Novel method for efficiently breeding astropteris contorta through green globular bodies GGBs

Abstract

The invention provides a novel method for efficiently breeding astropteris contorta through green globular bodies GGBs, and belongs to the technical field of biology. The new method comprises the following implementation steps: obtaining explants, disinfecting and sterilizing spores, aseptically germinating the spores into prothalli, proliferating the prothalli, inducing and growing young sporophytes, inducing and proliferating green globular bodies GGBs, regenerating plants, and acclimatizing and transplanting. According to the method, all culture processes of gametophyte generations can be omitted, propagation can be directly carried out in sporophyte generations, the production cycle of astropteris auriculata seedlings is greatly shortened, a complete artificial rapid propagation technical system of astropteris auriculata is created, the tissue culture process can be simplified, the artificial in-vitro propagation efficiency is greatly improved, and the method is suitable for large-scale popularization and application. And the multiplication coefficient is far higher than that of other reported species of the same family. The seedling method of the high-multiplication-coefficient green globular small bodies GGBs of the astropteris convolvulata can be used for cultivating seedlings with genetic stability and excellent quality so as to improve the artificial planting efficiency, and has the characteristics of low cost, short period, high quality, high survival rate and the like.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a new method for efficiently breeding star ferns through green spherical bodies (GGBs). Background Art

[0002] Microsorum punctatum (L.) Copel. is a Polypodiaceae plant, mainly distributed in Gansu, Taiwan, Hainan, Guizhou, Yunnan and other provinces in China. It mainly grows on tree trunks or walls in shady places in plain areas. Most Polypodiaceae plants have high ornamental value due to their different plant and leaf shapes, and can be used for indoor hanging plants or garden greening. In addition, studies have pointed out that star ferns contain vitamin C, vitamin E and polyphenol compounds, and are potential natural sources of antioxidants and anti-inflammatory compounds.

[0003] Among endangered plants, ferns that appeared 300 million years ago are the most important and a valuable source of genes. According to the International Union for Conservation of Nature (IUCN), more than half of the 12,000 existing fern species are threatened with extinction due to overexploitation, improper agricultural practices, urbanization and pollution. Star ferns have a low spore reproduction rate, a narrow habitat, and their natural habitats have been destroyed. Their wild populations are very rare, and research on artificial breeding is urgently needed.

[0004] Like all vascular plants, the life cycle of ferns alternates between two different stages or generations: the diploid sporophyte generation and the haploid gametophyte generation. The former is mainly vegetative growth, and later turns to reproductive growth, producing haploid spores through meiosis; the latter mainly performs sexual reproduction functions, produces male and female gametes, and then completes fertilization to form a new generation of sporophytes. In ferns and other more primitive plants, the gametophyte stage, from spore germination to fertilization, is completely independent of the diploid sporophyte. The development of the gametophyte begins with the germination of the spore, and then undergoes the transition from filamentous to lamellar bodies, and differentiates into a slightly asymmetrical heart-shaped prothallus. The reproductive organs, the antheridia and archegonium, are produced on the mature gametophyte, and the sporophyte is produced through fertilization. After the sporophyte develops on the gametophyte, the gametophyte gradually turns yellow and dies. The sporophyte is a relatively dominant life stage in most fern groups, and it has evolved many anatomical and physiological characteristics, which are more helpful in regulating the transport of nutrients. Generally speaking, ferns are easier to reproduce and cultivate than other plant groups due to their long-term adaptation to various environmental conditions. However, in terms of artificial breeding, the spore germination and gametophyte transformation to sporophyte stage of ferns are relatively difficult, and are also a research hotspot for this group. Environmental factors play an important role in spore germination and gametophyte transformation to sporophyte stage. Changes in environmental conditions may affect chemical signal transduction and metabolic activities inside and outside cells, thereby affecting biological development; in addition, it takes a long growth cycle to complete this process, and asexual reproduction is inefficient.

[0005] Some scholars have studied the proliferation of the gametophyte generation of ferns and found that some of the proliferation difficulties can be solved by homogenizing the gametophyte culture; however, there are still problems such as difficulty in inducing sporophyte formation in vitro, no proliferation of sporophytes or low proliferation coefficient, especially the most effective induction of green globular bodies (GGBs) is still rare. At present, the germplasm resources of star ferns are mainly wild. Due to the long gametophyte generation, the sporophyte population is difficult to recover once it is destroyed. The biological research of star ferns is almost blank, and there is no report on artificial breeding. Therefore, it is urgent to seek a new artificial breeding method with low cost, short time, high quality and survival rate, which has become an inevitable choice for the sustainable utilization and large-scale reproduction of star ferns. Summary of the invention

[0006] The purpose of the present invention is to provide a new method for efficient breeding of green spherical bodies GGBs of star ferns, so as to solve the problem of shortage of artificial breeding methods of star ferns at present, and to lay a technical foundation for protecting wild resources and developing artificial cultivation, and at the same time, it can also provide a certain reference for in vitro rapid propagation research of other ferns.

[0007] The present invention is achieved through the following technical solutions:

[0008] A new method for efficiently breeding star ferns through green spherical bodies (GGBs), comprising the following steps: obtaining explants, disinfecting and sterilizing spores, aseptic germination of spores into prothallus, proliferation of prothallus, induction and growth of young sporophytes, induction and proliferation of green spherical bodies (GGBs), plant regeneration, and seedling hardening and transplanting.

[0009] Furthermore, the novel method for efficiently breeding star ferns through green spherical bodies (GGBs) comprises the following steps:

[0010] (1) Explant acquisition: Select healthy sporophyte plants of Pteris ternifolia with good growth potential and no pests or diseases, and take fresh reproductive leaves;

[0011] (2) Disinfection: Use a clean blade to scrape off the mature spores on the back of the reproductive leaves and disinfect them;

[0012] (3) Spore germination: The spores disinfected in step (2) are inoculated into a culture medium A, wherein the culture medium A comprises the following raw materials:

[0013] 1 / 2MS basic culture medium

[0014] sucrose

[0015] Agar powder

[0016] By controlling the light intensity, temperature and illumination time for cultivation, a heart-shaped prothallus can be obtained;

[0017] (4) Prothallus proliferation: The prothallus mass in step (3) is divided into pieces of appropriate size and inoculated into culture medium B, wherein the culture medium B comprises the following raw materials:

[0018] MS basic culture medium

[0019] 6-Benzylaminopurine

[0020] α-Naphthylacetic acid

[0021] Kinetin

[0022] sucrose

[0023] Agar powder

[0024] Control the light intensity, temperature and illumination time to carry out the proliferation and cultivation of the prothallus;

[0025] (5) Induction of young spores: When the protothallus has proliferated to a sufficient amount for various experiments on inducing young spores, it is divided into 1.0 cm 2 The size was transferred into culture medium C, which included the following raw materials:

[0026] 1 / 4MS basic culture medium

[0027] sucrose

[0028] Agar powder

[0029] Control the light intensity, temperature, and illumination time, and add sterile water regularly to help the fusion of male and female gametes, thereby inducing young sporophytes;

[0030] (6) Induction and proliferation culture of green spherical bodies GGBs: The young sporophytes were transferred into fresh culture medium D, which contained the following raw materials:

[0031] MS basic culture medium

[0032] 6-Benzylaminopurine

[0033] α-Naphthylacetic acid

[0034] sucrose

[0035] Agar powder

[0036] Control the light intensity, temperature and illumination time to induce and proliferate green spheroids;

[0037] (7) Plant regeneration: The induced green spherical GGBs aggregates were divided into 2-3 groups and transferred to culture medium A to obtain complete sporophyte plants;

[0038] (8) Hardening and transplanting: The rooted sporophyte plants in step (7) are placed at room temperature for hardening for 3 days, and then the seedlings are removed from the culture medium. The root culture medium is washed with clean water, and the roots are soaked in a carbendazim solution. The seedlings are then transplanted to pine bark sterilized at high temperature to keep warm and moisturize the bark, thereby obtaining transplanted seedlings.

[0039] Furthermore, the disinfection method in step (2) is to pack the scraped spores into 3 mL sterile centrifuge tubes, disinfect them in a 0.1% mass concentration mercury solution for 8-10 minutes, shake them continuously and mix them thoroughly; then pour them into an empty bottle with a high-pressure sterilized filter paper at the bottle mouth for filtration, and wash them with sterile water for 4-6 times; use sterilized scissors to divide the filter paper into 4 parts, and touch each filter paper in reverse on the culture medium A so that the spores on the filter paper can be stained with the surface of the culture medium as much as possible.

[0040] Furthermore, the culture medium A comprises the following raw materials:

[0041] 1 / 2MS basic culture medium

[0042] Sucrose 30000mg / L

[0043] Agar powder 4700mg / L;

[0044] The culture medium B includes the following raw materials:

[0045] MS basic culture medium

[0046]

[0047] The culture medium C comprises the following raw materials:

[0048] 1 / 4MS basic culture medium

[0049] Sucrose 30000mg / L

[0050] Agar powder 4700mg / L;

[0051] The culture medium D comprises the following raw materials:

[0052] 1 / 2MS basic culture medium

[0053]

[0054] The pH values ​​of the culture media A, B, C and D are 5.4-5.8.

[0055] Furthermore, in step (8), the mass concentration of carbendazim is 0.1-0.5%, the size of the shredded pine bark is 1.5×1.5 cm, the disinfection method is boiling water for 2-3 hours, and the heat preservation and moisture retention conditions are a culture temperature of 20-25° C. and a humidity of 70%.

[0056] The present invention has at least the following beneficial effects:

[0057] (1) The present invention uses tissue culture technology to achieve year-round production in a culture room, which not only saves land resources but also improves economic benefits, overcoming the difficulty of traditional breeding methods that cannot achieve year-round production;

[0058] (2) After the prothallus stage of the haploid generation is completed and the young sporophyte is induced to the green spherical body GGBs, the entire culture process can be simplified to two culture media, D and A, thereby omitting all the culture stages of the long haploid generation and completing the entire breeding process directly in the sporophyte generation; therefore, through the present invention, a large number of seedlings can be obtained in a short time, and the cost is low, the cycle is short, and it is easy to standardize and operate in a factory, which effectively improves the quality of seedlings and can provide high-quality seedlings of uniform standards for large-scale promotion of planting;

[0059] (3) The present invention overcomes the slow growth characteristic of ferns and has the characteristics of high reproduction efficiency. A culture cycle can be completed in 120 days, and the proliferation coefficient can reach more than 32.0;

[0060] (4) Since the test tube seedlings of the present invention are derived from green globose bodies GGBs, which are similar to somatic embryos of angiosperms and have bipolar growth characteristics, the regenerated plants have strong seedlings and thick roots, and the survival rate of seedling hardening and transplanting is high. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 This is a diagram of aseptic germination of star fern spores;

[0062] Figure 2 This is a diagram of the proliferation of the prothallus of the star fern;

[0063] Figure 3 This is a diagram of the formation of male and female gametophytes, fertilization of male and female gametes, and sporophyte induction in the star fern;

[0064] Figure 4 This is a diagram of the induced development of green globose bodies GGBs in the star fern;

[0065] Figure 5 This is a diagram of the proliferation, regeneration and plant acclimatization of the green globose bodies (GGBs) of the star fern;

[0066] Figure 6 This is a diagram of the extracorporeal regeneration of the star fern.

[0067] Figure 1 Middle: AD scale bar = 2 cm, E and H scale bar = 50 μm, F and G scale bar = 100 μm; Figure 1 -A, B, C, D diagram of spore germination process in sterile bottles; Figure 1 -E: spores begin to germinate 20 days after inoculation, i.e., primary rhizoids and primitive cells are produced; Figure 1 -F is a picture of the filamentous stage after 30 days of culture, in which the primitive cells develop into cylindrical shapes with thin walls, bulging side walls, and no branches; Figure 1 -G, about 45 days after culture, the cells are arranged in a single row at the base, a wedge-shaped apical cell group appears at the top, and rhizoids begin to appear, which is the lamellar body stage; Figure 1 -H is a picture of the mature prothallus stage, which is elongated into a heart shape after 70 days of cultivation, with symmetrical broad wings, and multiple adventitious roots appearing on both wings and the base of the petiole.

[0068] Figure 2 Middle: AD ruler = 2 cm; Figure 2 -A is the image of the prothallus cluster after being divided and cultured for 15 days, the volume of which increases significantly and the newly formed prothallus is emerald green; Figure 2 -B is the picture of the prothallus mass continuing to grow larger after 30 days of culture, with a tight texture and larger top leaves; Figure 2 -C is the figure where the protothalli further enlarged after 45 days of culture, and due to its high density, the edges and bases began to turn yellow and wilt; Figure 2 -D After 60 days, the protothallus mass occupies the entire surface of the culture bottle, the yellowing phenomenon worsens, some protothallus die and turn black, at the same time, rhizoids appear, and the texture becomes loose. At this time, the proliferation coefficient can reach above 9.5.

[0069] Figure 3Middle: A scale bar = 500 μm, B and D scale bars = 40 μm, C scale bar = 200 μm, EH scale bar = 2 cm; Figure 3 -A shows that the prothallus is heart-shaped, bilaterally symmetrical, and emerald green after 30 days of culture, and the concave part above is the growth point; Figure 3 -B shows the appearance of multiple protruding or spherical spermatids with handles near the rhizoid, and the iconic cap cell layer structure can be seen; Figure 3 -C and D show 7-9 rows of archegonium below the growth point of the prothallus. Each archegonium is fusiform, narrow in the upper part and swollen in the middle and lower part, with two rows of obvious neck groove cells. Figure 3 -E is a picture in which sterile water is added to the culture medium and cultured for 20 days, where single-leaf young sporophytes can be seen growing on the surface of the prothallus mass, and the prothallus is still emerald green; Figure 3 -F is the image after 60 days of culture, the young sporophytes produced first mostly have 2-3 leaves, while young sporophytes continue to appear, while the prothallus still shows signs of continuing proliferation; Figure 3 -G is a picture of a large number of young sporophytes appearing on the prothallus after 90 days of culture. The prothallus basically stops proliferating, and many rhizophores appear on the surface, turning yellow-green. At this time, there are as many as 50-55 young spores in each bottle, and the average occurrence coefficient is about 10.0; Figure 3 -H is a picture of young sporophyte rooting culture or direct induction of green spherical bodies GGBs.

[0070] Figure 4 Middle: A and E scale bars = 1 cm, BD and H scale bars = 1 mm, F-scale bar = 500 μm, G scale bar = 5 mm; Figure 4 -A is a diagram showing the transfer of young spores induced from prothallus 60 days later into D medium; Figure 4 -B and C are pictures of green spherical bodies that are difficult to separate on the tender epidermis of the base rhizome after 30 days of culturing the young sporophyte; Figure 4 -D and E are the green spheroids that were peeled off and transferred to fresh culture medium for 30 days and then differentiated into green spheroid GGBs aggregates. The proliferation coefficient at this time can reach about 32.0; Figure 4 -F is a picture of a green spherical body with a single leaf growing on the surface; Figure 4 -G and H are separate single green spherical bodies. GGB continues to be cultured and gradually differentiates into young sporophytes.

[0071] Figure 5 Middle: AD ruler = 1 cm, E ruler = 5 cm, FH ruler = 3 cm; Figure 5 -A and B are the proliferation images of green spherical bodies GGBs; Figure 5 -C is a picture of a single green spherical body GGB peeled off and cultured for 20 days to grow a large number of two-leaf young sporophytes; Figure 5-D shows that after 30 days of culture, the sporophyte further enlarges, the leaves gradually increase, and the growth is very good; Figures E, F, G, and H show the spore domestication process. After 60 days of domestication, the survival rate reaches more than 90%. DETAILED DESCRIPTION

[0072] The present invention is further described below in conjunction with specific examples and the accompanying drawings, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art, and the reagents, raw materials and equipment used are all commercially available.

[0073] Example 1

[0074] A new method for efficiently breeding star ferns through green globose bodies (GGBs) comprises the following steps:

[0075] (1) Obtaining explants: Select healthy and healthy star fern plants with good growth potential and no pests or diseases, and take their young leaves;

[0076] (2) Disinfection: Use a clean blade to scrape off the mature spores on the back of the reproductive leaves in step (1) and place them in 3 mL sterile centrifuge tubes. Sterilize them in a 0.1% mercuric chloride solution for 8 min, shaking continuously to mix thoroughly. Then pour them into an empty bottle with a high-pressure sterilized filter paper at the mouth and filter them. Wash them with sterile water for 4 times. Use sterilized scissors to divide the filter paper into 4 equal parts. Touch each filter paper lightly on the culture medium A in the reverse direction to make the spores on the filter paper stick to the surface of the culture medium as much as possible.

[0077] (3) Spore germination: The spores disinfected in step (2) were inoculated into the following culture medium A for aseptic germination:

[0078] 1 / 2MS basic culture medium

[0079] Sucrose 30000mg / L

[0080] Agar powder 4700mg / L

[0081] The pH value of medium A is 5.4

[0082] Culture conditions: Culture is carried out under the conditions of light intensity of 1500-2000lx, light time of 10h / d, temperature of 23±2℃. Spores begin to germinate 20 days after inoculation, that is, primary rhizoids and primitive cells are produced; after 30 days of culture, the primitive cells develop into a cylindrical shape with thin walls, bulging side walls, and no branches, which is the filamentous body stage; after about 45 days of culture, the cells are arranged in a single row at the base, a wedge-shaped apical cell group appears at the top, and rhizoids begin to appear, which is the lamellar body stage; after 70 days of culture, it elongates into a heart shape with symmetrical broad wings, and multiple rhizoids appear on both wings and the base of the petiole, which is the mature prothallus stage.

[0083] (4) Prothallus proliferation: Divide the prothallus mass in step (3) into 1.0 cm 2 After the size is reached, inoculate into the following medium B:

[0084] MS basic culture medium

[0085]

[0086] The pH value of medium B is 5.4

[0087] Culture conditions: Culture was carried out under the conditions of light intensity of 1500-2000lx, light time of 10h / d, temperature of 23±2℃. After 15d of culture, the volume of small clusters of protothallus increased significantly, and the new protothallus was emerald green; after 30d of culture, the protothallus clusters continued to increase in size, the texture was compact, and the top leaves became larger; after 45d of culture, the protothallus clusters increased further, and due to their excessive density, yellowing and withering began to occur at the edges and bases; after 60d of culture, the protothallus clusters occupied the entire surface of the culture bottle, the yellowing phenomenon worsened, some protothallus died and turned black, rhizoids appeared, and the texture became loose; at this time, the protothallus proliferation coefficient reached 9.77.

[0088] (5) Induction of young spores: When the protothallus has proliferated to a sufficient size for various experiments on inducing young spores, it is divided into 1.0 cm 2 Transfer the size into the following medium C:

[0089] 1 / 4MS basic culture medium

[0090] Sucrose 30000mg / L

[0091] Agar powder 4700mg / L

[0092] The pH of medium C is 5.4

[0093] Culture conditions: Cultured under the conditions of 1500-2000lx illumination, 10h / d illumination time, and 23±2℃ temperature control. After 30 days of culture, the prothallus is heart-shaped, bilaterally symmetrical, emerald green, and the concave part above is the growth point. Near the rhizoid, there are multiple protruding or spherical spermatids with handles, and the iconic cap cell layer structure can be seen. Below the growth point of the prothallus, there are 7-9 rows of archegonium, each of which is fusiform, narrow in the upper part, and swollen in the middle and lower parts, with obvious two rows of neck groove cells. After 10 days of culture, it begins to Sterile water was injected into the plant at a cycle of 15 days. After 20 days of culture with sterile water, single-leaf young sporophytes were seen growing on the surface of the prothallus group, and the prothallus was still emerald green. After 60 days of culture, the young sporophytes produced first had 2-3 leaves, and young sporophytes continued to appear, while the prothallus still showed signs of continued proliferation. After 90 days of culture, a large number of young sporophytes appeared on the prothallus, and the prothallus basically stopped proliferating. More rhizoids appeared on the surface and turned yellow-green. At this time, there were as many as 50-55 young spores in each bottle, and the average occurrence coefficient was about 10.25. At this time, the sporophytes were acclimated and transplanted.

[0094] (6) Induction of green spherical bodies (GGBs): Transfer young sporophytes into the following medium D:

[0095] MS basic culture medium

[0096]

[0097] The pH value of medium D is 5.4

[0098] Culture conditions: Culture was carried out under the conditions of light intensity of 1500-2000lx, light time of 10h / d, temperature of 23±2℃. After 30 days of culture of young spores induced by 60 days of prothallus culture, green spherical bodies that were difficult to separate appeared on the tender epidermis of the rhizome at the base. At this time, the green spherical bodies were peeled off and transferred to fresh culture medium for culture for 30 days, and then differentiated into green spherical body GGBs aggregates, and the proliferation coefficient at this time reached 32.15. The separated single green spherical body GGB continued to be cultured and gradually differentiated into young spores.

[0099] (7) Plant regeneration: The induced green spherical GGBs aggregates were divided into 2-3 groups and transferred to fresh culture medium A. After culturing for 30 days under the conditions of light intensity of 1500-2000 lx, light duration of 10 h / d, and temperature of 23±2°C, complete sporophyte plants were obtained.

[0100] (8) Hardening and transplanting: The rooted plants obtained in step (7) are hardened at room temperature for 3 days, and then the seedlings are removed from the culture medium. The root culture medium is washed with clean water, and the root culture medium is soaked in a 0.1% carbendazim solution. The seedlings are then transplanted into 1.5×1.5 cm crushed pine bark that has been boiled in boiling water for 2.5 hours. The seedlings are then cultured at 20° C. and 70% humidity for 60 days to obtain transplanted seedlings.

[0101] Example 2

[0102] A new method for efficiently breeding star ferns through green globose bodies (GGBs) comprises the following steps:

[0103] (1) Obtaining explants: Select healthy plants with good growth potential and no pests or diseases, and take their young leaves.

[0104] (2) Disinfection: Use a clean blade to scrape off the mature spores on the back of the reproductive leaves in step (1) and place them in a 3 mL sterile centrifuge tube. Sterilize them in a 0.1% mercuric chloride solution for 9 minutes, shaking continuously to mix thoroughly. Then pour them into an empty bottle with a high-pressure sterilized filter paper at the mouth and filter them. Wash them with sterile water 4-6 times. Use sterilized scissors to divide the filter paper into 4 equal parts. Touch each filter paper against the medium A in reverse so that the spores on the filter paper are as close to the surface of the medium as possible.

[0105] (3) Spore germination: The spores disinfected in step (2) were inoculated into the following culture medium A for aseptic germination:

[0106] 1 / 2MS basic culture medium

[0107] Sucrose 30000mg / L

[0108] Agar powder 4700mg / L.

[0109] The pH value of medium A is 5.6

[0110] Culture conditions: Culture is carried out under the conditions of light intensity of 1500-2000lx, light time of 10h / d, temperature of 23±2℃. Spores begin to germinate 20 days after inoculation, that is, primary rhizoids and primitive cells are produced; after 30 days of culture, the primitive cells develop into a cylindrical shape with thin walls, bulging side walls, and no branches, which is the filamentous body stage; after about 45 days of culture, the cells are arranged in a single row at the base, a wedge-shaped apical cell group appears at the top, and rhizoids begin to appear, which is the lamellar body stage; after 70 days of culture, it elongates into a heart shape with symmetrical broad wings, and multiple rhizoids appear on both wings and the base of the petiole, which is the mature prothallus stage.

[0111] (4) Prothallus proliferation: Divide the prothallus mass in step (3) into 1.0 cm 2 After the size is reached, inoculate into the following medium B:

[0112] MS basic culture medium

[0113]

[0114] The pH value of medium B is 5.6

[0115] Culture conditions: Culture was carried out under the conditions of light intensity of 1500-2000lx, light time of 10h / d, temperature of 23±2℃. After 15d of culture, the volume of small clusters of protothallus increased significantly, and the new protothallus was emerald green; after 30d of culture, the protothallus clusters continued to increase in size, the texture was compact, and the top leaves became larger; after 45d of culture, the protothallus clusters increased further, and due to their excessive density, yellowing and withering began to occur at the edges and bases; after 60d of culture, the protothallus clusters occupied the entire surface of the culture bottle, the yellowing phenomenon worsened, some protothallus died and turned black, and rhizoids appeared, and the texture became loose; at this time, the protothallus proliferation coefficient reached 9.75.

[0116] (5) Induction of young spores: When the protothallus has proliferated to a sufficient size for various experiments on inducing young spores, it is divided into 1.0 cm 2 Transfer the size into the following medium C:

[0117] 1 / 4MS basic culture medium

[0118] Sucrose 30000mg / L

[0119] Agar powder 4700mg / L

[0120] The pH value of medium C is 5.6

[0121] Culture conditions: Cultured under the conditions of 1500-2000lx, 10h / d of light, and 23±2℃. After 30 days of culture, the prothallus is heart-shaped, bilaterally symmetrical, and emerald green, with the concave notch above being the growth point. Near the rhizoid, multiple protruding or spherical spermatids with handles appear, and the iconic cap cell layer structure can be seen. Below the growth point of the prothallus, 7-9 rows of archegonium are produced, each of which is fusiform, narrow in the upper part, and swollen in the middle and lower parts, with obvious two rows of neck groove cells. After 10 days of culture, the prothallus begins to grow at a rate of 1 Sterile water was injected into the plant at a cycle of 5 days. After 20 days of culture with sterile water, single-leaf young sporophytes could be seen growing on the surface of the prothallus, which was still emerald green. After 60 days of culture, the young sporophytes produced first had 2-3 leaves, and young sporophytes continued to appear, while the prothallus still showed signs of continued proliferation. After 90 days of culture, a large number of young sporophytes appeared on the prothallus, which basically stopped proliferating, with more rhizoids appearing on the surface and turning yellow-green. At this time, there were as many as 51-57 young spores in each bottle, with an average occurrence coefficient of about 10.45. At this time, the sporophytes could be directly domesticated and transplanted.

[0122] (6) Induction of green spherical bodies (GGBs): Transfer young sporophytes into the following medium D:

[0123] MS basic culture medium

[0124]

[0125] The pH value of medium D is 5.6

[0126] Culture conditions: Culture was carried out under the conditions of light intensity of 1500-2000lx, light time of 10h / d, and temperature of 23±2℃. After 30 days of culture of the young spores induced by the prothallus 60 days later, green spherical bodies that were difficult to separate appeared on the young epidermis of the rhizome at the base. At this time, the green spherical bodies were peeled off and transferred to fresh culture medium for culture for 30 days, and then differentiated into green spherical body GGBs aggregates, and the proliferation coefficient at this time could reach 32.10. The separated single green spherical body GGB continued to be cultured and gradually differentiated into young spores.

[0127] (7) Plant regeneration: The induced green spherical GGBs aggregates were divided into 2-3 groups and transferred to fresh culture medium A. After culturing for 30 days under the conditions of light intensity of 1500-2000 lx, light duration of 10 h / d, and temperature of 23±2°C, complete sporophyte plants were obtained.

[0128] (8) Hardening and transplanting: The rooted plants obtained in step (7) were hardened at room temperature for 3 days, and then the seedlings were removed from the culture medium. The root culture medium was washed with clean water, and the root culture medium was soaked in a 0.3% carbendazim solution. The seedlings were then transplanted into 1.5×1.5 cm crushed pine bark that had been boiled in boiling water for 2.5 hours. The seedlings were cultured at 22° C. and 70% humidity for 60 days to obtain transplanted seedlings.

[0129] Example 3

[0130] A new method for efficiently breeding star ferns through green globose bodies (GGBs) comprises the following steps:

[0131] (1) Obtaining explants: Select healthy plants with good growth potential and no pests or diseases, and take their young leaves.

[0132] (2) Disinfection: Use a clean blade to scrape off the mature spores on the back of the reproductive leaves in step (1) and place them in a 3 mL sterile centrifuge tube. Sterilize them in a 0.1% mercuric chloride solution for 10 min, shaking continuously to mix thoroughly. Then pour them into an empty bottle with a high-pressure sterilized filter paper at the mouth and filter them. Wash them with sterile water 4-6 times. Use sterilized scissors to divide the filter paper into 4 equal parts. Touch each filter paper against the culture medium A in reverse so that the spores on the filter paper can be as close to the surface of the culture medium as possible.

[0133] (3) Spore germination: The spores disinfected in step (2) were inoculated into the following culture medium A for aseptic germination:

[0134] 1 / 2MS basic culture medium

[0135] Sucrose 30000mg / L

[0136] Agar powder 4700mg / L

[0137] The pH value of medium A is 5.8

[0138] Culture conditions: Culture is carried out under the conditions of light intensity of 1500-2000lx, light time of 10h / d, temperature of 23±2℃. Spores begin to germinate 20 days after inoculation, that is, primary rhizoids and primitive cells are produced; after 30 days of culture, the primitive cells develop into a cylindrical shape with thin walls, bulging side walls, and no branches, which is the filamentous body stage; after about 45 days of culture, the cells are arranged in a single row at the base, a wedge-shaped apical cell group appears at the top, and rhizoids begin to appear, which is the lamellar body stage; after 70 days of culture, it elongates into a heart shape with symmetrical broad wings, and multiple rhizoids appear on both wings and the base of the petiole, which is the mature prothallus stage.

[0139] (4) Prothallus proliferation: Divide the prothallus mass in step (3) into 1.0 cm 2 After the size is reached, inoculate into the following medium B:

[0140] MS basic culture medium

[0141]

[0142] The pH value of medium B is 5.8

[0143] Culture conditions: Culture was carried out under the conditions of light intensity of 1500-2000lx, light time of 10h / d, temperature of 23±2℃. After 15d of culture, the volume of small clusters of protothallus increased significantly, and the new protothallus was emerald green; after 30d of culture, the protothallus clusters continued to increase in size, the texture was compact, and the top leaves became larger; after 45d of culture, the protothallus clusters increased further, and due to their excessive density, yellowing and withering began to occur at the edges and bases; after 60d of culture, the protothallus clusters occupied the entire surface of the culture bottle, the yellowing phenomenon worsened, some protothallus died and turned black, and rhizoids appeared, and the texture became loose; at this time, the protothallus proliferation coefficient could reach 9.55.

[0144] (5) Induction of young spores: When the protothallus has proliferated to a sufficient size for various experiments on inducing young spores, it is divided into 1.0 cm 2 Transfer the size into the following medium C:

[0145] 1 / 4MS basic culture medium

[0146] Sucrose 30000mg / L

[0147] Agar powder 4700mg / L

[0148] The pH value of medium C is 5.8

[0149] Culture conditions: Cultured under the conditions of 1500-2000lx, 10h / d of light intensity, and 23±2℃. After 30 days of culture, the prothallus is heart-shaped, bilaterally symmetrical, and emerald green, with the concave part above being the growth point. Near the rhizoid, multiple protruding or spherical spermatids with handles appear, and the iconic cap cell layer structure can be seen. Below the growth point of the prothallus, 7-9 rows of archegonium are produced, each of which is fusiform, narrow in the upper part, and swollen in the middle and lower parts, with obvious two rows of neck groove cells. After 10 days of culture, it begins to grow in a Sterile water is injected into the plant at a cycle of 15 days. After 20 days of culture with sterile water, single-leaf young sporophytes can be seen growing on the surface of the prothallus group, and the prothallus is still emerald green. After 60 days of culture, the young sporophytes produced first mostly have 2-3 leaves, and young sporophytes continue to appear, while the prothallus still shows signs of continued proliferation. After 90 days of culture, a large number of young sporophytes appear on the prothallus, and the prothallus basically stops proliferating. More rhizoids appear on the surface and turn yellow-green. At this time, there are as many as 50-54 young spores in each bottle, and the average occurrence coefficient is about 10.25. At this time, the sporophytes can be directly domesticated and transplanted.

[0150] (6) Induction of green spherical bodies (GGBs): Transfer young sporophytes into the following medium D:

[0151] MS basic culture medium

[0152]

[0153] The pH value of medium D is 5.8

[0154] Culture conditions: Culture was carried out under the conditions of light intensity of 1500-2000lx, light time of 10h / d, and temperature of 23±2℃. After 30 days of culture of the young spores induced by the prothallus 60 days later, green spherical bodies that were difficult to separate appeared on the young epidermis of the rhizome at the base. At this time, the green spherical bodies were peeled off and transferred to fresh culture medium for culture for 30 days, and then differentiated into green spherical body GGBs aggregates, and the proliferation coefficient at this time could reach 32.15. The separated single green spherical body GGB continued to be cultured and gradually differentiated into young spores.

[0155] (7) Plant regeneration: The induced green spherical GGBs aggregates were divided into 2-3 groups and transferred to fresh culture medium A. After culturing for 30 days under the conditions of light intensity of 1500-2000 lx, light time of 10 h / d, and temperature controlled at 23±2°C, complete sporophyte plants were obtained.

[0156] (8) Hardening and transplanting: The rooted plants in step (7) are placed in the bottles for hardening at room temperature for 3 days, and then the seedlings are removed from the culture medium. The root culture medium is washed with clean water, and the root culture medium is soaked in a 0.5% carbendazim solution. The seedlings are then transplanted into 1.5×1.5 cm crushed pine bark that has been boiled in boiling water for 2.5 hours. The seedlings are then cultured at 25° C. and 70% humidity for 60 days to obtain transplanted seedlings.

[0157] Technical principle of the present invention:

[0158] 1. In the artificial breeding of ferns, although mature spores are a commonly used source of explants, the series of steps from spore germination to prothallus formation and proliferation, as well as conversion to sporophyte after fertilization are very cumbersome, and the culture conditions required for each step are different; in addition, in the life cycle of ferns, reproductive cells form ovophytes (female gametophytes) and spermatophytes (male gametophytes) through spore germination; this life cycle feature is not as easy to induce callus as seed plants. Therefore, it is quite difficult to produce regenerated plants in vitro from spores. In addition, the sporophyte of ferns lacks ovules and nutritional tissues during growth, which also limits its callus formation and cultivation. The present invention solves the above problems, and the spore germination to prothallus formation and proliferation are successfully completed, and the prothallus obtains a satisfactory proliferation coefficient.

[0159] 2. The present invention solves the key problem of fertilization of male and female gametophytes in the bottle of star fern by adding sterile water at regular intervals, and can induce a large number of young sporophytes on the prothallus in the bottle, which is a key node connecting the previous and the next; and the appearance of green spherical bodies GGBs is the basis for producing high reproductive lines.

[0160] 3. The present invention adopts two approaches ( Figure 6 ) to achieve sporophyte regeneration. First, the prothallus group is regarded as the "callus tissue" in angiosperms, and appropriate culture conditions are given to enable it to efficiently transform into young sporophytes, while the young sporophytes on the prothallus are regarded as "clustered buds"; second, the sporophyte is used as a material to induce the production of green spherical bodies GGBs aggregates, thereby differentiating the sporophyte. From the results, in the production and application of star ferns, in addition to the cultivation of the gametophyte generation such as spore germination, when the young sporophyte appears, the green spherical bodies GGBs are differentiated through the young epidermis of its rhizome; the appearance of green spherical bodies GGBs greatly improves the proliferation coefficient, and the cultivation of the gametophyte generation can be completely omitted to achieve the purpose of regenerating sporophyte plants through repeated induction of GGBs in the sporophyte generation. In this way, the artificial rapid propagation cycle of star ferns has been greatly shortened, and it has the characteristics of small variation, low cost, and high efficiency.

[0161] The above-mentioned embodiments only express the specific implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the protection scope of the present application. It should be pointed out that, for ordinary technicians in this field, several adjustments and improvements can be made without departing from the technical solution concept of the present application, and these all belong to the protection scope of the present application.

Claims

1. A new method for efficient propagation of star ferns through green globose bodies (GGBs). It is characterized in that The implementation steps of the new method are: obtaining explants, disinfecting and sterilizing spores, aseptic germination of spores into prothallus, proliferation of prothallus, induction and growth of young sporophytes, induction and proliferation of green spherical bodies GGBs, plant regeneration, and seedling hardening and transplanting.

2. The novel method for efficiently breeding star ferns through green spherical bodies (GGBs) according to claim 1, It is characterized in that The specific implementation steps of the new method are: (1) Explant acquisition: Select healthy sporophyte plants of Pteris ternifolia with good growth potential and no pests or diseases, and take fresh reproductive leaves; (2) Disinfection: Use a clean blade to scrape off the mature spores on the back of the reproductive leaves and disinfect them; (3) Spore germination: The spores disinfected in step (2) are inoculated into a culture medium A, wherein the culture medium A comprises the following raw materials: 1 / 2MS basic culture medium sucrose Agar powder By controlling the light intensity, temperature and illumination time for cultivation, a heart-shaped prothallus can be obtained; (4) Prothallus proliferation: The prothallus mass in step (3) is divided into pieces of appropriate size and inoculated into culture medium B, wherein the culture medium B comprises the following raw materials: MS basic culture medium 6-Benzylaminopurine α-Naphthylacetic acid Kinetin sucrose Agar powder Control the light intensity, temperature and illumination time to carry out the proliferation and cultivation of the prothallus; (5) Induction of young sporophytes: When the protothallus proliferates to a sufficient amount for various experiments on inducing young sporophytes, it is divided into 1.0 cm 2 The size was transferred into culture medium C, which included the following raw materials: 1 / 4MS basic culture medium sucrose Agar powder Control the light intensity, temperature, and illumination time, and add sterile water regularly to help the fusion of male and female gametes, thereby inducing young sporophytes; (6) Induction and proliferation culture of green spherical bodies GGBs: The young sporophytes were transferred into fresh culture medium D, which contained the following raw materials: MS basic culture medium 6-Benzylaminopurine α-Naphthylacetic acid sucrose Agar powder Control the light intensity, temperature and illumination time to induce and proliferate green spheroids; (7) Plant regeneration: The induced green spherical GGBs aggregates were divided into 2-3 groups and transferred to culture medium A to obtain complete sporophyte plants; (8) Hardening and transplanting: The rooted sporophyte plants in step (7) are placed at room temperature for hardening for 3 days, and then the seedlings are removed from the culture medium. The root culture medium is washed with clean water, and the roots are soaked in a carbendazim solution. The seedlings are then transplanted to pine bark sterilized at high temperature to keep warm and moisturize the bark, thereby obtaining transplanted seedlings.

3. The novel method for efficiently breeding star ferns through green spherical bodies (GGBs) according to claim 2, It is characterized in that The disinfection method in step (2) is as follows: the scraped spores are divided into 3 mL sterile centrifuge tubes, and disinfected in a mercury solution with a mass concentration of 0.1% for 8-10 minutes, during which time they are shaken continuously to mix thoroughly; then the spores are poured into an empty bottle with a high-pressure sterilized filter paper placed at the bottle mouth for filtration, and washed with sterile water for 4-6 times; the filter paper is divided into 4 parts with sterilized scissors, and each part of the filter paper is lightly touched on the culture medium A in the reverse direction to allow the spores on the filter paper to be as much as possible on the surface of the culture medium.

4. The novel method for efficiently breeding star ferns through green spherical bodies (GGBs) according to claim 2, It is characterized in that The culture medium A comprises the following raw materials: 1 / 2MS basic culture medium Sucrose 30000mg / L Agar powder 4700mg / L.

5. The novel method for efficiently breeding star ferns through green spherical bodies (GGBs) according to claim 2, It is characterized in that The culture medium B includes the following raw materials: MS basic culture medium 6. The novel method for efficiently breeding star ferns through green spherical bodies (GGBs) according to claim 2, It is characterized in that The culture medium C comprises the following raw materials: 1 / 4MS basic culture medium Sucrose 30000mg / L Agar powder 4700mg / L.

7. The novel method for efficiently breeding star ferns through green spherical bodies (GGBs) according to claim 2, It is characterized in that The culture medium D comprises the following raw materials: 1 / 2MS basic culture medium 8. The novel method for efficiently breeding star ferns through green spherical bodies (GGBs) according to any one of claims 2 to 7, It is characterized in that The pH value of the culture medium is 5.4-5.

8.

9. The novel method for efficiently breeding star ferns through green spherical bodies (GGBs) according to claim 2, It is characterized in that The mass concentration of carbendazim in step (8) is 0.1-0.5%, the size of the crushed pine bark is 1.5×1.5 cm, and the disinfection method is boiling water for 2-3 hours.

10. The novel method for efficiently breeding star ferns through green globose bodies (GGBs) according to claim 2, It is characterized in that The heat preservation and moisture retention conditions in step (8) are a culture temperature of 20-25° C. and a humidity of 70%.

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