A method for improving the germination rate of stevia seeds

Abstract

The application discloses a method for improving the germination rate of stevia seed, and specifically comprises the following steps: selecting full and healthy stevia seed with consistent size for standby; disinfecting the stevia seed with 10% sodium hypochlorite solution for 5-15 minutes, then rinsing with distilled water for multiple times and filtering dry; preparing different concentrations of salicylic acid to pretreat the stevia seed; taking equal amounts of salicylic acid with different concentrations into at least three portions, and adding equal amounts of the disinfected stevia seed into each portion for seed soaking; after the seed soaking, rinsing with distilled water for 3-5 times, and performing seed germination test by adopting the culture dish filter paper method; starting observation and recording from the day when the stevia seed is placed on the bed; and comparing the germination of the stevia seed. The method for improving the germination rate of stevia seed has the advantages of convenience, high efficiency, non-toxicity, non-residue, low cost, high economic benefit, and wide application prospect in modern efficient agricultural production.

Description

Technical Field

[0001] This invention relates to seed treatment technology in the field of agricultural technology, specifically, to a method for improving the germination rate of stevia seeds by using the plant endogenous hormone salicylic acid as a plant growth regulator. Background Technology

[0002] Stevia rebaudiana (Bertoni) Hemsl., native to subtropical South America, is a perennial herb belonging to the Asteraceae family and the Stevia genus. Steviosides extracted from stevia are hailed as "natural saccharin" and the world's "third sugar source." They are a natural, high-sweetness, low-calorie, novel, green, and health-promoting sweetener, an ideal substitute for sucrose and beet sugar. The glycosides possess excellent anti-cancer activity and can lower blood sugar levels in type II diabetes and control hypertension, showing great potential for development into pharmaceutical and health products. Currently, they are widely used in the food, beverage, pharmaceutical, and daily chemical industries, with a very broad market prospect. Therefore, steviol glycosides, as a novel sugar source, have extremely considerable economic and social value. How to increase stevia yield and glycoside content has become the key to the development of the stevia industry.

[0003] Stevia seeds are achenes, composed of four parts: pappus, germination coat, seed coat, and embryo. The fruit is spindle-shaped, about 3–4 mm long and 0.5–0.8 mm wide. The pericarp is blackish-brown with 5–6 raised, whitish-brown longitudinal stripes, with grooves between the stripes. The pericarp is densely covered with bristles. The apex has 20–22 light brown pappus hairs, with sharp spines on the pappus skin. Stevia seed quality varies greatly, with a generally low failure rate of 25–60%, and in some areas as high as 90%. The seeds are small, with a thousand-seed weight of only 0.25–0.4 grams. The seeds have no dormancy period, small cotyledons, degenerated endosperm, and very little stored nutrients. The outer skin is loose, easily permeable to water and air, leading to increased respiration and loss of vitality. Germination rates are mostly between 23–60%. Stevia seed production suffers from low germination rates, difficulty in emergence, and uneven emergence, severely restricting stevia yield. Seed germination and seedling establishment are critical periods in crop growth, and the quality of seed germination directly affects crop growth and economic benefits. Therefore, there is an urgent need for an effective method to improve the germination rate of stevia seeds in order to increase stevia yield. Summary of the Invention

[0004] In view of this, the present invention provides a method for improving the germination rate of stevia seeds.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A method to improve the germination rate of stevia seeds, the specific steps are as follows:

[0007] (1) Gently rub the stevia seeds by hand to remove the pappus, remove the chaff and impurities by winnowing, select plump, healthy stevia seeds of uniform size, rinse them with distilled water and set aside.

[0008] (2) Disinfect the rinsed stevia seeds with 10% sodium hypochlorite solution for 5-15 minutes, then rinse them several times with distilled water and drain.

[0009] (3) Prepare salicylic acid at different concentrations to pretreat stevia seeds; the concentrations of the salicylic acid are 0, 0.03, 0.05, 0.10, 0.50, and 1.00 mmol / L.

[0010] (4) Take at least three equal amounts of salicylic acid of different concentrations, add equal amounts of sterilized stevia seeds, and soak the seeds in a 25℃ constant temperature water bath for 12h, 24h, and 36h respectively.

[0011] (5) After soaking the seeds, rinse them 3 to 5 times with distilled water and conduct seed germination tests using the petri dish filter paper method;

[0012] (6) Germination index determination: observation and recording began from the day the stevia seeds were placed in the seedbed; after the soaking was completed, the seeds of each group were evenly sown onto a culture dish with more than 3 layers of filter paper. The filter paper was moistened with distilled water. 100 seeds were sown in each dish. Water was sprayed every day to keep the moisture content of the filter paper at 65% to 75%. After the dish was covered, it was placed in an artificial constant temperature incubator for cultivation. The cultivation temperature was 25℃, the humidity was 85%, the light intensity was 12h / d, and the light intensity was 1250Ix. Each treatment was repeated at least 3 times, and germination was carried out in a constant temperature incubator with humidity.

[0013] (7) Compare the germination of stevia seeds: the germination standard is that the sprout length exceeds half the seed length. The number of sprouts is recorded at regular intervals every day. The germination period begins when the first seed sprouts. The number of sprouted seeds is recorded every day thereafter. If no seeds sprout for 3 to 5 consecutive days, it is considered as no germination and the germination period ends. The germination rate is calculated on the 7th day.

[0014] As can be seen from the above technical solution, compared with the prior art, the present invention discloses a method for improving the germination rate of stevia seeds. The present invention uses salicylic acid (SA) solutions of different concentrations to pretreat seeds. The pretreatment method is simple, easy to operate, and pollution-free, and can significantly improve the germination rate, germination potential, germination index, and vigor index of stevia seeds. Salicylic acid maintains the stability of the cell membrane structure of stevia seeds by increasing the content of soluble proteins, soluble sugars, and proline and other osmotic substances in stevia seeds, activating the ROS scavenging enzyme system, and regulating the activity of α-amylase, thereby alleviating the inhibitory effect of adverse conditions on the germination and seedling growth of stevia seeds to a certain extent, improving the stress resistance of stevia seeds and seedlings, and promoting the germination of stevia seeds and seedling growth. The method for improving the germination rate of stevia seeds adopted in the present invention has the advantages of being convenient, efficient, non-toxic, residue-free, and low-cost, with high economic benefits, convenient for agricultural production, and has broad application prospects in modern high-efficiency agricultural production. Detailed Implementation

[0015] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0016] Example 1

[0017] Example 1: Determining the optimal concentration and best pretreatment time

[0018] Gently rub the stevia seeds by hand to remove the pappus, and use winnowing to remove chaff and impurities. Select plump, healthy stevia seeds of uniform size, weigh out 20g of seeds into 54 portions, rinse each portion with distilled water 4 times, and rinse thoroughly. Then disinfect with 10% sodium hypochlorite solution for 10 minutes, rinse with distilled water 3 times, and drain.

[0019] Different concentrations of salicylic acid were prepared to pretreat stevia seeds. The concentrations of salicylic acid were 0, 0.03, 0.05, 0.10, 0.50, and 1.00 mmol / L. 100 mL of salicylic acid of different concentrations was taken in 3 (3 replicates) x 3 (3 pretreatment times) portions, and an equal amount of sterilized stevia seeds was added to each portion. The seeds were soaked in a constant temperature water bath at 25℃ for 12, 24, and 36 h, respectively. After soaking, the seeds were rinsed 3 times with distilled water.

[0020] Seed germination experiments were conducted using the petri dish filter paper method: After soaking, seeds from each group were evenly sown onto petri dishes lined with three layers of filter paper. The filter paper was moistened with distilled water. 100 seeds were sown in each dish. Water was sprayed daily to maintain the moisture content of the filter paper at 65%–75%. The dishes were then covered and placed in a constant temperature incubator for germination while maintaining humidity. The incubation temperature was 25℃, humidity was 85%, light intensity was 12h / d, and light intensity was 1250 Ix.

[0021] Germination index determination was carried out from the day the stevia seeds were placed in the seedbed. Germination was defined as the sprout length exceeding half the seed length. The number of germinated seeds was recorded daily. The germination of the first seed was the beginning of germination. The number of germinated seeds was recorded daily thereafter. Seeds that did not germinate for 3-5 consecutive days were considered to have not germinated and were considered to have ended germination. The germination rate was calculated on the 7th day. The results are shown in Table 1.

[0022] Table 1. Germination rate of stevia seeds

[0023]

[0024] As shown in Table 1, the germination rates of stevia seeds were similar when soaked in 0.05 mmol / L salicylic acid for 24 h and 36 h. Based on the principle of saving time, soaking in 0.05 mmol / L salicylic acid for 24 h was chosen.

[0025] Example 2

[0026] Two thousand stevia seeds of the Huinong No. 3 variety, propagated by the Gansu Provincial Academy of Agricultural Engineering Technology, were randomly divided into 1,000 equal portions. One thousand 1,000 seeds were soaked in a 0.05 mmol / L salicylic acid solution; the other thousand 1,000 seeds served as a control, soaked in water at a 1:10 ratio at 25°C for 24 hours, then rinsed thoroughly and air-dried. Seed germination was tested using the petri dish filter paper method. The treated seeds were evenly sown onto the filter paper in petri dishes (90 mm in diameter) moistened with distilled water to a moisture content of 60% and lined with three layers of filter paper. Each dish contained 100 seeds, with three replicates. The filter paper was kept moist by spraying water once daily. The dishes were then covered and placed in a constant temperature incubator. The incubation conditions were 25°C, 85% humidity, 12 h / d light, and 1250 Ix light intensity. Germination was carried out under constant temperature and humidity conditions in the incubator. Record the number of seeds germinated daily for each treatment and calculate the germination rate using the following formula:

[0027] Germination rate = (SN1 / SN0) × 100%,

[0028] Where: SN1 – number of germinated seeds; SN0 – total number of seeds tested;

[0029] The results are shown in Table 2.

[0030] Table 2

[0031]

[0032] As shown in Table 2, soaking seeds in a 0.05 mmol / L salicylic acid solution for 24 hours can significantly improve the germination rate of stevia seeds.

[0033] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method of increasing the germination rate of Stevia rebaudiana seeds, characterized by, The specific steps are as follows: (1) Rub the stevia seed with hands, remove the pappus, remove the chaff and impurities by air selection, select the full and healthy stevia seed with uniform size, rinse clean with distilled water, and reserve; (2) Disinfect the rinsed stevia seed with 10% sodium hypochlorite solution for 5-15 minutes, rinse with distilled water for multiple times, and filter dry; (3) Prepare different concentrations of salicylic acid for pretreatment of the stevia seed; (4) Take at least 3 portions of equal amount of salicylic acid with different concentrations, add equal amount of the disinfected stevia seed, and immerse in a constant temperature water bath pot at 25 DEG C for 12h, 24h and 36h respectively; (5) After the seed immersion, rinse with distilled water for 3-5 times, and perform seed germination test by using the petri dish filter paper method; (6) Perform germination index determination, and start observation and recording from the day when the stevia seed is placed on the bed; (7) Compare the germination of the stevia seed; The concentration of the salicylic acid in step (3) is 0, 0.03, 0.05, 0.10, 0.50 and 1.00 mmol / L; The specific steps of the petri dish filter paper method for seed germination test in step (5) are as follows: evenly sow the seed after seed immersion on the petri dish with more than 3 layers of filter paper, the filter paper is moistened with distilled water, sow 100 seeds per dish, spray water every day, keep the water content of the filter paper at 65%-75%, cover the dish cover, and place in an artificial constant temperature box for culture; the culture temperature is 25 DEG C, the humidity is 85%, the light illumination is 12h / d, and the light intensity is 1250Ix; repeat each treatment at least 3 times, and keep moist in the constant temperature box for germination; The specific method of the germination index determination in step (6) is as follows: take the standard of the bud length exceeding half of the seed length as the germination standard, record the germination number at fixed time every day, the first seed germination is the seed germination initial stage, record the germination seed number every day, continuously for 3-5 days, and take the non-germination as non-germination, which is the germination end stage; record the germination rate on the 7th day.

Images