A method for saving the national first-class rare and endangered plant Adiantum scabra from extinction

Abstract

The present invention relates to a method for saving the national Class I rare and endangered plant, Adiantum spp., from extinction. The method first involves physically treating the Adiantum spp. plants, primarily by uniformly isolating the Adiantum spp. plants that have undergone pre-double treatment under physical space isolation and special environmental condition control. After completing the uniform physical space isolation and special environmental condition control, when the shoots grow to a height of 0.5 cm to 2.5 cm, the shoots of the Adiantum spp. plants are sprayed with a chromosome mutagenesis mixed solvent solution (0.4% to 0.8% amoxifen + 0.01% to 0.05% colchicine + 1 to 3 mg / L CaCl2·2H2O + 0.5 to 1.0 g / L P2O5 + 1.0 g / L to 2 g / L aloe extract), and a control group is set up at the same time. The present invention induces new varieties with both excellent appearance and traits in the shortest possible time, achieving the preservation, creation, and utilization of national Class I rare and endangered plant germplasm resources and protecting biodiversity. At the same time, a new method is provided to avoid the extinction of the germplasm resources of the national first-class rare and endangered plant, the iron wire of lotus leaves.

Description

Technical Field

[0001] The invention relates to a method for saving a national Class I rare and endangered plant, Adiantum nudiflorum, from extinction, and belongs to the technical field of biological induction. Background Art

[0002] Adiantum nelumboides is the only simple-leaf fern in the genus Adiantum of the family Pteridaceae distributed in Asia. It is endemic to China and a relict plant of the Quaternary. It is known as the "Chinese Treasure" among the fern treasures.

[0003] The Adiantum saccharina (Adiantum saccharina) is found in an ecologically fragile area with a very narrow distribution range, limited to Wanxian, Fuling, and Shizhu counties in the Three Gorges region of the Yangtze River. It is a Class I nationally protected wild plant. The IUCN Red List of Threatened Species lists Adiantum saccharina as Critically Endangered (CR). To prevent the extinction of rare and endangered plants, conservation efforts primarily include establishing nature reserves, implementing ex situ conservation, strengthening publicity and education, and improving the legal system. However, these methods all have drawbacks, including high investment, long timeframes, extensive resource utilization, and difficulty guaranteeing effective conservation. Summary of the Invention

[0004] In view of the problems existing in the existing technology, the present invention provides a method for saving the national first-class rare and endangered plant Adiantum nucifera and preventing it from extinction, realizing the preservation, creation and utilization of national first-class rare and endangered plant germplasm resources, maintaining biodiversity, implementing the "Yangtze River Protection Law", and directly contributing to the protection of the Yangtze River.

[0005] The technical solution of the present invention is:

[0006] A method for saving the national Class I rare and endangered plant Adiantum spheniscus from extinction, the method comprising the following steps:

[0007] (1) Plant physical treatment: The Adiantum plants that have been doubled are uniformly isolated in physical space and subjected to special environmental conditions;

[0008] (2) Plant chemical reagent treatment: After completing step 1), when the shoots grow to a height of 0.5 cm to 2.5 cm, the buds of the Adiantum spp. plant are sprayed with a chromosome mutagenesis mixed solvent solution; after spraying, the water mist is preferably attached to the surface of the stem segment and does not flow, and finally wrapped with absorbent cotton soaked in purified water to create a dark environment while moisturizing;

[0009] (3) After the seedlings were induced to undergo a growth cycle of 10-12 months, new individual plants germinated, and after vegetative growth and reproductive growth, they were tested on the molecular biology experimental platform of the Southwest China Wildlife Germplasm Resource Bank of the Kunming Institute of Botany, Chinese Academy of Sciences, using a flow cytometer (instrument model: BD FACScalibur) to measure the genome size of the target plant. The test confirmed that a new variety of Adiantum spp. "Yunxia Treasure No. 1" with obvious polyploidy characteristics was obtained, which will be used for the conservation and breeding of rare and endangered planting resources of Adiantum spp.

[0010] Preferably, in step 1), physical space isolation is performed, that is, all the Adiantum plants are placed uniformly in a certain space, and the plants are arranged from north to south according to their growth height, and the sprouting direction of the plants is perpendicular to the plant direction, that is, from east to west, to ensure that the plants are in the same direction as the earth's rotation direction.

[0011] Preferably, in the step 1), the plants are subjected to special environmental conditions, that is, all the Adiantum plants are intermittently supplemented with light, the supplementary light time is from 18:00 to 24:00 every day, once every 2 hours, the interval time is 10 to 30 minutes, the light intensity is 2500lx to 4000lx, and the air humidity is 70% to 90%, so as to facilitate the germination of the young shoots of Adiantum as soon as possible.

[0012] Preferably, in step 2), the mixed solvent solution is: 0.4%-0.8% amoxifen + 0.01%-0.05% colchicine + 1-3 mg / L CaCl2·2H2O + 0.5-1.0 g / L P2O5 + 1.0 g / L-2 g / L aloe extract.

[0013] Preferably, in the step 2), the polyploidy inducing mixed solvent treatment is performed once every 2 days, for a total of 3 spraying times. The spraying method is to use a 100ml small spray pot for each spraying, adjust the pot mouth to the best mist-like state, and spray around the tender shoots of the lotus leaf fern, focusing on the curved part of the top of the tender shoot. After spraying, it is best if the water mist adheres to the surface of the stem segment and does not flow. Finally, wrap it with absorbent cotton soaked in pure water to create a dark environment while moisturizing.

[0014] The results of polyploidy in this invention are different from those of other plants. Typically, new varieties of polyploidy exhibit enlarged leaves, which is a desirable appearance trait, especially in crops and vegetables. However, enlarged leaves are unsuitable for ferns to survive in the wild. This method, however, results in smaller leaves, which is exactly what ferns need, enhancing their resistance to extreme weather conditions such as heat and drought.

[0015] The present invention has the following beneficial effects:

[0016] 1. Pretreatment operations such as physical space isolation and special environmental conditions control are simple. After the seedlings are mutagenized, they can be induced into new varieties after a growth cycle of 10-12 months. The induction rate can reach 93.3%, which is shorter than the previous induction time limit and has a significant induction effect.

[0017] 2. Physical space isolation ensures that the plants rotate in the same direction as the earth, conforms to the weather and promotes the formation of group effects. It also makes it easier to water and fertilize the plants and promote the growth of young shoots.

[0018] 3. Chemical agents + nutrients + aloe vera extract have fully integrated solution components to avoid excessive volatilization of effective inducing components, thereby ensuring that they can effectively promote changes in plant chromosome multiplicity, produce polyploidy, and form new fern varieties with low evaporation from plant leaves and strong resistance to extreme climate changes such as high temperature and drought.

[0019] 4. Through a combined approach of physical and chemical treatment, new varieties with superior appearance and traits can be induced in the shortest possible time, creating a new path for plant conservation and effectively preventing extinction disasters. This enables the preservation, creation, and utilization of national Class I rare and endangered plant germplasm resources, safeguarding biodiversity and having significant ecological and social benefits. Cultivating new varieties can effectively avoid the aforementioned shortcomings, cultivating new characteristics not inherent in existing plants, improving stress resistance and adapting to more complex environmental changes, thus creating a new path for plant conservation and effectively preventing extinction disasters. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Comparison of the vegetative growth stages of the new variety "Yunxia Treasure No. 1" and the original variety (the new variety is on the left, the original variety is on the right);

[0021] Figure 2 Comparison of the reproductive growth stages of the new variety "Yunxia Treasure No. 1" and the original variety (the new variety is on the left, the original variety is on the right);

[0022] Figure 3 Appearance of the new variety "Yunxia Treasure No. 1";

[0023] Figure 4 Appearance of the original species;

[0024] Figure 5 Comparison of the number of sporangia in the new variety "Yunxia Treasure No. 1" and the original variety (the new variety is on the left, the original variety is on the right);

[0025] Figure 6 Diagram of the leaf growth stages of the new variety "Yunxia Treasure No. 1";

[0026] Figure 7Comparison of the vegetative and reproductive growth stages of the new variety "Yunxia Treasure No. 1" and the original variety (a is the vegetative growth period, b is the front of the leaf during the reproductive growth period, and c is the back of the leaf during the reproductive growth period. In a, b, and c, the new variety is on the left and the original variety is on the right);

[0027] Figure 8 A comparison of leaf sizes between the new variety "Yunxia Treasure No. 1" and the original variety (the top of the picture is the new variety, the bottom is the original variety);

[0028] Figure 9 Photos of the new variety "Yunxia Treasure No. 1" being mass-produced;

[0029] Figure 10 Cell density map, scatter plot, and histogram of the original species and the new variety "Yunxia Treasure No. 1"; Group Figure Adiantum+M001 shows the cell density map (a), scatter plot (b), and histogram (c) of the original species of Adiantum; Group Figure 14230-1+M002 shows the cell density map (d), scatter plot (e), and histogram (f) of the new variety "Yunxia Treasure No. 1"; Group Figure 14230-2+M003 shows the cell density map (g), scatter plot (h), and histogram (i) of the new variety "Yunxia Treasure No. 1"; Group Figure 14230-3+M004 shows the cell density map (j), scatter plot (k), and histogram (l) of the new variety "Yunxia Treasure No. 1". DETAILED DESCRIPTION

[0030] Example 1

[0031] A method for saving the national Class I rare and endangered plant Adiantum scabra from extinction:

[0032] In order to effectively promote the change of chromosome multiplicity and cultivate new plant varieties, the physical treatment of the Adiantum plants is first carried out, mainly to isolate the Adiantum plants that have been pre-doubled and place them in a physical space and control special environmental conditions. The first is to isolate the physical space, that is, to place all the Adiantum plants in a certain space. This experimental culture room is oriented north-south (other similar culture rooms or isolation rooms can be adjusted according to actual conditions). The plants are arranged from north to south according to their growth height. The direction of plant sprouting is perpendicular to the direction of the plant, that is, from east to west, to ensure that the plant is in the same direction as the direction of the earth's rotation, in line with the weather and to promote the formation of group effects. It is also convenient for watering, fertilizing and promoting the germination of tender shoots. Secondly, after completing the unified physical space isolation, the plants are subject to special environmental conditions control, that is, all the Adiantum plants are intermittently supplemented with light. The supplementary light time is 18:00 every day, once every 2 hours, with an interval of 20 minutes, a light intensity of 2800lx, and an air humidity of 80%, so as to facilitate the germination of the young shoots of Adiantum as soon as possible.

[0033] The study of chemical treatment of Adiantum plants involved spraying a chromosomal mutagenesis mixture (0.5% amifosylphos + 0.03% colchicine + 2mg / L CaCl2·2H2O + 0.8g / L P2O5 + 1.0g / L to 2g / L aloe extract) on sprouts growing to 0.5cm to 2.5cm in height after uniform physical isolation and special environmental conditions. A control group (no spraying of the inducing mixture) was also established. Each treatment included 30 pots of plants, and the experiment was replicated three times. The polyploidy-inducing mixture was sprayed once every two days for a total of three times. The spraying method is to use a 100ml small spray bottle each time, adjust the mouth of the bottle to the best mist state, and spray around the tender shoots of the lotus leaf fern, focusing on spraying the curved part of the top of the tender shoot. After spraying, the water mist should adhere to the surface of the stem segment without flowing. Finally, wrap it with absorbent cotton soaked in pure water to create a dark environment while moisturizing.

[0034] After the seedlings are mutagenized, they undergo a 10-12 month growth cycle, sprouting new plants. Following vegetative and reproductive growth, the target plants are tested using a flow cytometer (BD FACScalibur) at the Southwest China Wildlife Germplasm Bank's Molecular Biology Laboratory at the Kunming Institute of Botany, Chinese Academy of Sciences, to determine their genome size. The resulting varieties exhibit distinct polyploid traits and are used for the conservation and propagation of rare and endangered Adiantum spp. This method offers significant time advantages over other crop and breeding methods. For example, the super rice developed by Professor Yuan Longping typically takes anywhere from three to five years to several decades. Another method involves placing seeds in a space laboratory for mutagenesis breeding, which typically takes five to ten years. Detailed experimental results from various treatments are shown in Tables 1-2 below.

[0035] Table 1 Induction of the new polyploid variety “Yunxia Treasure No. 1” under different treatments of Adiantum serrata

[0036]

[0037]

[0038] Table 2 Comparison between the new polyploid variety of Adiantum serrata “Yunxia Treasure No. 1” and the original species

[0039]

[0040] As shown in Table 2, there is no significant difference in plant crown width and height between the new variety and the original species. The main significant differences are in leaf size and spore sacs. The plant crown width of the new variety is 26.8 cm, while that of the original species is 25.7 cm. The plant crown width has a mutation rate of 1.1 cm, and the mutation rate is 4.3%. The plant height of the new variety is 18.8 cm, while that of the original species is 19.1 cm. The plant height has a mutation rate of 0.3 cm, and the mutation rate is 1.6%. The longest horizontal axis of the leaf of the new variety is 2.3 cm, while that of the original species is 6.5 cm. The mutation rate of the horizontal axis of the leaf is 4.2 cm, and the mutation rate is 64.6%. The longest vertical axis of the leaf of the new variety is 1.5 cm, while that of the original species is 3.6 cm. The mutation rate of the horizontal axis of the leaf is 2.1 cm, and the mutation rate is 58.3%. The leaf area per plant of the new variety is 4.15 cm. 2 , original species 33.17cm 2 The area variation is 29.02 cm 2 , the mutation rate was 87.5%; the number of sporangia in the new variety was 25, and that in the original variety was 38, the difference in the number of sporangia was 13, and the mutation rate was 34.2%; the longest sporangium in the new variety was 0.38cm, and that in the original variety was 0.85cm, the difference in the number of mutagenia was 0.47cm, and the mutation rate was 55.3%; the widest sporangium in the new variety was 0.22cm, and that in the original variety was 0.47cm, the difference in the number of mutagenia was 0.09cm, and the mutation rate was The new variety has an average sporangium length of 0.22cm, while the original variety has an average sporangium length of 0.47cm. The difference in mutation amount is 0.25cm, and the mutation rate is 53.2%. The new variety has an average sporangium width of 0.17cm, while the original variety has an average sporangium width of 0.26cm. The difference in mutation amount is 0.09cm, and the mutation rate is 34.6%. The total area of ​​single leaf sporangium of the new variety is 99.04mm2, while the total area of ​​single leaf sporangium of the original variety is 471.99mm2. 2 , the difference in mutation amount is 372.95mm 2 The mutation rate is 79.0%; the average area of ​​the single leaf capsule of the new variety is 3.96mm 2 The average area of ​​the original species single leaf sporangium is 12.42mm 2 , the difference in mutation amount is 8.46mm 2 , the mutagenesis rate was 68.1%.

[0041] Flow cytometry detection and genome size calculation

[0042] Using maize A188 as an internal reference, suspensions of the test sample and internal reference sample were mixed in appropriate proportions. The stained cell nuclear suspensions were analyzed using a BD FACScalibur flow cytometer, using 488nm blue light excitation to measure the fluorescence intensity of propidium iodide emission. 10,000 particles were collected for each assay. The coefficient of variation (CV%) was maintained within 5%. Modifit 3.0 analysis software was used for graphical analysis.

[0043] Propidium iodide is a fluorescent dye that uniformly intercalates into double-stranded nucleic acid base pairs, allowing it to specifically stain DNA. Under 488nm excitation light, the fluorescence emitted by propidium iodide can be detected by flow cytometry. Furthermore, the amount of PI intercalated during the staining process is directly proportional to the amount of DNA, so the fluorescence intensity can indicate the relative content of genomic DNA. By observing the peak fluorescence of the PI-DNA complex in the test sample and control plant, the ratio of the DNA content of the two plants can be calculated. This ratio is then multiplied by the C value of the internal reference plant to calculate the C value of the test plant. The calculation formula is: DNA content of the test sample = internal reference DNA content × fluorescence intensity of the test sample / fluorescence intensity of the internal reference sample.

[0044] Table 3 Statistics of flow cytometry test results of the original species of Adiantum serrata and the new variety “Yunxia Treasure No. 1”

[0045] Sample number variety Internal reference selection Internal reference fluorescence intensity Fluorescence intensity of the sample to be tested Ratio Genome (Gb) Adiantum Original species corn 33.94 85.24 2.51 5.78 14230-1 Spore leaves New Varieties corn 34.84 96.01 2.76 6.34 14230-2 leaves New Varieties corn 32.73 90.24 2.76 6.34 14230-3 leaves New Varieties corn 33.29 90.79 2.73 6.27

[0046] The above embodiments are merely preferred technical solutions of the present invention and should not be construed as limiting the present invention. The embodiments and features in the embodiments of this application may be arbitrarily combined with each other unless they conflict. The scope of protection of the present invention shall be the technical solutions described in the claims, including equivalent alternatives to the technical features of the technical solutions described in the claims. Equivalent alternatives and improvements within this scope are also within the scope of protection of the present invention.

Claims

1. A method for saving the national Class I rare and endangered plant Adiantum spheniscus from extinction, characterized in that: The method comprises the following steps: (1) Physical treatment of plants: The pre-doubled treatment of Adiantum plants was uniformly isolated in physical space and under special environmental conditions. Physical space isolation means that all Adiantum plants were uniformly placed in a certain space, and the plants were arranged from north to south according to their growth height. The sprouting direction of the plants was perpendicular to the plant direction, that is, from east to west, to ensure that the plants were in the same direction as the earth's rotation direction. Special environmental conditions were controlled for the plants, that is, all Adiantum plants were intermittently supplemented with light. The supplementing time was from 18:00 to 24:00 every day, once every 2 hours, and the interval time was 10 to 30 minutes. The light intensity was 2500lx to 4000lx, and the air humidity was 70% to 90%, so as to facilitate the germination of the young shoots of Adiantum as soon as possible. (2) Plant chemical reagent treatment: After completing step 1), when the shoots grow to a height of 0.5 cm to 2.5 cm, spray the shoots of the Adiantum nerii plant with a polyploidy induction mixed solvent. The mixed solvent is: 0.4%-0.8% ammonium sulfamate + 0.01%-0.05% colchicine + 1-3 mg / L CaCl2·2H2O + 0.5-1.0 g / L P2O5 + 1.0 g / L-2 g / L aloe extract. After spraying, the water mist should adhere to the surface of the stem without flowing. Finally, wrap it with absorbent cotton soaked in pure water to create a dark environment while moisturizing. (3) After the seedlings are induced to undergo a growth cycle of 10-12 months, new individual plants germinate, and after vegetative growth and reproductive growth, the polyploid induction rate is over 70%, which can be used for the preservation and breeding of rare and endangered planting resources of Adiantum oleifera.

2. The method for saving the national Class I rare and endangered plant Adiantum nerii from extinction according to claim 1, characterized in that: In the step 2), the polyploidy inducing mixed solvent treatment is applied once every 2 days, for a total of 3 spraying times. The spraying method is to use a 100 ml small spray bottle for each spraying, adjust the mouth of the bottle to the best mist-like state, and spray around the tender shoots of the lotus leaf fern, focusing on the curved part of the top of the tender shoot. After spraying, the water mist should adhere to the surface of the stem segment without flowing. Finally, wrap it with absorbent cotton soaked in pure water to create a dark environment while moisturizing.

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