Method for improving sowing survival rate of fritillaria taipaiensis seeds

Abstract

The invention discloses a method for increasing the sowing survival rate of fritillaria taipaiensis seeds, and belongs to the technical field of fritillaria taipaiensis breeding. Fritillaria taipaiensis seeds are embedded with an embedding material and then subjected to variable-temperature stratification treatment, all components in the embedding material act together, and the survival rate of the fritillaria taipaiensis seeds is increased; the morphological after-ripening and physiological after-ripening of the seeds are sequentially promoted by combining temperature-variable stratification treatment, so that the fritillaria taipaiensis seeds can better reach a germination state, the seed vitality is improved, the sowing survival rate of the fritillaria taipaiensis seeds is improved, and the fritillaria taipaiensis seed sowing method has important significance in promoting the industrial development of fritillaria taipaiensis.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of Fritillaria taipaiensis breeding, and particularly relates to a method for improving the seed sowing survival rate of Fritillaria taipaiensis. BACKGROUND

[0002] Fritillaria taipaiensis P. Y. Li is a perennial herb of the Fritillaria genus of the Liliaceae family. Fritillaria taipaiensis has the effects of moistening the lung to stop coughing, reducing phlegm to relieve asthma, and resolving nodules and swelling, and is widely used in clinical treatment of lung heat and dry cough, lung yin deficiency, etc. Its commodity has a long history of use as Fritillaria cirrhosa in the folk. In recent years, the wild resources of Fritillaria taipaiensis have been overexploited, and the breeding environment has been severely damaged, which seriously restricts the development of the Fritillaria taipaiensis industry.

[0003] The breeding method of Fritillaria taipaiensis includes sexual reproduction and asexual reproduction. The asexual reproduction is whole or split bulb planting, which has the advantage of fast breeding speed, but needs a large amount of bulb as breeding material, and is difficult to be popularized on a large scale when the seed source is insufficient, and the breeding coefficient is low. The sexual reproduction is seed propagation, and the seed yield of Fritillaria taipaiensis is high, with 200-250 seeds per flower. When seed propagation is adopted, the breeding material is sufficient, and large-scale cultivation is facilitated. However, the seed embryo of newly harvested Fritillaria taipaiensis seed is in the original embryo state, has a dormancy phenomenon, and needs to complete morphological after-ripening and physiological after-ripening through sand storage and stratification before germination and emergence. Under the conventional sand storage and stratification method, it takes about 90 days for Fritillaria taipaiensis seed to complete morphological after-ripening, and 180-210 days to complete physiological after-ripening. Long-term stratification treatment not only increases the time and labor cost, but also easily causes seed embryo death and reduces the seed sowing and breeding efficiency of Fritillaria taipaiensis.

[0004] Therefore, it is necessary to find a method for improving the seed sowing survival rate of Fritillaria taipaiensis to solve the problem of long time and high labor cost caused by the conventional sand storage and stratification treatment of Fritillaria taipaiensis seed, and the problem of seed embryo death and reduced seed sowing and breeding efficiency. SUMMARY

[0005] In view of this, the purpose of the present application is to provide a method for improving the seed sowing survival rate of Fritillaria taipaiensis to solve the problem of long time and high labor cost caused by the conventional sand storage and stratification treatment of Fritillaria taipaiensis seed, and the problem of seed embryo death and reduced seed sowing and breeding efficiency.

[0006] The present application solves the above technical problems through the following technical means:

[0007] A method for improving the seed sowing survival rate of Fritillaria taipaiensis, the method comprising the following steps:

[0008] (1) Seed disinfection: select the seeds of Fritillaria taipaiensis free of diseases and insect pests, and soak them in 0.1wt% potassium permanganate solution for 12-15min, then wash them with clean water for 2-3 times to obtain the disinfected seeds;

[0009] (2) Seed embedding: embed the disinfected seeds with embedding materials to obtain embedded seeds;

[0010] (3) Stratification treatment: mix the embedded seeds with the substrate, and then perform variable-temperature stratification treatment to obtain the seeds to be sown;

[0011] (4) Sowing: sow the seeds to be sown, and then perform routine management.

[0012] Further, the embedding material in step (2) comprises the following raw materials:

[0013] glucose, sodium caseinate, gibberellin, sodium alginate, hyaluronic acid, calcium peroxide, polyvinyl alcohol, L-asparagine, p-toluenesulfonic acid, 3-aminobenzoic acid, borax.

[0014] Further, the preparation method of the embedded seeds is as follows:

[0015] A: dissolve glucose in water to prepare a 40wt% glucose solution; dissolve sodium caseinate in water to prepare a 5wt% sodium caseinate solution; mix the sodium caseinate solution with the glucose solution to obtain a sodium caseinate-glucose mixture, then stir the sodium caseinate-glucose mixture at a speed of 200-300r / min, and drop 20wt% citric acid solution into the stirring sodium caseinate-glucose mixture at a speed of 1 drop / s until the pH of the sodium caseinate-glucose mixture is adjusted to 4.5, continue to stir at a speed of 200-300r / min for 10min after the dropping is completed, then stand for 1-2h, centrifuge to obtain the precipitate, and dry the precipitate to obtain sodium caseinate-loaded glucose particles;

[0016] B: dissolve sodium caseinate in water to prepare a 5wt% sodium caseinate solution, add gibberellin to the sodium caseinate solution and stir to mix uniformly to obtain a sodium caseinate-gibberellin mixture, stir the sodium caseinate-gibberellin mixture at a speed of 200-300r / min, and drop 20wt% citric acid solution into the stirring sodium caseinate-gibberellin mixture at a speed of 1 drop / s until the pH of the sodium caseinate-gibberellin mixture is adjusted to 4.5, continue to stir at a speed of 200-300r / min for 10min after the dropping is completed, then stand for 1-2h, centrifuge to obtain the precipitate, and dry the precipitate to obtain sodium caseinate-loaded gibberellin particles;

[0017] C: The sterilized seeds are soaked in 1wt% sodium alginate solution for 10-30 minutes, then taken out and mixed with casein-glucose particles in an amount of 50% of the sterilized seeds, and then dried to obtain the first-embedded seeds;

[0018] D: Hyaluronic acid is dissolved in water to prepare a 1wt% hyaluronic acid solution, and calcium peroxide is uniformly dispersed to obtain a hyaluronic acid-calcium peroxide mixture. The second-embedded seeds are obtained by uniformly spraying the hyaluronic acid-calcium peroxide mixture in an amount of 30% of the first-embedded seeds onto the surface of the first-embedded seeds and then drying.

[0019] E: Polyvinyl alcohol is added to dimethyl sulfoxide, stirred and dissolved at 90°C, then L-asparagine and p-toluenesulfonic acid are added, and the reaction is continuously stirred for 3-4 hours. After the reaction is completed, the pH is adjusted to 7, and then naturally cooled to room temperature. The modified polyvinyl alcohol is dried to obtain a modified polyvinyl alcohol solution. The modified polyvinyl alcohol solution is prepared by adding 10 times the mass of water, heating to 90°C, stirring and dissolving, and then cooling to 40-50°C. 3-aminobenzoic acid is added, the pH is adjusted to 7, and the reaction is stirred for 3-4 hours. After the reaction is completed, the reaction is naturally cooled to room temperature, and sodium caseinate-glucosinolate particles are added and mixed uniformly to obtain a polyvinyl alcohol composite glue solution. The second-embedded seeds are added to the polyvinyl alcohol composite glue solution, stirred and mixed for 3-5 minutes, then taken out and placed in a 0.5wt% borax solution to gel the surface polyvinyl alcohol composite glue solution. Then, the seeds are taken out, washed with water for 2-3 times, and then dried at 40°C for 30 minutes to obtain the embedded seeds.

[0020] Further, in step A, the mass ratio of the glucose solution to the sodium caseinate solution is (80-100):(30-50).

[0021] Further, in step B, the mass ratio of sodium caseinate to gibberellin is (20-40):(0.1-0.2).

[0022] Further, in step D, the mass ratio of the hyaluronic acid solution to calcium peroxide is (50-100):(30-60).

[0023] Further, in step E, the mass ratio of polyvinyl alcohol, L-asparagine, and p-toluenesulfonic acid is (10-20):(2-4):(0.1-0.2).

[0024] Further, in step E, the mass ratio of the modified polyvinyl alcohol solution, 3-aminobenzoic acid, and sodium caseinate-glucosinolate particles is (100-200):(2-5):(20-40).

[0025] Further, the step (3) matrix is obtained by mixing sawdust, humus soil and sand in a mass ratio of 1:1:2, and then spraying 0.1wt% potassium permanganate solution for disinfection; the mass ratio of the embedded seeds to the matrix is 1:(3-5).

[0026] Further, the temperature-variable stratification treatment method in the step (3) is as follows:

[0027] After the embedded seeds are mixed with the mixed seeds, the mixture is first subjected to warm stratification treatment at 15-20℃ for 60-70 days, and then subjected to low-temperature stratification treatment at 4℃ for 80-100 days to obtain the seeds to be sowed, during which the water content of the matrix is maintained at 60-70%.

[0028] During the sand storage stratification process, the seeds of F. przewalskii need to complete morphological after-ripening and physiological after-ripening in sequence to reach the germination state and ensure the seed germination rate. Although both are after-ripening processes, the cores of morphological after-ripening and physiological after-ripening are not exactly the same. The core of morphological after-ripening is to promote the morphological development of the embryo, while the core of physiological after-ripening is to remove dormancy and activate the physiological activity of the embryo. In the morphological after-ripening process, the morphological development of the embryo needs to be carried out in a suitable temperature, humidity and aerobic environment; in the physiological after-ripening process, in addition to a suitable temperature, humidity and aerobic environment, the stimulation of plant hormones is also crucial. In the conventional method, the seeds of F. przewalskii are directly mixed into sand for low-temperature stratification, and the fixed low-temperature environment cannot promote the morphological development of the embryo well. In the later physiological after-ripening process, the stimulation of gibberellin is lacking, and the effect is not good. Moreover, the stratification time period is long, the effect is not ideal, and the seed germination rate of F. przewalskii is limited. Therefore, in order to shorten the seed sand storage stratification time, improve the sand storage stratification efficiency, and further improve the seed germination rate of F. przewalskii, the seeds of F. przewalskii are subjected to temperature-variable stratification treatment after being embedded, so that the seeds of F. przewalskii can efficiently complete morphological after-ripening in a more suitable temperature, humidity and aerobic environment, and further efficiently complete physiological after-ripening in a suitable temperature, humidity, aerobic environment and the stimulation of gibberellin, thereby effectively shortening the sand storage stratification time, improving the stratification efficiency, and improving the seed germination rate.

[0029] Specifically, the Fritillaria taipaiensis seed is embedded step by step to obtain the embedded seed, wherein the first embedding is to adhere the casein loaded glucose particles to the surface of the seed by using sodium alginate as the adhesive; the second embedding is to spray the mixture of hyaluronic acid and calcium peroxide; finally, the second embedded seed is put into the polyvinyl alcohol composite glue solution added with casein loaded gibberellin particles, adhered to the surface of the seed, and then put into the borax solution to gel, so as to obtain the embedded seed, wherein the hyaluronic acid in the second embedded seed has good film forming property, and then carries the calcium peroxide to firmly adhere to the surface of the seed, and then the calcium peroxide is slowly hydrolyzed and oxygen is released to ensure sufficient aerobic environment in the after-ripening; in combination with the suitable temperature in the early warm stratification and the humidity of the sand, the after-ripening of the seed is ensured to be more suitable in temperature, humidity and aerobic environment, so as to accelerate the after-ripening of the embryo, and then with the extension of the stratification time, the glucose in the casein loaded glucose gradually releases from the casein, enters the polyvinyl alcohol gel in the outermost layer through the hyaluronic acid film, and since the polyvinyl alcohol gel is prepared by adding 3-aminobenzene boronic acid for reaction to introduce the borate ester bond with glucose sensitivity, the polyvinyl alcohol gel is disintegrated when meeting glucose in the later period, so as to release the gibberellin in the casein loaded gibberellin particles, and the gibberellin acts on the seed to inhibit the formation of abscisic acid of the seed, improve the enzyme activity of the seed, thereby break the dormancy, activate the physiological activity of the embryo, and thus better complete the physiological after-ripening of the Fritillaria taipaiensis seed.

[0030] Although the embedded seed provides more suitable environmental conditions to accelerate the after-ripening process and shorten the after-ripening time, the stratification time of the Fritillaria taipaiensis seed is still relatively long, in order to ensure that the gibberellin can effectively play a role in the later physiological after-ripening process, the embedded seed with three-layer embedding structure is prepared in the application, the glucose in the innermost layer is first released from the casein, then enters the polyvinyl alcohol gel layer with good stability through the hyaluronic acid film, and then the gibberellin in the casein loaded gibberellin is released to act on the seed after the gel structure of the polyvinyl alcohol gel is disintegrated, so as to effectively prolong the release time of the gibberellin and ensure the concentration of the gibberellin in the later physiological after-ripening stage to ensure the effect of the gibberellin.

[0031] In addition, the application adopts polyvinyl alcohol with strong stability as the outer gel layer to embed and load gibberellin, so that the embedding stability can be ensured during the stratification process for a long time. Although polyvinyl alcohol has strong stability and can maintain a stable state during the stratification process of seeds for a long time, it has strong water absorption and is easy to absorb excessive water to swell and block the pores, causing seed oxygen deficiency. Therefore, the application adopts L-asparagine to treat polyvinyl alcohol. L-asparagine is connected with polyvinyl alcohol in the form of ester bond, so as to reduce the hydrophilicity of polyvinyl alcohol, prevent polyvinyl alcohol from absorbing too much water, and at the same time, L-asparagine can adjust the charge property of polyvinyl alcohol, enhance the repulsive force of polyvinyl alcohol molecular chain bond, and thus increase the porosity of the three-dimensional network structure of polyvinyl alcohol hydrogel, so as to ensure the circulation of oxygen.

[0032] Beneficial effects:

[0033] 1. The application adopts the method of warm stratification and low temperature stratification to treat the seeds of Fritillaria taipaiensis in turn. The seeds complete morphological after-ripening of the seed embryo under the treatment of warm stratification, and further complete physiological after-ripening to reach the germination state under the treatment of low temperature stratification. The temperature variation of the stratification treatment is more in line with the temperature change of the seeds in the natural environment, improves the adaptability of the seeds to the natural environment, and further improves the germination and survival rate of the seeds.

[0034] 2. The application adopts embedding material to embed the seeds before stratification treatment, so as to provide suitable humidity and more sufficient oxygen environment for the seeds, release gibberellin to inhibit the formation of abscisic acid in the seeds, promote the decomposition of the storage materials of the seeds, improve the activity of the seeds, and further complete the physiological after-ripening. Combined with the temperature variation stratification treatment, the after-ripening process is completed more quickly and efficiently to reach the germination state, so as to improve the seed survival rate of Fritillaria taipaiensis and has important significance for promoting the development of Fritillaria taipaiensis industry. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 : It is the picture of the seeds of Fritillaria taipaiensis collected by the application;

[0036] Figure 2 : It is the picture of the seedling condition obtained by sowing the seeds in the experimental group 1 of the application. DETAILED DESCRIPTION

[0037] The application will be described in detail below in combination with specific embodiments and drawings:

[0038] The application discloses a method for improving seed sowing survival rate of Fritillaria taipaiensis, and specifically relates to a method for preparing embedded seeds by embedding seeds and then performing temperature-variable stratification treatment.

[0039] Example 1: preparation of embedded seeds

[0040] A: 40wt% glucose solution is prepared by dissolving glucose in water; 5wt% sodium caseinate solution is prepared by dissolving sodium caseinate in water; 40g of the sodium caseinate solution is added into 90g of the glucose solution to mix uniformly to obtain a sodium caseinate-glucose mixture, then the sodium caseinate-glucose mixture is stirred at a speed of 250r / min, 20wt% citric acid solution is added dropwise into the stirred sodium caseinate-glucose mixture at a speed of 1 drop / s until the pH of the sodium caseinate-glucose mixture is adjusted to 4.5, after the dropwise addition is completed, the stirring is continued at a speed of 250r / min for 10min, then the mixture is left to stand for 1.5h, and the precipitate is obtained by centrifugation at a speed of 10000rpm for 15min, and the precipitate is dried at 45℃ to obtain sodium caseinate loaded glucose particles;

[0041] B: 5wt% sodium caseinate solution is prepared by dissolving 30g of sodium caseinate in water, 0.15g of gibberellin is added into the sodium caseinate solution to mix uniformly to obtain a sodium caseinate-gibberellin mixture, then the sodium caseinate-gibberellin mixture is stirred at a speed of 250r / min, 20wt% citric acid solution is added dropwise into the stirred sodium caseinate-gibberellin mixture at a speed of 1 drop / s until the pH of the sodium caseinate-gibberellin mixture is adjusted to 4.5, after the dropwise addition is completed, the stirring is continued at a speed of 250r / min for 10min, then the mixture is left to stand for 1.5h, and the precipitate is obtained by centrifugation at a speed of 10000rpm for 15min, and the precipitate is dried at 45℃ to obtain sodium caseinate loaded gibberellin particles;

[0042] C: the sterilized seeds are soaked in 1wt% sodium alginate solution for 20min, then taken out, and then mixed with sodium caseinate loaded glucose particles with a mass percentage of 50% of the sterilized seeds, and dried at 40℃ to obtain primary embedded seeds;

[0043] D: 1wt% hyaluronic acid solution is prepared by dissolving hyaluronic acid in water, 40g of calcium peroxide is uniformly dispersed in 70g of the hyaluronic acid solution to obtain a hyaluronic acid-calcium peroxide mixture, and the hyaluronic acid-calcium peroxide mixture with a mass percentage of 30% of the primary embedded seeds is uniformly sprayed on the surface of the primary embedded seeds, and then dried at 40℃ to obtain secondary embedded seeds;

[0044] E: 15 g of polyvinyl alcohol was added into 150 g of dimethyl sulfoxide, and after being stirred and dissolved at 90 °C, 3 g of L-asparagine and 0.15 g of p-toluenesulfonic acid were added, and the reaction was continuously stirred for 3.5 h. After the reaction was completed, the pH was adjusted to 7, and then the temperature was naturally cooled to room temperature. After being dried at 60 °C, the modified polyvinyl alcohol was obtained. The modified polyvinyl alcohol was added into 10 times the mass of water, and after being stirred and dissolved at 90 °C, 2.5 g of 3-aminobenzoic acid was added after the temperature was cooled to 45 °C. After the pH was adjusted to 7, the reaction was stirred for 3.5 h. After the reaction was completed, the temperature was naturally cooled to room temperature, and 30 g of sodium caseinate was added to load gibberellin particles and mixed uniformly to obtain a polyvinyl alcohol composite glue solution. The secondary embedded seeds were immersed in the polyvinyl alcohol composite glue solution, stirred and mixed for 4 min, then taken out and placed in a 0.5 wt% borax solution to gel the surface polyvinyl alcohol composite glue solution. Then, after being washed with water for 3 times, the embedded seeds were dried at 40 °C for 30 min.

[0045] Example 2: Preparation of embedded seeds for preparing secondary

[0046] A: A 40 wt% glucose solution was prepared by dissolving glucose in water; a 5 wt% sodium caseinate solution was prepared by dissolving sodium caseinate in water; 30 g of the sodium caseinate solution was added into 80 g of the glucose solution and mixed uniformly to obtain a sodium caseinate-glucose mixture, and then 20 wt% citric acid solution was added dropwise into the stirred sodium caseinate-glucose mixture at a rate of 1 drop per second until the pH of the sodium caseinate-glucose mixture was adjusted to 4.5. After the addition was completed, the stirring was continuously performed at a speed of 200 r / min for 10 min, and then the mixture was statically placed for 1 h. The precipitate was obtained by centrifugation at 10,000 rpm for 15 min, and then dried at 45 °C to obtain casein-loaded glucose particles;

[0047] B: 20 g of sodium caseinate was dissolved in water to prepare a 5 wt% sodium caseinate solution, and 0.1 g of gibberellin was added into the sodium caseinate solution and stirred and mixed uniformly to obtain a sodium caseinate-gibberellin mixture. Then, 20 wt% citric acid solution was added dropwise into the stirred sodium caseinate-gibberellin mixture at a rate of 1 drop per second until the pH of the sodium caseinate-gibberellin mixture was adjusted to 4.5. After the addition was completed, the stirring was continuously performed at a speed of 200 r / min for 10 min, and then the mixture was statically placed for 1 h. The precipitate was obtained by centrifugation at 10,000 rpm for 15 min, and then dried at 45 °C to obtain casein-loaded gibberellin particles;

[0048] C: The sterilized seeds were soaked in a 1 wt% sodium alginate solution for 10 min, then taken out, and then mixed with casein-loaded glucose particles at 50% of the mass of the sterilized seeds, and then dried at 40 °C to obtain primary embedded seeds;

[0049] D: Dissolve hyaluronic acid in water to prepare a 1 wt% hyaluronic acid solution, add 30 g of calcium peroxide to 50 g of the hyaluronic acid solution to uniformly disperse to obtain a hyaluronic acid-calcium peroxide mixed solution, take 30% of the first embedding seed mass of the hyaluronic acid-calcium peroxide mixed solution, uniformly spray the hyaluronic acid-calcium peroxide mixed solution onto the surface of the first embedding seed, and then dry at 40°C to obtain a second embedding seed;

[0050] E: Add 10 g of polyvinyl alcohol to 100 g of dimethyl sulfoxide, heat to 90°C, stir to dissolve, then add 2 g of L-asparagine and 0.1 g of p-toluenesulfonic acid, continue to stir for 3 h, adjust the pH to 7 after the reaction is completed, and then naturally cool to room temperature, and dry at 60°C to obtain modified polyvinyl alcohol; add the modified polyvinyl alcohol to 10 times the mass of water, heat to 90°C, stir to dissolve to prepare 100 g of a modified polyvinyl alcohol solution, add 2 g of 3-aminobenzoic acid after cooling to 40°C, adjust the pH to 7, and stir for 3 h, then naturally cool to room temperature and add 20 g of sodium caseinate to load gibberellin particles to obtain a polyvinyl alcohol composite glue solution, immerse the second embedding seed in the polyvinyl alcohol composite glue solution, stir for 3 min, then take it out and immerse it in a 0.5 wt% borax solution to gel the surface polyvinyl alcohol composite glue solution, then wash it with water for 2 times, and then dry it at 40°C for 30 min to obtain an embedding seed.

[0051] Example 3: Preparation of a three-embedding seed

[0052] A: Dissolve glucose in water to prepare a 40 wt% glucose solution; dissolve sodium caseinate in water to prepare a 5 wt% sodium caseinate solution; mix 50 g of the sodium caseinate solution with 100 g of the glucose solution to obtain a sodium caseinate-glucose mixed solution, then continuously stir the sodium caseinate-glucose mixed solution at a speed of 300 r / min, and continuously add 20 wt% citric acid solution to the stirred sodium caseinate-glucose mixed solution at a rate of 1 drop per second until the pH of the sodium caseinate-glucose mixed solution is adjusted to 4.5, continue to stir at a speed of 300 r / min for 10 min after the addition is completed, then stand for 2 h, centrifuge at 10,000 rpm for 15 min to obtain a precipitate, and dry at 45°C to obtain casein-loaded glucose particles;

[0053] B: 40 g sodium caseinate was dissolved in water to prepare a 5 wt% sodium caseinate solution, 0.2 g gibberellin was added to the sodium caseinate solution and stirred to mix uniformly to obtain a sodium caseinate-gibberellin mixture, while stirring the sodium caseinate-gibberellin mixture at a speed of 300 r / min, 20 wt% citric acid solution was added dropwise to the stirred sodium caseinate-gibberellin mixture at a rate of 1 drop / s until the pH of the sodium caseinate-gibberellin mixture was adjusted to 4.5, after the addition was completed, the stirring was continued at a speed of 300 r / min for 10 min, then it was left to stand for 2 h, and then the precipitate was obtained by centrifugation at 10,000 rpm for 15 min, and the sodium caseinate-gibberellin particles were obtained by drying at 45℃;

[0054] C: The sterilized seeds were soaked in a 1 wt% sodium alginate solution for 30 min, then taken out, and then mixed with sodium caseinate-glucose particles at 50% of the mass of the sterilized seeds, and then dried at 40℃ to obtain the first-embedded seeds;

[0055] D: Hyaluronic acid was dissolved in water to prepare a 1 wt% hyaluronic acid solution, 60 g of calcium peroxide was added to 100 g of the hyaluronic acid solution to disperse uniformly to obtain a hyaluronic acid-calcium peroxide mixture, and the hyaluronic acid-calcium peroxide mixture at 30% of the mass of the first-embedded seeds was uniformly sprayed onto the surface of the first-embedded seeds, and then dried at 40℃ to obtain the second-embedded seeds;

[0056] E: 20 g of polyvinyl alcohol was added to 200 g of dimethyl sulfoxide, heated to 90℃ and stirred to dissolve, then 4 g of L-asparagine and 0.2 g of p-toluenesulfonic acid were added, and the stirring was continued for 4 h, then the pH was adjusted to 7, and then the reaction was allowed to cool to room temperature naturally, and then dried at 60℃ to obtain modified polyvinyl alcohol; the modified polyvinyl alcohol was added to 10 times the mass of water, heated to 90℃ and stirred to dissolve to prepare a 200 g modified polyvinyl alcohol solution, then cooled to 50℃, and then 5 g of 3-aminobenzoic acid was added, the pH was adjusted to 7, and then the reaction was stirred for 4 h, then the reaction was allowed to cool to room temperature naturally, and then 40 g of sodium caseinate-gibberellin particles were added and mixed uniformly to obtain a polyvinyl alcohol composite glue solution, the second-embedded seeds were immersed in the polyvinyl alcohol composite glue solution, stirred and mixed for 5 min, then taken out and placed in a 0.5 wt% borax solution to gel the surface polyvinyl alcohol composite glue, then washed with water for 3 times, and then dried at 40℃ for 30 min to obtain the embedded seeds.

[0057] Comparative Example 1: Preparation of embedded seeds

[0058] In contrast to Example 1, the only difference is that in Comparative Example 1, the original Step A is missing during the preparation of the embedded seeds, that is, the seeds are not embedded with sodium caseinate-glucose particles during the preparation of the embedded seeds, which is shown as follows:

[0059] A: The same as Step B of Example 1;

[0060] B: The sterilized seeds were soaked in 1wt% sodium alginate solution for 20 min, then taken out and dried at 40℃ to obtain the primary embedding seeds;

[0061] C: The same as step D of example 1;

[0062] D: The same as step E of example 1.

[0063] Preparation of embedding seeds

[0064] In comparison with example 1, the only difference is that the step B is missing in the preparation of embedding seeds in comparative example 2, and the gibberellin is directly added instead of being loaded by sodium caseinate in the preparation of embedding seeds, which is shown as follows:

[0065] A: The same as example 1;

[0066] B: The same as step C of example 1;

[0067] C: The same as step D of example 1;

[0068] D: 15g polyvinyl alcohol was added into 150g dimethyl sulfoxide, and after being stirred and dissolved at 90℃, 3g L-asparagine and 0.15g p-toluenesulfonic acid were added, and the reaction was continuously stirred for 3.5h. After the reaction was completed, the pH was adjusted to 7, and then the temperature was naturally cooled to room temperature. After drying at 60℃, modified polyvinyl alcohol was obtained. The modified polyvinyl alcohol was added into 10 times mass of water, and after being stirred and dissolved at 90℃, 2.5g 3-aminobenzoic acid was added after the temperature was cooled to 45℃. After the pH was adjusted to 7, the reaction was stirred for 3.5h. After the reaction was completed, the temperature was naturally cooled to room temperature, and 0.15g gibberellin particles were added and mixed uniformly to obtain polyvinyl alcohol composite glue solution. The secondary embedding seeds were immersed in the polyvinyl alcohol composite glue solution, stirred and mixed for 4min, then taken out and placed in 0.5wt% borax solution to gel the surface polyvinyl alcohol composite glue solution. Then, after being washed with water for 3 times, the embedding seeds were dried at 40℃ for 30min.

[0069] Preparation of embedding seeds

[0070] The same as example 1, the only difference is that in the preparation of embedding seeds in comparative example 3, calcium peroxide is not added in step D, but hyaluronic acid solution is directly used for spraying to prepare secondary embedding seeds, which is shown as follows:

[0071] A~C: The same as example 1;

[0072] D: Hyaluronic acid was dissolved in water to prepare 1wt% hyaluronic acid solution. 30% of the hyaluronic acid solution of the mass of the primary embedding seeds was uniformly sprayed onto the surface of the primary embedding seeds, and then dried at 40℃ to obtain the secondary embedding seeds;

[0073] E: Same as Example 1.

[0074] Comparative Example 4: Preparation of the embedded seeds

[0075] In contrast to Example 1, the only difference is that in Comparative Example 4, the original Step D is missing in the preparation of the embedded seeds, i.e. the secondary embedding of the seeds with the hyaluronic acid-calcium peroxide mixture is not used, which is shown as follows:

[0076] A-C: Same as Example 1;

[0077] D: 15 g of polyvinyl alcohol was added to 150 g of dimethyl sulfoxide, and after being dissolved by stirring at 90°C, 3 g of L-asparagine and 0.15 g of p-toluenesulfonic acid were added, and the stirring reaction was continued for 3.5 h. After the reaction was completed, the pH was adjusted to 7, and then it was naturally cooled to room temperature. After being dried at 60°C, modified polyvinyl alcohol was obtained. The modified polyvinyl alcohol was added to 10 times its mass of water, and after being dissolved by stirring at 90°C, it was cooled to 45°C, and 2.5 g of 3-aminobenzoic acid was added. After the pH was adjusted to 7, the stirring reaction was continued for 3.5 h. After the reaction was completed, it was naturally cooled to room temperature, and 30 g of sodium caseinate loaded with gibberellin particles was added and mixed uniformly to obtain a polyvinyl alcohol composite glue solution. The primary embedded seeds were immersed in the polyvinyl alcohol composite glue solution, and after being stirred and mixed for 4 min, they were taken out and placed in a 0.5 wt% borax solution to gel the surface polyvinyl alcohol composite glue. Then, after being washed with water for 3 times, the embedded seeds were dried at 40°C for 30 min.

[0078] Comparative Example 5: Preparation of the embedded seeds

[0079] In contrast to Example 1, the only difference is that in Comparative Example 5, L-asparagine is not added to modify the polyvinyl alcohol in Step E during the preparation of the embedded seeds, but a conventional polyvinyl alcohol is used instead, which is shown as follows:

[0080] A-D: Same as Example 1;

[0081] E: The polyvinyl alcohol was added to 10 times its mass of water, and after being dissolved by stirring at 90°C, it was cooled to 45°C, and 2.5 g of 3-aminobenzoic acid was added. After the pH was adjusted to 7, the stirring reaction was continued for 3.5 h. After the reaction was completed, it was naturally cooled to room temperature, and 30 g of sodium caseinate loaded with gibberellin particles was added and mixed uniformly to obtain a polyvinyl alcohol composite glue solution. The secondary embedded seeds were immersed in the polyvinyl alcohol composite glue solution, and after being stirred and mixed for 4 min, they were taken out and placed in a 0.5 wt% borax solution to gel the surface polyvinyl alcohol composite glue. Then, after being washed with water for 3 times, the embedded seeds were dried at 40°C for 30 min.

[0082] Comparative Example 6: Preparation of the embedded seeds

[0083] In contrast to Example 1, the only difference is that in Comparative Example 6, no 3-aminobenzenboronic acid is added to treat the polyvinyl alcohol in step E during the preparation of the embedding seed, which is shown as follows:

[0084] A-D: Same as Example 1;

[0085] E: 15 g of polyvinyl alcohol is added to 150 g of dimethyl sulfoxide, and after being stirred and dissolved at 90°C, 3 g of L-asparagine and 0.15 g of p-toluenesulfonic acid are added, and the stirring reaction is continued for 3.5 h. After the reaction is completed, the pH is adjusted to 7, and then it is naturally cooled to room temperature. After being dried at 60°C, the modified polyvinyl alcohol is obtained. The modified polyvinyl alcohol is added to 10 times the mass of water, and after being stirred and dissolved at 90°C, it is naturally cooled to room temperature and 30 g of sodium caseinate is added to load the giberellin particles and mix uniformly to obtain a polyvinyl alcohol composite glue solution. The secondary embedding seed is immersed in the polyvinyl alcohol composite glue solution, and after being stirred and mixed for 4 min, it is taken out and placed in a 0.5 wt% borax solution to gel the surface polyvinyl alcohol composite glue solution. Then, after being washed with water for 3 times, it is dried at 40°C for 30 min to obtain the embedding seed.

[0086] Comparative Example 7: Preparation of embedding seed

[0087] In contrast to Example 1, the only difference is that in Comparative Example 7, during the preparation of the embedding seed, the pH is adjusted to 8 when 3-aminobenzenboronic acid is added for reaction in step E, and the remaining steps are the same as Example 1.

[0088] Example 4: Method for improving the seed germination rate of Fritillaria taipaiensis

[0089] (1) Seed disinfection: select Fritillaria taipaiensis seeds that are free of pests and diseases and have plump particles, immerse them in a 0.1 wt% potassium permanganate solution for 15 min, and then wash them with water for 3 times to obtain disinfected seeds;

[0090] (2) Seed embedding: prepare the disinfected seeds into embedding seeds according to the method of Example 1;

[0091] (3) Stratification treatment: mix sawdust, humus soil, and sand in a mass ratio of 1:1:2, spray them with a 0.1 wt% potassium permanganate solution to disinfect the substrate, and then mix the embedding seeds and the substrate in a mass ratio of 1:5 for variable-temperature stratification treatment. Specifically, first, warm stratification treatment is performed at 20°C for 60 days, and then low-temperature stratification treatment is performed at 4°C for 80 days. During the treatment, the water content of the substrate is maintained at about 65%. After the stratification treatment is completed, the treated seeds are obtained.

[0092] (4) Sowing: sow the treated seeds according to the conventional method, and then perform watering and other management according to the conventional method.

[0093] Experiment: Fritillaria taipaiensis seed sowing experiment

[0094] 1. Germination potential detection experiment of the embedded seeds prepared in Example 1 and Comparative Examples 1-7 after temperature stratification treatment, and field sowing germination rate detection experiment, the experiment was carried out in the Fritillaria taipaiensis planting base of Tangkou Village, Zhutou Town, Taibai County, Shaanxi Province, the experiment was divided into 9 groups: experimental group 1, control group 1-7, blank control group;

[0095] 2. The embedded seeds prepared in Example 1 and Comparative Examples 1-7 were subjected to temperature stratification treatment according to the method in step (3) of Example 4, and the seeds to be sown of experimental group 1, control group 1-7 were obtained; the seeds to be sown of the blank control group were prepared by directly preparing the disinfected seeds according to the method in step (1) of Example 4 and then subjected to temperature stratification treatment according to the method in step (3) of Example 4, and each group was subjected to temperature stratification treatment of 60 days at 20°C and then 80 days at 4°C, and the seeds selected for each group were full and uniform in size;

[0096] 3. Germination potential detection: paper bed was used as germination bed, specifically the culture dish was surface disinfected with 75% ethanol, then 2 layers of filter paper were laid on the bottom of the culture dish, distilled water was added to the culture dish to make the filter paper absorb water, the seeds to be sown were evenly laid in the culture dish, and then the culture dish was placed in a 24°C incubator for germination test, the paper bed needed to be kept moist during the germination period, the germination was based on the embryo root breaking through the seed, the number of germination was recorded regularly, 50 seeds were used for each group of experimental seeds, the germination potential of each group of seeds to be sown was detected on the 10th day, 3 repeated experiments were carried out, and the average data are shown in Table 1;

[0097] 4. Field sowing germination rate detection: the seeds to be sown obtained from experimental group 1, control group 1-7 and blank control group were sown according to the same method, 50 seeds of each group were sown, after 30 days of sowing, the field sowing germination rate of each group of seeds was detected, 3 repeated experiments were carried out, and the average data are shown in Table 1.

[0098] Table 1

[0099] Experiment group 1 Control group 1 Control group 2 Control group 3 Control group 4 Control group 5 Control group 6 Control group 7 Blank control Germination potential (%) 85.3 80.0 82.7 83.3 78.7 84.0 82.0 82.7 68.7 Germination rate (%) 94.7 84.7 88.7 89.3 83.3 90.0 87.3 88.0 78.7

[0100] According to the data analysis of Table 1:

[0101] (1) In the experimental group 1, the seeds are treated by warm temperature stratification for 60 days and low temperature stratification for 80 days, and the germination potential of the seeds reaches 85.3% on the 10th day, and the germination rate reaches 94.7% after 30 days of field sowing. In the blank control group, the seeds are not embedded and the germination potential of the seeds is only 68.7% after 10 days of sowing after sand storage stratification, and the germination rate is only 78.7% after field sowing, which shows that the seeds embedded according to the method of the application can effectively promote the after-ripening of the seeds, and the seeds obtained have high vigor and high germination rate.

[0102] (2) In the control group 1, the seeds are not embedded with casein loaded glucose particles during preparation, which cannot guarantee the efficient release of gibberellin in the later period, resulting in poor physiological after-ripening effect and poor seed vigor, which reduces the germination potential and germination rate. In the control group 2, the seeds are not embedded with sodium caseinate during preparation, but directly added, and the polyvinyl alcohol itself has a slow release property, which leads to a large amount of unembedded gibberellin released in the early stage, reducing the concentration of gibberellin during the physiological after-ripening period, resulting in poor effect and reducing the germination potential and germination rate of the seeds.

[0103] (3) In the control group 3, no calcium peroxide is added during the preparation of the embedded seeds, and the low oxygen concentration in the sand storage leads to slow physiological metabolism of the seeds, incomplete after-ripening, and reduced seed vigor and germination rate. In the control group 4, the embedded seeds are not prepared with a mixture of hyaluronic acid and calcium peroxide, which on the one hand leads to low oxygen concentration in the sand storage, resulting in low physiological metabolism of the seeds, and on the other hand, the lack of barrier property of the hyaluronic acid film leads to rapid release of glucose into the polyvinyl alcohol, which breaks the borate ester bond, making the gibberellin release faster, resulting in low concentration of gibberellin during the physiological after-ripening period, reducing the effect, incomplete seed after-ripening, and obvious reduction in germination potential and germination rate.

[0104] (4) In the control group 5, no L-asparagine is added to modify the polyvinyl alcohol during the preparation of the embedded seeds, and the strong water absorption of the polyvinyl alcohol leads to excessive water absorption of the substrate, which inhibits the completion of the after-ripening process of the embedded seeds. In the control group 6, no 3-aminobenzoic acid is added to treat the polyvinyl alcohol during the preparation of the embedded seeds, which fails to introduce glucose sensitivity, resulting in limited release of gibberellin during the physiological after-ripening process, reducing the effect, incomplete seed after-ripening, and reduced germination potential and germination rate. In the control group 7, the pH of the polyvinyl alcohol and 3-aminobenzoic acid reaction is 8, which forms a gel with a pH that is too high for the environment required by the seeds, inhibiting the after-ripening process of the seeds.

[0105] The above examples are only used to illustrate the technical solutions of the present application but not limit the present application. Although the present application is described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or equivalently replaced without departing from the purpose and scope of the present application, and all should be covered in the scope of the claims of the present application. The technical, shape and structure parts not described in detail in the present application are all known technologies.

Claims

1. A method for improving the seed germination rate of Fritillaria taipaiensis, characterized in that, The method is as follows: (1) seed disinfection: select the seed of Fritillaria taipaiensis without disease and insect pests, and soak the seed in 0.1wt% potassium permanganate solution for 12-15 min, then wash with water for 2-3 times to obtain the disinfected seed; (2) seed embedding: embed the disinfected seed with embedding material to obtain the embedded seed; (3) stratification treatment: mix the embedded seed and the substrate, and then perform the variable-temperature stratification treatment to obtain the seed to be sowed; (4) sowing: sow the seed to be sowed, and then perform the routine method for management.

2. The method for improving the seed sowing survival rate of Fritillaria taipaiensis according to claim 1, characterized in that, The embedding material in step (2) comprises the following raw materials: glucose, sodium caseinate, gibberellin, sodium alginate, hyaluronic acid, calcium peroxide, polyvinyl alcohol, L-asparagine, p-toluenesulfonic acid, 3-aminobenzoic acid, borax.

3. The method for improving the seed sowing survival rate of Fritillaria taipaiensis according to claim 2, characterized in that, The preparation method of the embedded seed is as follows: A: dissolve glucose in water to prepare 40wt% glucose solution; dissolve sodium caseinate in water to prepare 5wt% sodium caseinate solution; mix the sodium caseinate solution with the glucose solution to obtain sodium caseinate-glucose mixed solution, then stir the sodium caseinate-glucose mixed solution at a speed of 200-300r / min, and drop 20wt% citric acid solution into the stirring sodium caseinate-glucose mixed solution at a speed of 1 drop / s until the pH of the sodium caseinate-glucose mixed solution is adjusted to 4.5, continue to stir at a speed of 200-300r / min for 10 min after the dropping is completed, then stand for 1-2h, centrifuge to obtain the precipitate, and dry the precipitate to obtain sodium caseinate-loaded glucose particles; B: dissolve sodium caseinate in water to prepare 5wt% sodium caseinate solution, add gibberellin to the sodium caseinate solution, stir and mix to obtain sodium caseinate-gibberellin mixed solution, then stir the sodium caseinate-gibberellin mixed solution at a speed of 200-300r / min, and drop 20wt% citric acid solution into the stirring sodium caseinate-gibberellin mixed solution at a speed of 1 drop / s until the pH of the sodium caseinate-gibberellin mixed solution is adjusted to 4.5, continue to stir at a speed of 200-300r / min for 10 min after the dropping is completed, then stand for 1-2h, centrifuge to obtain the precipitate, and dry the precipitate to obtain sodium caseinate-loaded gibberellin particles; C: soak the disinfected seed in 1wt% sodium alginate solution for 10-30 min, then take out, and then mix the disinfected seed with 50% sodium caseinate-loaded glucose particles of the mass of the disinfected seed, and dry to obtain the first embedded seed; D: dissolve hyaluronic acid in water to prepare 1wt% hyaluronic acid solution, and disperse calcium peroxide in the hyaluronic acid solution to obtain hyaluronic acid-calcium peroxide mixed solution, take 30% hyaluronic acid-calcium peroxide mixed solution of the mass of the first embedded seed, uniformly spray the hyaluronic acid-calcium peroxide mixed solution on the surface of the first embedded seed, and dry to obtain the second embedded seed; E: add polyvinyl alcohol to dimethyl sulfoxide, stir and dissolve at a temperature of 90℃, then add L-asparagine and p-toluenesulfonic acid, continuously stir for 3-4h, adjust the pH to 7 after the reaction is completed, naturally cool to room temperature, and dry to obtain modified polyvinyl alcohol. The modified polyvinyl alcohol is added into 10 times mass of water, heated to 90℃, stirred and dissolved to prepare a modified polyvinyl alcohol solution, cooled to 40-50℃, then 3-aminobenzoic acid is added, the pH is adjusted to 7, and stirred for 3-4h, then cooled to room temperature, and casein sodium is added to load gibberellin particles, mixed uniformly to obtain a polyvinyl alcohol composite glue solution, the secondary embedding seeds are added into the polyvinyl alcohol composite glue solution, stirred and mixed for 3-5min, then taken out, placed into a 0.5wt% borax solution to gel the surface polyvinyl alcohol composite glue solution, then taken out, washed with water for 2-3 times, and dried at 40℃ for 30min to obtain the embedded seeds.

4. The method for improving the seed sowing survival rate of Fritillaria taipaiensis according to claim 3, characterized in that, The mass ratio of the glucose solution to the casein sodium solution in step A is (80-100):(30-50).

5. The method for improving the seed sowing survival rate of Fritillaria taipaiensis according to claim 4, characterized in that, The mass ratio of the casein sodium to the gibberellin in step B is (20-40):(0.1-0.2).

6. The method for improving the seed sowing survival rate of Fritillaria taipaiensis according to claim 5, characterized in that, The mass ratio of the hyaluronic acid solution to the calcium peroxide in step D is (50-100):(30-60).

7. The method for improving the seed sowing survival rate of Fritillaria taipaiensis according to claim 6, characterized in that, The mass ratio of the polyvinyl alcohol, L-asparagine and p-toluenesulfonic acid in step E is (10-20):(2-4):(0.1-0.2).

8. The method for improving the seed sowing survival rate of Fritillaria taipaiensis according to claim 7, characterized in that, The mass ratio of the modified polyvinyl alcohol solution, 3-aminobenzoic acid and casein sodium loaded gibberellin particles in step E is (100-200):(2-5):(20-40).

9. The method for improving the seed sowing survival rate of Fritillaria taipaiensis according to claim 1, characterized in that, The substrate in step (3) is prepared by mixing sawdust, humus soil and sand at a mass ratio of 1:1:2, then spraying 0.1wt% potassium permanganate solution for sterilization; the mass ratio of the embedded seeds to the substrate is 1:(3-5).

10. The method for improving the seed sowing survival rate of Fritillaria taipaiensis according to claim 9, characterized in that, The temperature-variable stratification treatment method in step (3) is as follows: After the embedded seeds are mixed with the mixed seeds, they are first subjected to warm stratification treatment at 15-20℃ for 60-70 days, then subjected to low-temperature stratification treatment at 4℃ for 80-100 days to obtain the seeds to be sowed, and the substrate moisture content is kept at 60-70% during the treatment.

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