to prepare a nitrogen treatment solution; take agar, trace elements, sucrose, organic matter, iron salt and the above prepared nitrogen treatment solution to prepare an MS medium; add 6-benzyladenine, naphthalene acetic acid and activated carbon to the above prepared MS medium to prepare a propagation medium; The trace elements are prepared by dissolving 22.3 mg of MnSO4·4H2O, 8.6 mg of ZnSO4·7H2O, 6.2 mg of H3BO3, 0.83 mg of KI, 0.025 mg of CuSO4·5H2O, 0.025 mg of CoCl2·6H2O and 0.25 mg of Na2MoO4·2H2O in 1 L of water; the organic matter is prepared by dissolving 2 mg of glycine, 0.1 mg of VB1, 0.5 mg of VB6, 0.5 mg of VB5 and 100 mg of myo-inositol in 1 L of water; and the iron salt is prepared by dissolving 37.3 mg of Na2·EDTA and 27.8 mg of FeSO4·7H2O in 1 L of water; S2, culturing Jinfo dog: Take the Jinfo dog tissue culture seedlings prepared in step S1 and place them in the propagation medium prepared in step S1 for subculture; take the Jinfo dog tissue culture seedlings obtained by subculture and place them in the propagation medium for continuous culture.

2. The method for increasing the multiplication rate of tissue culture plantlets and promoting their sporophyte propagation of Cinnamomum camphora according to claim 1, characterized in that, In the nitrogen treatment solution in step S1, the concentration of NH4NO3 is 0.825-6.6 g / L; the concentration of KH2PO4 is 3.4 g / L; the concentration of KNO3 is 38 g / L; the concentration of CaCl2 is 8.8 g / L; and the concentration of MgSO4 is 3.61 g / L.

3. The method for increasing the multiplication rate of tissue culture seedlings of Cynodon natalensis and promoting their sporophyte propagation according to claim 2, characterized in that, The concentration of NH4NO3 in the nitrogen treatment solution in step S1 is 0.825 g / L.

4. The method for increasing the multiplication rate of tissue culture plantlets and promoting their sporophyte propagation of Cinnamomum camphora according to claim 1, characterized in that, In the MS medium in step S1, the concentration of agar is 4.5 g / L; the concentration of trace elements is 1 mL / L; the concentration of sucrose is 20 g / L; the concentration of organic matter is 10 mL / L; the concentration of the nitrogen treatment solution is 50 mL / L; and the concentration of iron salt is 10 mL / L.

5. The method for increasing the multiplication rate of tissue culture plantlets and promoting their sporophyte propagation of Cinnamomum camphora according to claim 1, characterized in that, In the propagation medium in step S1, the concentration of 6-benzyladenine is 1.5 mg / L; the concentration of naphthalene acetic acid is 0.5 mg / L; and the concentration of activated carbon is 1 mg / L.

6. The method for increasing the propagation multiple of tissue culture seedlings of Canis lupus familiaris and promoting its sporogony according to claim 1, characterized in that, In step S2, the Jinfo dog tissue culture seedlings are prepared as follows: 10 mg of Jinfo dog spore powder is weighed into a 1.5 mL centrifuge tube, soaked in distilled water for 1 h, centrifuged to remove the supernatant, the lower layer of the precipitate is sterilized with 70% alcohol for 30 s, centrifuged and the supernatant is removed, the lower layer of the precipitate is sterilized with 4% NaClO solution for 15 min, centrifuged and the supernatant is removed, the lower layer of the precipitate is washed with sterile water for 4-6 times, centrifuged again, 1.5 mL of sterile water is added to the obtained precipitate, and mixed evenly to obtain a spore suspension; the spore suspension is inoculated into 1 / 2 MS medium, 1 mL of sterile spore suspension is inoculated into each bottle, and protoplasts are formed after 60-90 days of culture; when the protoplasts grow into heart-shaped, they are transferred to new medium for continuous culture, and the Jinfo dog tissue culture seedlings are obtained.

7. The method for increasing the multiplication rate of tissue culture plantlets and promoting their sporophyte propagation of Cinnamomum camphora according to claim 1, characterized in that, The subculture in step S2 is specifically as follows: selecting well-grown and sterile Cui green gametophyte groups on an ultra-clean workbench, dividing the groups into 1 cm x 1 cm small protocorm groups, inoculating 5 or 6 protocorm groups into each bottle of Jinmao dog propagation culture medium, and then culturing at a constant temperature after inoculation. The propagation culture medium comprises: MS + agar or carrageenan + sucrose + IAA + 6-BA + activated carbon without agar and sucrose.

8. The method for increasing the multiplication rate of tissue culture plantlets and promoting their sporophyte propagation of Cineraria axiarum according to any one of claims 1 or 6 or 7, characterized in that, The culture conditions of step S2 are as follows: a temperature of 25±2 ℃, a humidity of 50%, a light intensity of 1000 Lx, and a light time of 12 h / d.

9. The method for increasing the multiplication rate of tissue culture plantlets and promoting their sporophyte propagation of Cinnamomum camphora according to claim 1, characterized in that, The continued culturing time in step S2 is 60-90 days.

10. The method for increasing the multiplication rate of tissue culture plantlets and promoting their sporophyte propagation of Cinnamomum camphora according to claim 9, characterized in that, The continued culturing time in step S2 is 60 days.

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