A method for the mass production of cycas revoluta spores

Abstract

The application provides a method for breeding Cycas revoluta spores, comprising the following steps: 1) a first nutrient medium comprising Cycas revoluta spores is subjected to light treatment at a temperature of 24-26 DEG C and a light intensity of 1000-6500 lux for 24 hours per day to obtain prothallus; and 2) a second nutrient medium comprising the prothallus is subjected to light treatment at a temperature of 24-26 DEG C and a light intensity of 1000-3500 lux for 24 hours per day to obtain a sporophyte. The method for breeding Cycas revoluta spores provided by the application greatly shortens the time for forming sporophytes by limiting the temperature, light intensity and light time of the first nutrient medium comprising Cycas revoluta spores and the second nutrient medium comprising the prothallus, solves the problems that the time for breeding and raising seedlings from spores of ferns is relatively long and in-vitro breeding and differentiation are difficult, and provides technical support for the breeding of ferns.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of plant cultivation, and particularly relates to a method for spore breeding of Cycas revoluta. BACKGROUND

[0002] Cycas revoluta, also known as red fern, is a single evergreen soil cycas large herbaceous fern of Cycas in the family of Blechnaceae, which is distributed in Guangdong, Guangxi, Hainan, Fujian, Taiwan, Yunnan and Guizhou, and usually grows on sunny slopes with an altitude of 450-1700 meters. Cycas revoluta is a surviving plant of the Devonian period in the Paleozoic era, which reproduces by spores, has large leaves and developed xylem, and its stem can be thickened, so it is an intermediate type between ferns and gymnosperms. Therefore, the study of Cycas revoluta is of great significance for the study of plant species evolution and flora. At the same time, it has extremely high ornamental value due to its beautiful tree shape, tender red leaves and old green leaves. Cycas revoluta not only has ornamental value, but also has medicinal value. Its stem has the effects of clearing heat and detoxifying, promoting blood circulation and removing blood stasis, and antibiosis and astringency. However, due to the relatively strict requirement for the growth environment and the biological characteristics of Cycas revoluta itself, although the distribution of Cycas revoluta is relatively wide, the number of plants is relatively small. In addition, the change of the environment of wild resources makes the wild Cycas revoluta face extinction. Therefore, in 1999, Cycas revoluta was listed in the National Key Protected Wild Plants List (the first batch), and the protection level is the national II-level key protection. In the Red List of Endangered Species of the World (IUCN), Cycas revoluta is listed as vulnerable (VU). Therefore, it is of great significance to protect and breed Cycas revoluta.

[0003] Cycas revoluta mainly reproduces by spores, and there are few reports on artificial spore breeding of Cycas revoluta in the prior art. Patent document CN102613075B and doctoral dissertation Biological Research on in vitro Breeding of Adiantum Raddianum show that the spore germination of ferns needs about 15-20 days, and the spore formation needs 40-45 days, which indicates that the seedling raising time of ferns by spore reproduction is relatively long, and the in vitro breeding differentiation is difficult. SUMMARY

[0004] The main purpose of the present application is to provide a method for spore breeding of Cycas revoluta, which solves the problems of long seedling raising time of ferns by spore reproduction and difficult in vitro breeding differentiation, and provides technical support for the breeding of ferns.

[0005] The present application provides a method for spore breeding of Cycas revoluta, which comprises the following steps:

[0006] 1) A first nutrient medium comprising spores of Cycas revoluta is subjected to light treatment at a temperature of 24-26℃ and a light intensity of 1000-6500 lux for 24h / d to obtain prothallus;

[0007] 2) the second nutrient medium comprising the prothallus is subjected to light treatment at a temperature of 24-26℃ and a light intensity of 1000-3500 lux for 24h / d to obtain a sporophyte.

[0008] The method for breeding the spores of the Cycas revoluta as described above, wherein the first nutrient medium comprises a substrate and a first nutrient solution; and the second nutrient medium comprises a substrate and a second nutrient solution.

[0009] The substrate is prepared by a method comprising the following steps: mixing peat, vermiculite, river sand and perlite in a volume ratio of 80-100:30-50:15-25:15-25 to form a substrate precursor, subjecting the substrate precursor to solution sterilization treatment with a sterilization solution, and then subjecting the solution sterilization system to high-temperature sterilization treatment at 120-125℃.

[0010] The sterilization solution comprises 400-600 times of chlorothalonil and 1400-1600 times of an algicide, and the volume ratio of the substrate precursor to the sterilization solution is 3-5:1-2.

[0011] The method for breeding the spores of the Cycas revoluta as described above, wherein the first nutrient solution or the second nutrient solution comprises a nutrient solution functional component.

[0012] The nutrient solution functional component comprises at least one of gibberellin, 1 / 4MS nutrient solution, 6-BA and NAA.

[0013] The method for breeding the spores of the Cycas revoluta as described above, wherein the first nutrient solution comprises water and a first nutrient solution functional component.

[0014] The first nutrient solution functional component comprises at least one of gibberellin and 1 / 4MS nutrient solution.

[0015] The method for breeding the spores of the Cycas revoluta as described above, wherein the first nutrient solution comprises gibberellin and water, and the mass-volume ratio of the gibberellin to the water is 0.8-1.0mg:800-1000mL; or,

[0016] The first nutrient solution comprises 1 / 4MS nutrient solution and water, and the volume ratio of the 1 / 4MS nutrient solution to the water is 50-60:800-1000; or,

[0017] The first nutrient solution comprises gibberellin, 1 / 4MS nutrient solution and water, and the mass-volume ratio of the gibberellin, the 1 / 4MS nutrient solution and the water is 0.8-1.0mg:50-60mL:800-1000mL.

[0018] The method for breeding the spores of the Cycas revoluta as described above, wherein the mass-volume ratio of the spores of the Cycas revoluta to the first nutrient medium is 0.004-0.006g:80-100mL.

[0019] The second nutrient solution comprises water and a second nutrient solution functional component.

[0020] The second nutrient solution functional component comprises at least one of gibberellin, 1 / 4MS nutrient solution, 6-BA and NAA.

[0021] The second nutrient solution comprises water, gibberellin, 6-BA and NAA, and the mass-volume ratio of gibberellin, 6-BA, NAA and water is 0.8-1.0 mg:0.4-0.6 mg:0.08-0.1 mg:800-1000 mL; or,

[0022] The second nutrient solution comprises 1 / 4MS nutrient solution and water, and the volume ratio of 1 / 4MS nutrient solution and water is 50-60:800-1000; or,

[0023] The second nutrient solution comprises gibberellin, 6-BA, NAA, 1 / 4MS nutrient solution and water, and the mass-volume ratio of gibberellin, 6-BA, NAA, 1 / 4MS nutrient solution and water is 0.8-1.0 mg:0.4-0.6 mg:0.08-0.1 mg:50-60 mL:800-1000 mL.

[0024] The method for breeding spores of Cycas revoluta as described above, wherein the volume ratio of the substrate and the first nutrient solution is 350-400:200-250; and the volume ratio of the substrate and the second nutrient solution is 350-400:200-250.

[0025] The method for breeding spores of Cycas revoluta as described above, wherein the light intensity in step 1) is 3000-3500 lux; and the light intensity in step 2) is 3000-3500 lux.

[0026] The present application provides a method for breeding spores of Cycas revoluta, which greatly shortens the time for forming sporophytes by limiting the temperature, light intensity and illumination time of a first nutrient substrate comprising spores of Cycas revoluta and a second nutrient substrate comprising prothalli, solves the problems of long time for breeding and culturing seedlings of spores of ferns and difficult differentiation of in vitro breeding, and provides technical support for breeding of ferns. BRIEF DESCRIPTION OF DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the related art, the accompanying drawings needed to be used in the description of the embodiments of the present application or the related art are briefly introduced below. Obviously, the accompanying drawings in the following description are only some embodiments of the present application, and other accompanying drawings can be obtained by those skilled in the art without any creative effort on the basis of these accompanying drawings.

[0028] Figure 1 Photos of spore germination for Example 2;

[0029] Figure 2 Photos of spore germination for Example 4;

[0030] Figure 3 Photos of prothallus formation for Example 2;

[0031] Figure 4 Photos of prothallus formation for Example 4;

[0032] Figure 5 Photos of sporophyte formation for Example 13;

[0033] Figure 6 Photos of sporophyte formation for Example 15;

[0034] Figure 7 Photos of no sporophyte formation after 60 days for Comparative Example 7;

[0035] Figure 8 Photos of no sporophyte formation after 60 days for Comparative Example 8. DETAILED DESCRIPTION

[0036] In order to make the objects, technical solutions and advantages of the present application clearer, the following will combine the embodiments of the present application to make a clear and complete description of the technical solutions in the embodiments of the present application. Obviously, the described embodiments are some but not all of the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0037] The present application provides a method for breeding Cycas revoluta spores, comprising the following steps:

[0038] 1) A first nutrient medium comprising Cycas revoluta spores is subjected to light treatment at a temperature of 24-26℃ and a light intensity of 1000-6500 lux for 24h / d to obtain prothallus;

[0039] 2) A second nutrient medium comprising the prothallus is subjected to light treatment at a temperature of 24-26℃ and a light intensity of 1000-3500 lux for 24h / d to obtain sporophyte.

[0040] It can be understood that Cycas revoluta belongs to the category of spore plants as a fern, and it reproduces by using spores.

[0041] In the embodiments of the present application, Cycas revoluta spores are sowed, and after a series of light treatments, sporophyte is finally obtained.

[0042] Specifically, in step 1), the first nutrient medium comprising the cycas revoluta spores is subjected to light treatment at a temperature of 24-26℃ and a light intensity of 1000-6500 lux for 24h / d to obtain the prothallus. The cycas revoluta spores can be collected, for example, in July every year, when the cycas revoluta spores are dark brown, i.e. the spores are mature. Before sowing, the seedling box can be subjected to sterilization treatment, for example, immersed in a 3% potassium permanganate solution for 2h for sterilization treatment, then the first nutrient medium is loaded into the sterilized seedling box, and the cycas revoluta spores are sown into the first nutrient medium, the temperature is set to 24-26℃, the light intensity is set to 1000-6500 lux, and the first nutrient medium sown with the cycas revoluta spores is placed under light of the above conditions for irradiation. The researchers found that the first nutrient medium is subjected to light treatment for 24h / d, and spore germination can be observed on the 4th-7th day, i.e. dark green small pieces can be seen, and the prothallus is formed on the 8th-14th day, i.e. green small stolon can be seen with the naked eye.

[0043] In step 2), the second nutrient medium comprising the prothallus is subjected to light treatment at a temperature of 24-26℃ and a light intensity of 1000-3500 lux for 24h / d to obtain the sporophyte. After the prothallus is formed, the first nutrient medium is replaced with the second nutrient medium, and the second nutrient medium with the prothallus is subjected to light treatment at a temperature of 24-26℃ and a light intensity of 1000-3500 lux for 24h / d. The researchers found that under the above light treatment conditions, sporophyte formation can be observed on the 31st-43rd day, which greatly shortens the time for sporophyte formation compared with other ferns (the sporophyte formation of ferns in the prior art takes about 60 days), and also solves the problem of difficult in vitro breeding and differentiation of ferns (the prothallus to sporophyte formation in the in vitro breeding of ferns takes about 100 days).

[0044] The sporophyte is subjected to light treatment at a temperature of 24-26℃ and a light intensity of 1000-3500 lux for 24h / d, and the height of the sporophyte can reach 0.8-2.2cm on the 60th day.

[0045] The method for breeding cycas revoluta spores of the present embodiment greatly shortens the time for sporophyte formation by limiting the temperature, light intensity and light time of the first nutrient medium comprising the cycas revoluta spores and the second nutrient medium comprising the prothallus, solves the problems of long breeding time and difficult in vitro breeding and differentiation of fern spores, and provides technical support for the breeding of ferns.

[0046] In some embodiments of the present application, the first nutrient medium comprises a substrate and a first nutrient solution; and the second nutrient medium comprises a substrate and a second nutrient solution.

[0047] The substrate is prepared by a method comprising the following processes: mixing peat, vermiculite, river sand, and perlite in a volume ratio of 80-100:30-50:15-25:15-25 to form a substrate precursor, performing solution sterilization treatment on the substrate precursor with a sterilization solution, and then performing high-temperature sterilization treatment on the solution sterilization system at 120-125°C.

[0048] The sterilization solution comprises 400-600 times of chlorothalonil and 1400-1600 times of algicide, and the volume ratio of the substrate precursor to the sterilization solution is 3-5:1-2.

[0049] In an embodiment, the first nutrient substrate comprises a substrate and a first nutrient solution, the second nutrient substrate comprises a substrate and a second nutrient solution, the first nutrient solution and the second nutrient solution can be the same or different, and the substrates can be the same.

[0050] The substrate in the first nutrient substrate and the second nutrient substrate can be prepared by the following processes: mixing peat, vermiculite, river sand, and perlite in a volume ratio of 80-100:30-50:15-25:15-25 to form a substrate precursor, which can provide a good growth environment for plants and promote the healthy growth of plants. The vermiculite and perlite have good water absorption and air permeability, which can adjust the moisture content and porosity of the substrate precursor; the peat can be 0-10 mm peat, which contains rich organic matter and nutrient elements such as nitrogen, phosphorus, and potassium, and can provide the required nutrient components for plants; the river sand can provide mineral substances such as calcium and magnesium. The substrate precursor is then subjected to solution sterilization treatment and high-temperature sterilization treatment to completely eliminate various pathogens and algae in the substrate precursor, thereby avoiding the damage caused by pathogens and algae to plant growth and reducing the risk of plant diseases and pests. The sterilization solution used in the solution sterilization treatment can be obtained by mixing 400-600 times of chlorothalonil and 1400-1600 times of algicide, and the volume ratio of the substrate precursor to the sterilization solution is 3-5:1-2. The above sterilization solution is sprayed on the substrate precursor and stirred uniformly to sterilize and remove algae, and then the solution sterilization system is subjected to high-temperature sterilization treatment at 120-125°C. The high-temperature sterilization treatment can use a high-temperature sterilization bag to seal the solution sterilization system and perform complete sterilization in a 120°C high-temperature sterilization pot.

[0051] In an embodiment, the first nutrient solution or the second nutrient solution comprises water, which can provide the required basic nutrients for the growth of sago fern spores into prothallus and the growth of prothallus into sporophyte.

[0052] The substrate prepared by the method in the present embodiment can provide the required nutrient components for plant growth, avoid the damage caused by pathogens and algae to plant growth, reduce the risk of plant diseases and pests, and solve the problem of complex sterilization process in the in vitro cultivation of ferns.

[0053] In some embodiments of the present application, the first nutrient solution or the second nutrient solution comprises a nutrient solution functional component;

[0054] The nutrient solution functional component comprises at least one of gibberellin, 1 / 4MS nutrient solution, 6-BA, and NAA.

[0055] In an embodiment, the first nutrient solution or the second nutrient solution comprises a nutrient solution functional component, and the nutrient solution functional component comprises at least one of gibberellin, 1 / 4MS nutrient solution, 6-BA, and NAA. Gibberellin, as a plant hormone, can promote the growth and development of plants; 1 / 4MS nutrient solution is a commonly used plant tissue culture medium, which contains a large amount of nutrients necessary for plant growth, including carbohydrates, amino acids, vitamins, inorganic salts, and growth factors, etc., and provides comprehensive nutritional support for the growth and development of plant tissues; 6-BA (6-benzylaminopurine) is a common plant growth regulator, which can promote the division and growth of plant cells; and NAA (naphthalene acetic acid) is a plant growth regulator, which can promote the formation and growth of roots.

[0056] The first nutrient solution or the second nutrient solution in the present embodiment can be added at the bottom of the seedling box, and the substrate can absorb the nutrient solution through the bottom water absorption method to provide the spores with the required nutrients for growing into thalli, thereby reducing labor and cost, and avoiding damage to young prothalli caused by the commonly used watering method.

[0057] The first nutrient solution or the second nutrient solution in the present embodiment further comprises a nutrient solution functional component, wherein the nutrient solution functional component comprises at least one of gibberellin, 1 / 4MS nutrient solution, 6-BA, and NAA, which can improve the growth effect of plants.

[0058] In some embodiments of the present application, the first nutrient solution comprises water and a first nutrient solution functional component;

[0059] The first nutrient solution functional component comprises at least one of gibberellin and 1 / 4MS nutrient solution.

[0060] The first nutrient solution in the present embodiment comprises water and a first nutrient solution functional component, wherein the first nutrient solution functional component is at least one of gibberellin and 1 / 4MS nutrient solution, i.e., the first nutrient solution comprises water and gibberellin, or the first nutrient solution comprises water and 1 / 4MS nutrient solution, or the first nutrient solution comprises water, gibberellin, and 1 / 4MS nutrient solution.

[0061] The first nutrient solution in the present embodiment comprises water and a first nutrient solution functional component, wherein the first nutrient solution functional component comprises at least one of gibberellin and 1 / 4MS nutrient solution, which can provide the spores of cycas fern with nutrients for growing into prothalli, thereby shortening the time required for the spores of cycas fern to grow into prothalli.

[0062] In some embodiments of the present application, the first nutrient solution comprises gibberellin and water, and the mass-volume ratio of gibberellin and water is 0.8-1.0 mg: 800-1000 mL; or,

[0063] The first nutrient solution comprises 1 / 4MS nutrient solution and water, and the volume ratio of 1 / 4MS nutrient solution and water is 50-60: 800-1000; or,

[0064] The first nutrient solution comprises gibberellin, 1 / 4MS nutrient solution and water, and the mass-volume ratio of gibberellin, 1 / 4MS nutrient solution and water is 0.8-1.0 mg: 50-60 mL: 800-1000 mL.

[0065] The three components in the first nutrient solution and the mass-volume ratio of each component in the present embodiment can further provide the required nutrients for the growth of the cycas revoluta spore into prothallus, and shorten the time required for the growth of the cycas revoluta spore into prothallus.

[0066] In some embodiments of the present application, the mass-volume ratio of the cycas revoluta spore and the first nutrient substrate is 0.004-0.006 g: 80-100 mL.

[0067] In the present embodiment, the mass-volume ratio of the cycas revoluta spore and the first nutrient substrate is controlled to be 0.004-0.006 g: 80-100 mL, which is in a suitable range, can promote the germination and growth of the cycas revoluta spore, can ensure that the first nutrient substrate provides sufficient nutrients for the growth of the cycas revoluta spore, and can also avoid excessive growth of the spore due to excessive nutrition of the substrate.

[0068] In some embodiments of the present application, the second nutrient solution comprises water and a second nutrient solution functional component.

[0069] The second nutrient solution functional component comprises at least one of gibberellin, 1 / 4MS nutrient solution, 6-BA and NAA.

[0070] The second nutrient solution in the embodiment includes water and a second nutrient solution functional component, wherein the second nutrient solution functional component includes at least one of gibberellin, 1 / 4MS nutrient solution, 6-BA, and NAA, i.e., the second nutrient solution includes water and gibberellin, or the second nutrient solution includes water and 1 / 4MS nutrient solution, or the second nutrient solution includes water and 6-BA, or the second nutrient solution includes water and NAA, or the second nutrient solution includes water, 6-BA, and NAA, or the second nutrient solution includes water, gibberellin, and NAA, or the second nutrient solution includes water, gibberellin, and 6-BA, or the second nutrient solution includes water, 1 / 4MS nutrient solution, and 6-BA, or the second nutrient solution includes water, gibberellin, and 1 / 4MS nutrient solution, or the second nutrient solution includes water, NAA, and 1 / 4MS nutrient solution, or the second nutrient solution includes water, gibberellin, 6-BA, and NAA, or the second nutrient solution includes water, 1 / 4MS nutrient solution, 6-BA, and NAA, or the second nutrient solution includes water, 1 / 4MS nutrient solution, gibberellin, and NAA, or the second nutrient solution includes water, 1 / 4MS nutrient solution, gibberellin, and 6-BA, or the second nutrient solution includes water, gibberellin, 6-BA, NAA, and 1 / 4MS nutrient solution.

[0071] The second nutrient solution in the embodiment includes water and a second nutrient solution functional component, wherein the second nutrient solution functional component includes at least one of gibberellin, 1 / 4MS nutrient solution, 6-BA, and NAA, and can provide nutrients for the protocorms, and shorten the time required for the protocorms to grow into sporophytes.

[0072] In some embodiments of the present application, the second nutrient solution includes gibberellin, 6-BA, NAA, and water, and the mass-volume ratio of gibberellin, 6-BA, NAA, and water is 0.8-1.0 mg:0.4-0.6 mg:0.08-0.1 mg:800-1000 mL; or,

[0073] The second nutrient solution includes 1 / 4MS nutrient solution and water, and the volume ratio of 1 / 4MS nutrient solution and water is 50-60:800-1000; or,

[0074] The second nutrient solution includes water, 1 / 4MS nutrient solution, gibberellin, 6-BA, and NAA, and the mass-volume ratio of gibberellin, 6-BA, NAA, 1 / 4MS nutrient solution, and water is 0.8-1.0 mg:0.4-0.6 mg:0.08-0.1 mg:50-60 mL:800-1000 mL.

[0075] The three components in the second nutrient solution and the mass-volume ratio of each component in the embodiment can further provide the required nutrients for the protocorms, and shorten the time required for the protocorms to grow into sporophytes.

[0076] In some embodiments of the present application, the volume ratio of the substrate and the first nutrient solution is 350-400:200-250; the volume ratio of the substrate and the second nutrient solution is 350-400:200-250.

[0077] In the present embodiment, the volume ratio of the substrate and the first nutrient solution is 350-400:200-250, and the volume ratio of the substrate and the second nutrient solution is 350-400:200-250, so that the nutrients can be uniformly distributed, the plants can obtain sufficient nutrients, the conditions of excessive nutrients or insufficient nutrients can be avoided, the nutrient utilization rate is improved, and a good growth environment is provided for the plants.

[0078] In some embodiments of the present application, the light intensity in step 1) is 3000-3500 lux; and the light intensity in step 2) is 3000-3500 lux.

[0079] In the present embodiment, the light intensity in step 1) is limited to 3000-3500 lux, and the light intensity in step 2) is limited to 3000-3500 lux, as a preferred scheme, the cycas revoluta spore breeding is carried out according to the limited light intensity in the present embodiment, so that the time for the cycas revoluta spore to grow into a sporophyte can be further shortened. Under this condition, the time for the cycas revoluta spore to grow into a sporophyte is 31-33 days from sowing.

[0080] Hereinafter, the technical scheme of the present application will be further described in combination with specific embodiments.

[0081] Embodiment 1

[0082] The method for breeding cycas revoluta spores in the present embodiment comprises the following steps:

[0083] 1) Spore collection: the spores are collected when they are dark brown in July;

[0084] 2) A seedling raising box with a bottom and a cover is selected and soaked in 3% potassium permanganate for 2h for sterilization;

[0085] 3) 0-10mm peat, vermiculite, river sand and perlite are mixed in a volume ratio of 2:1:0.5:0.5 to form a substrate precursor, the substrate precursor is sprayed with a sterilization solution formed by 500 times of chlorothalonil and 1500 times of algaecide, and stirred uniformly, and then subjected to high-temperature sterilization treatment at 120℃ to obtain the substrate, wherein the volume ratio of the substrate precursor and the sterilization solution is 5:2;

[0086] 4) adding water, gibberellin and 1 / 4MS nutrient solution to form a first nutrient solution at the bottom of the seedling box, then putting the substrate obtained in step 3) into the seedling box to soak until the substrate is wet, sowing the cyatheaspores, wherein the mass-volume ratio of gibberellin, 1 / 4MS nutrient solution and water is 1.0 mg:55 mL:1.0 L, the volume ratio of the substrate and the first nutrient solution is 400:250, the first nutrient solution and the substrate form a first nutrient substrate, and the mass-volume ratio of the cyatheaspores and the first nutrient substrate is 0.005 g:100 mL;

[0087] 5) putting the first nutrient substrate sowed with the cyatheaspores into 25℃, recorded as temperature 1, and light intensity of 3000-3500 lux, recorded as light intensity 1, for light treatment of 24 h / d, recorded as light treatment time 1, and observing and counting the time of spore germination and prothallial formation.

[0088] 6) after the prothallial formation, pouring out the remaining first nutrient solution in the seedling box, and adding water, 1 / 4MS nutrient solution, gibberellin, 6-BA and NAA to form a second nutrient solution in the seedling box, and the mass-volume ratio of gibberellin, 6-BA, NAA, 1 / 4MS nutrient solution and water is 1.0 mg:0.5 mg:0.1 mg:55 mL:1.0 L, and the volume ratio of the substrate and the second nutrient solution is 400:250, and putting the second nutrient substrate with the prothallia into 25℃, recorded as temperature 2, and light intensity of 3000-3500 lux, recorded as light intensity 2, for light treatment of 24 h / d, recorded as light treatment time 2, and observing and counting the time of gametophyte formation.

[0089] Example 2-44

[0090] Example 2-44 is basically the same as the method for breeding cyatheaspores in Example 1, except that one or more of the breeding conditions are changed. The specific parameters are shown in Table 1 and Table 2.

[0091] Comparative Example 1-9

[0092] Comparative Example 1-9 is basically the same as the method for breeding cyatheaspores in Example 1, except that one or more of the breeding conditions are changed. The specific parameters are shown in Table 1 and Table 2.

[0093] Figure 1 The photos of spore germination of Example 2;

[0094] As can be seen from Figure 1 , the first nutrient solution is water, which can promote the germination of cyatheaspores.

[0095] Figure 2 The photos of spore germination of Example 4;

[0096] As can be seen from Figure 2As can be seen, the first nutrient solution is 1 / 4 MS nutrient solution and water, which can promote the germination of Cycas revoluta spores.

[0097] Figure 3 A photograph of the prothallus formed in Example 2;

[0098] from Figure 3 As can be seen, the first nutrient solution is water, which can promote the formation of prothallus.

[0099] Figure 4 A photograph of the prothallus formed in Example 4;

[0100] from Figure 4 As can be seen from the data, the first nutrient solution is 1 / 4 MS nutrient solution and water, which can promote the formation of prothallus.

[0101] Figure 5 A photograph of the sporophyte formation in Example 13;

[0102] from Figure 5 As can be seen, the second nutrient solution is water, which can promote sporophyte formation.

[0103] Figure 6 A photograph of the sporophyte formation in Example 15;

[0104] from Figure 6 As can be seen, the second nutrient solution is 1 / 4 MS nutrient solution and water, which can promote sporophyte formation.

[0105] Figure 7 This is a photograph of Comparative Example 7 after 60 days when no sporophytes formed;

[0106] from Figure 7 As can be seen from the data, temperature 2 was 20℃ at night and 30℃ during the day, and no sporophytes were formed after 60 days.

[0107] Figure 8 This is a photograph of Comparative Example 8 after 60 days where no sporophytes have formed;

[0108] from Figure 8 As can be seen, when the light treatment time was 12h / d, no sporophytes were formed after 60 days.

[0109] Table 1

[0110]

[0111]

[0112]

[0113]

[0114] Table 2

[0115]

[0116]

[0117]

[0118] As shown in Tables 1-2, compared with the comparative examples, the method for spore propagation of Cycas revoluta provided by the present invention significantly shortens the time for sporophyte formation by limiting the temperature, light intensity, and light duration of the first nutrient substrate including Cycas revoluta spores and the second nutrient substrate including prothallium. This solves the problems of long propagation and seedling cultivation time of fern spores and difficulty in differentiation through in vitro propagation, and provides technical support for the propagation of fern plants.

[0119] As can be seen from the comparison between Example 1 and Comparative Examples 1-9, the propagation method according to the present invention can shorten the time for sporophyte formation.

[0120] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method of breeding Cycas revoluta spores, characterized by, The method comprises the following steps: 1) a first nutrient medium comprising the spores of Cycas revoluta is kept at a constant temperature of 24-26 DEG C and light intensity of 3000-3500 lux for 24 hours per day to obtain prothallus; 2) a second nutrient medium comprising the prothallus is kept at a constant temperature of 24-26 DEG C and light intensity of 3000-3500 lux for 24 hours per day to obtain sporophyte; The first nutrient medium comprises a substrate and a first nutrient solution; the second nutrient medium comprises a substrate and a second nutrient solution; The first nutrient solution comprises gibberellin, 1 / 4MS nutrient solution and water, and the mass-volume ratio of gibberellin, 1 / 4MS nutrient solution and water is 0.8-1.0 mg: 50-60 mL: 800-1000 mL; The second nutrient solution comprises gibberellin, 6-BA, NAA, 1 / 4MS nutrient solution and water, and the mass-volume ratio of gibberellin, 6-BA, NAA, 1 / 4MS nutrient solution and water is 0.8-1.0 mg: 0.4-0.6 mg: 0.08-0.1 mg: 50-60 mL: 800-1000 mL; The mass-volume ratio of the spores of Cycas revoluta and the first nutrient medium is 0.004-0.006 g: 80-100 mL; The volume ratio of the substrate and the first nutrient solution is 350-400: 200-250; the volume ratio of the substrate and the second nutrient solution is 350-400: 200-250.

2. The method of breeding Cycas revoluta spores according to claim 1, characterized by, The substrate is prepared by a method comprising the following process: peat, vermiculite, river sand and perlite are mixed in a volume ratio of 80-100: 30-50: 15-25: 15-25 to form a substrate precursor, the substrate precursor is subjected to solution sterilization treatment with a sterilization solution, and then the solution sterilization system is subjected to high-temperature sterilization treatment at 120-125 DEG C; The sterilization solution comprises 400-600 times of chlorothalonil and 1400-1600 times of algicide; the volume ratio of the substrate precursor and the sterilization solution is 3-5: 1-2.

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