A reagent formula and supporting method for breaking deep dormancy in perilla seeds

Abstract

This invention relates to a method for breaking the deep dormancy of perilla seeds, comprising the following steps: a cold treatment step, wherein the cold treatment step involves placing the perilla seeds at 0-10°C for 1-8 days; preferably, placing the perilla seeds at 4°C for 5 days; and a gibberellin soaking step, wherein the gibberellin soaking step involves soaking the cold-stored deep dormant perilla seeds in a gibberellin solution of 30-120 mg / L for 4-10 hours; preferably, soaking the cold-stored perilla seeds in a gibberellin solution of 50-100 mg / L for 6-10 hours; and more preferably, soaking the cold-stored perilla seeds in a gibberellin solution of 100 mg / L for 7 hours. The beneficial effects of this invention include the ability to treat perilla seeds with different dormancy levels, thus preserving the intrinsic quality of the perilla seeds to the greatest extent possible. This invention also provides a reagent for breaking the deep dormancy of perilla seeds.

Description

Technical Field

[0001] This invention belongs to the field of seed treatment technology, specifically relating to a reagent formula and supporting method for breaking deep dormancy in perilla seeds. Background Technology

[0002] Perilla (Perilla frutescens (L.) Britt.) is an annual herb belonging to the genus Perilla in the family Lamiaceae. Perilla seeds exhibit dormancy. In recent years, with the increasing demand for different types of perilla, year-round production and the cultivation of high-quality new varieties have received increasing attention. However, the dormancy characteristic of perilla seeds poses a significant obstacle and limitation to off-season cultivation (multiple cultivations throughout the year) and variety selection. Seeds with different depths of dormancy extend the breeding cycle and increase the difficulty of variety selection to varying degrees. Only by breaking the dormancy of perilla seeds can the required breeding cycle be significantly shortened, making the southern propagation and generation of perilla possible. Known technologies have proposed methods to break or remove the dormancy of perilla seeds. For example, Chinese patent CN112956303A discloses a reagent and application method for breaking perilla seed dormancy, which disinfects the seed coat. However, the broad applicability of the selected seeds (test samples) is questionable, and the minimum germination rate is still above 20%. Chinese patent CN107006161A discloses a method for breaking the dormancy of perilla seeds. This method requires cinnamon filtrate, centrifuged pig blood liquid, and calcium hydroxide reagent. The preparation process also requires adjusting pressure, pH, and centrifugation, making it neither applicable nor practical. Its effectiveness on deeply dormant perilla seeds is also unsatisfactory. Furthermore, the known technology has at least the following problems: 1. It uses sodium hypochlorite, which is harmful to humans, for disinfection; however, sodium hypochlorite is a carcinogen listed by the International Agency for Research on Cancer (IARC) of the World Health Organization; 2. Excessive breaking of dormancy affects the quality of perilla; 3. No corresponding method for breaking dormancy based on the different dormancy depths of perilla seeds has been found; 4. No experiments have been conducted on perilla seeds with all dormancy depths, and the effect on deeply dormant perilla germplasm is unsatisfactory. Summary of the Invention

[0003] To address at least one of the above-mentioned technical problems, one aspect of the present invention relates to a method for breaking the deep dormancy of perilla seeds, the method comprising the following steps:

[0004] The cold treatment step involves storing the perilla seeds at 0-10°C for 1-8 days; preferably, the perilla seeds are stored at 4°C for 5 days.

[0005] The gibberellin soaking step involves soaking the deeply dormant perilla seeds in 30-120 mg / L gibberellin for 4-10 hours; preferably, soaking the perilla seeds in 50-100 mg / L gibberellin for 6-10 hours; and more preferably, soaking the perilla seeds in 100 mg / L gibberellin for 7 hours.

[0006] A further method for breaking deep dormancy in perilla seeds includes a pretreatment step prior to the cold treatment step, the pretreatment step comprising:

[0007] First, break down the waxy layer of the perilla seeds with fine sand; preferably, mix the perilla seeds with fine sand thoroughly and knead or vortex the waxy layer of the perilla seeds.

[0008] The perilla seeds are then buried in moist fine sand; preferably, the perilla seeds are buried in dry fine sand, and an appropriate amount of water is added to keep the sand moist; more preferably, the amount of water added is such that there is no flowing water when tilted to the side.

[0009] A further method for breaking the deep dormancy of perilla seeds includes a collection and preservation step before the pretreatment step, the collection and preservation step comprising:

[0010] Select mature, healthy plants free from pests and diseases for seed collection; when most of the seeds are mature and the fruits have hardened and changed color, harvest the fruit spikes manually or mechanically or harvest the whole plant. After the morning dew has dissipated, harvest all at once and transport them to the site to dry in the sun. Threshing and cleaning, removing shriveled fruits and other impurities, then pack them in paper or cloth and store them in a dry, low-temperature environment.

[0011] A further method for breaking the deep dormancy of perilla seeds includes an ultrasonic treatment step between the cold treatment step and the gibberellin soaking step, the ultrasonic treatment step comprising:

[0012] Select 40kHz-60kHz ultrasonic treatment for 60-120 minutes.

[0013] A further method for breaking the deep dormancy of perilla seeds includes a germination step following the gibberellin soaking step, the germination step comprising:

[0014] After soaking the perilla seeds in gibberellin, place them in double-layered filter paper or gauze and germinate them under conditions of 20-25℃, 50-75% humidity, and 8-10 hours of light / 14-16 hours of darkness.

[0015] A further method for breaking deep dormancy in perilla seeds is characterized by comprising a PEG treatment step following the gibberellin soaking step, wherein the PEG treatment step comprises soaking perilla seeds in a PEG2000 solution of 30-120 mg / L for 6-8 hours.

[0016] A further method for breaking the deep dormancy of perilla seeds includes a dormancy degree assessment step following the cold treatment step. The dormancy degree assessment step includes:

[0017] Take a portion of the perilla seeds that have been cold-stored for 3 days from the cold treatment step and conduct a germination test. Calculate the germination rate by the degree of white sprouting of the perilla seeds. If the germination rate is greater than 85% after 1-2 days, stop cold storage of the other portion of seeds from the cold treatment step and do not need to do any further treatment steps.

[0018] A further method for breaking the deep dormancy of perilla seeds, wherein the reagent is a 30-120 mg / L gibberellin solution, the gibberellin solution containing a small amount of ethanol;

[0019] Preferably, the method for preparing the gibberellin solution is as follows: dissolve the gibberellin in a small amount of high-concentration ethanol, and then add purified water to make up to the required volume.

[0020] A further method to break the deep dormancy of perilla seeds is to soak them in a 100 mg / L PEG2000 solution for 6-8 hours.

[0021] The final aspect of this invention also provides corresponding treatment methods for perilla seeds with different dormancy depths.

[0022] The beneficial effect of this invention is that different treatments are applied to perilla leaves with different dormant states, which preserves the intrinsic quality of perilla to the greatest extent possible. Attached Figure Description

[0023] Figure 1 The effect of gibberellin as a single factor on the germination of perilla seeds at different dormancy depths;

[0024] In the figure, the vertical axis represents the germination rate, the horizontal axis represents the gibberellin concentration, and CK is the blank control.

[0025] Figure 2 Effects of combined treatments on the germination of perilla seeds at different dormancy depths;

[0026] Figure 3 Comparative photographs of water-treated and gibberellin-treated perilla germplasm;

[0027] Figure 4 for Figure 3 Photo of the seedlings being transplanted into a nutrient pot;

[0028] Figure 5 and Figure 6 Photographs of perilla germplasm treated with mycotoxin;

[0029] Figure 7 Photographs of perilla seed treatment according to an embodiment of the present invention. Detailed Implementation

[0030] The following embodiments further illustrate the content of the present invention, but should not be construed as limiting the present invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the present invention are within the scope of the present invention.

[0031] the term

[0032] Unless otherwise specified, the term "seed" in this invention refers to "germ material".

[0033] In this invention, the term "germstone" includes "seed".

[0034] The terms "seed" and "germ" in this invention may sometimes be used interchangeably.

[0035] Test Material Description

[0036] Perilla (Perilla frutescens (L.) Britt.) is an annual herb belonging to the genus Perilla in the family Lamiaceae. Perilla has a wide range of uses as both a food and a medicine. Medicinally, perilla leaves, stems, and seeds are the main medicinal materials. Edible uses include fresh perilla leaves for wrapping roasted meat, perilla leaves as a side dish in seafood cooking, perilla sprouts, perilla seeds as filling for mooncakes, perilla seeds for birdwatching, and perilla seeds as a barbecue seasoning. In addition, perilla has extensive industrial applications, serving as a cosmetic ingredient, a source of natural pigments, a high-sweetness sweetener (2000 times sweeter than sucrose), and a natural preservative.

[0037] Significant differences exist in the biological characteristics and growth habits of perilla seeds for different uses. Even based solely on appearance, the dormancy depth of perilla seeds of different sizes and color intensities varies significantly. Some seeds, especially those with larger diameters, exhibit almost no dormancy (for example, some seeds with a diameter of 1 mm or 1.5 mm germinate very quickly), while some germplasm materials exhibit dormancy depths ranging from 3 to 6 months. This invention utilizes representative perilla germplasm materials from across the country with different dormancy depths, namely perilla germplasm 1, perilla germplasm 2, perilla germplasm 3, perilla germplasm 4, and perilla germplasm 5, as detailed in the table below:

[0038] Perilla germplasm 1 Chongqing none Greater than 2mm White Double-sided green Perilla germplasm 2 Hebei shallow Greater than 2mm gray brown Single-sided purple Perilla germplasm 3 Anhui middle Approximately 1.5mm gray brown Single-sided purple Perilla germplasm 4 Zhejiang Extremely deep Approximately 1.5mm grayish brown Double-sided purple Perilla germplasm 5 Guangdong Deeper Less than 1mm grayish brown Single-sided purple

[0039] Existing technologies provide methods for processing perilla germplasm. Figure 3The image shows comparative photos of perilla treated with water and perilla treated with gibberellin. The top three are perilla germplasm materials treated with water, and the bottom ones are perilla germplasm materials treated with gibberellin, corresponding to the top three. It can be seen that the stems at the bottom are excessively elongated. Combined with... Figure 4 As shown, when transplanted into a nutrient pot, its stem is very long, thin, and weak. Figure 5 and Figure 6 Photographs of perilla germplasm treated with cypermethrin; it can be seen that after cypermethrin treatment, deformed and abnormally developed perilla plants appeared.

[0040] Existing technologies do not address the core characteristics of perilla seed dormancy. They can be simply summarized as "different germplasms, different degrees, different depths," applying the same method to seeds with varying dormancy depths without differentiation. This easily leads to the following problems: 1. Because different germplasms have different dormancy depths, the required reagent amounts and conditions differ. The gibberellin concentration described in existing technologies is not a guiding or reference value. Some technical solutions in this invention propose corresponding treatment methods for perilla seeds with different dormancy depths, maximizing the preservation of the perilla's intrinsic quality.

[0041] Test case

[0042] Experiment Example 1 studied the effect of single-factor gibberellin on perilla germplasm.

[0043] 1. Experimental materials: Perilla germplasm 1-5

[0044] 2. Reagents: Gibberellin, purified water, a small amount of ethanol (or liquor), filter paper, petri dishes, etc.

[0045] 3. Five concentration gradients were set for the reagent: 0 mg / L, 50 mg / L, 100 mg / L, 150 mg / L, and 200 mg / L;

[0046] 4. Preparation method: Dissolve gibberellin powder of different masses in a small amount of 75% ethanol (or white wine) until dissolved, and then add purified water to make up to the required volume.

[0047] 5. Experimental Methods: Perilla frutescens germplasm seeds (1-5) at different dormancy depths were set up in triplicate and soaked in gibberellin solutions of different concentrations (0 mg / L, 50 mg / L, 100 mg / L, 150 mg / L, 200 mg / L) for 6-8 hours. After rinsing with purified water, the seeds were placed in petri dishes lined with double layers of filter paper for several days. The number of germinating seeds and the germination rate were observed and recorded. Germination rate (%) = number of germinating seeds / total number of seeds;

[0048] 6. Test Results: See Figure 1 As shown.

[0049] (1) Some perilla seeds do not have dormancy and do not require additional treatment. For example, perilla germplasm 1 has a germination rate of more than 98%.

[0050] (2) Single gibberellin treatment has an effect on the germination rate of perilla seeds, and the overall trend is first rising, then falling and then rising again. However, single gibberellin treatment has a limited effect on breaking dormancy in most perilla germplasm seeds.

[0051] Experiment Example 2: Effects of Cold Treatment on Perilla Germplasm

[0052] 1. Experimental materials: Perilla germplasm 1-5

[0053] 2. Reagents: purified water, filter paper, petri dishes, etc.

[0054] 3. Experimental Methods: Perilla frutescens germplasm seeds (1-5) at different dormancy depths were set up in three replicates and soaked in purified water. They were then placed in a refrigerator for 0, 3, and 5 days of cold treatment. After removal, they were placed in petri dishes lined with double layers of filter paper for several days. The number of germinating seeds and the germination rate were observed and recorded. Germination rate (%) = number of germinating seeds / total number of seeds;

[0055] 4. Test results: See Table 1.

[0056] 5. Results Analysis

[0057] (1) The cold treatment step significantly improved the dormancy breaking of perilla germplasm 1, perilla germplasm 2, perilla germplasm 3 and perilla germplasm 5.

[0058] (2) The cold treatment step had no effect on breaking the dormancy of the deep dormancy of the Perilla germplasm 4.

[0059] Table 1: Effects of single-factor cold treatment on germination of perilla seeds at different dormancy depths

[0060]

[0061] Experimental Example 4: Combined treatment has a greater impact than single cold treatment.

[0062] 1. Experimental materials: Perilla germplasm 1-5

[0063] 2. Reagents: Gibberellin, purified water, a small amount of ethanol (or liquor), filter paper, petri dishes, etc.

[0064] 3. Experimental Method: Perilla frutescens germplasm seeds of different dormancy depths were divided into three replicates. The seeds were soaked in purified water and then placed in a refrigerator for 3 days (4℃). After removal, they were soaked in a 100 mg / L gibberellin solution for 6-8 hours, rinsed with purified water, and placed in petri dishes lined with double layers of filter paper for several days. The number of germinating seeds and the germination rate were observed and recorded. Germination rate (%) = number of germinating seeds / total number of seeds;

[0065] 4. Test Results: See Figure 2 As shown.

[0066] 5. Results Analysis:

[0067] (1) Cold treatment + gibberellin treatment can increase the germination rate of perilla germplasm 2, perilla germplasm 3, perilla germplasm 4 and perilla germplasm 5 by about 10%-25%.

[0068] (2) In an optional embodiment, placing perilla seeds at 0-10°C for 1-8 days also achieved similar technical effects.

[0069] Experimental Example 5: Combined Formula Enhances Highly Dormant Germplasm

[0070] 1. Experimental materials: Perilla germplasm 4

[0071] 2. Reagents: Gibberellin, PEG 2000 (dissolved in 80-90°C warm water), purified water, a small amount of ethanol (or white wine), filter paper, petri dishes, fine sand, etc.

[0072] 3. Experimental Methods: Deeply dormant Perilla frutescens germplasm 4 seeds were treated according to the methods shown in Table 2, with three replicates for each treatment. After treatment, the seeds were washed with purified water and placed in petri dishes lined with double-layered filter paper for several days. The number of germinated seeds and the germination rate were observed and recorded. Germination rate (%) = number of germinated seeds / total number of seeds; average germination rate (%) = Σ germination rate (%) / number of replicates; fertilization factor = average germination rate (%) / original germination rate (%); original germination rate (%) = number of germinated seeds / total number of seeds.

[0073] Table 2. Effects of formulation combinations on germination of deeply dormant perilla seeds.

[0074] Method 1 cs+c+g100, 6-8h 37.50 6.25 Method 2 sm+c+g100, 6-8h 34.17 5.69 Method 3 c+cs+p100, 6-8h 19.17 3.19 Method 4 c+cs+g100, 6-8h 50.00 8.33 Method 5 c+sm+g100, 6-8h 45.83 7.64

[0075] Wherein, sm represents the sand milling step, 6-8h represents the soaking step (gibberellin or PEG2000 soaking step); cs represents the ultrasonic step (40kHz-60kHz ultrasonic treatment, treatment for 60min-120min); c represents the cold treatment step (perilla seeds are placed at 4℃ for 5 days); p100 represents the treatment step with PEG2000 at a concentration of 100mg / L, and g100 represents the treatment step with gibberellin solution at a concentration of 100mg / L.

[0076] 4. Experimental Results and Analysis

[0077] (1) The germination rate of perilla germplasm 4 obtained by methods 4-5 is increased by 7-8 times, which is better than that of methods 1-3, among which method 4 is the best embodiment. The embodiments of the present invention are easier to implement and more operable.

[0078] (2) The difference between Method 4 and Method 1 is that the order of ultrasonic treatment and cold treatment is different, indicating that the first cold treatment has a greater impact on the germination rate.

[0079] (3) The difference between Method 4 and Method 2 is that Method 2 involves sand milling but not ultrasonic treatment. The difference between Method 4 and Method 3 is that Method 3 uses PEG2000 instead of gibberellin. The difference between Method 4 and Method 5 is that Method 5 uses sand milling instead of ultrasonic treatment. This indicates that sand milling, ultrasonic treatment and gibberellin have a synergistic effect on germination rate.

[0080] Experimental Example 6: Comparative Experiment

[0081] Perilla germplasm 1-5 were used, and the experiment was repeated using the method described in "202110125215.6 A reagent and application method for breaking the dormancy of perilla seeds". The gibberellin concentration was 200 mg / L. For experimental conditions not mentioned, refer to Experiment Example 1.

[0082] Experimental results: For perilla germplasm 1 and 2, excessive dormancy disruption treatment affected the quality of the perilla. For perilla germplasm 3-5, the average germination rate increase was only around 4 times.

[0083] Experiment 7 employed continuously upgraded supporting methods for germplasm at different dormant depths.

[0084] Experimental methods: 136 perilla germplasm accessions collected nationwide were tested;

[0085] Step 1: Pre-treatment to break dormancy of perilla seeds: Thoroughly mix and knead perilla seeds with fine sand, then vortex to break the waxy layer on the outer seed coat. Bury the perilla seeds in dry fine sand, add an appropriate amount of water to keep the sand moist, ensuring that there is no flowing water when tilted to one side. Store in a refrigerator at 4℃.

[0086] Step 2: Identification of dormancy level of perilla seeds: A germination test was conducted on a small number of perilla seeds that had been stored in a refrigerator at 4℃ for 3 days after step 2. After 1-2 days, the germination rate was calculated based on the degree of white sprouting of the perilla seeds. If the germination rate was greater than 85%, the seeds were removed and no further treatment was required. If the germination rate was less than 85%, further testing was conducted.

[0087] Step 3: Further treatment of deeply dormant seeds: If the germination rate in Step 3 is less than 85%, take out the perilla seeds after 5 days, wash them, and treat them with ultrasound at 40kHz-60kHz for 60-120 minutes. Then soak the perilla seeds in 50-100mg / L gibberellin for 6-10 hours.

[0088] Step 4: Germination of Deep Dormant Seeds: Germinate the perilla seeds from Step 3 by placing them in a breathable mesh bag and germinating them under conditions of 20-25℃ temperature, 50-75% humidity, and 8-10 hours of light / 14-16 hours of darkness.

[0089] Experimental results: Seeds with a diameter greater than 2mm accounted for approximately 20%, all of which germinated (100%); seeds with a diameter of around 1.5mm accounted for 50%, also all of which germinated (100%); and seeds with a diameter less than 1mm accounted for 30%, and all but two samples germinated, possibly due to poor seed quality. This demonstrates that our technical process is effective for almost all germplasm types. Figure 7 As shown, this method preserves the intrinsic quality of perilla to the greatest extent possible and is not affected by external factors (such as hormones like gibberellin).

[0090] Although the present invention has been described in detail above with general descriptions, specific embodiments, and experiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for selecting and treating seeds of perilla, characterized in that, The method includes the following steps: Collection and preservation steps: Select mature, healthy plants free from pests and diseases for seed collection; when most of the seeds are mature and the fruits have hardened and changed color, harvest the fruit spikes manually or mechanically or harvest the whole plant. After the morning dew has dissipated, harvest all at once and transport them to the site to dry. Threshing and cleaning, removing shriveled fruits and other impurities, then pack them in paper or cloth and store them in a dry, low-temperature environment. Step 1: Pre-treatment of perilla seeds to break dormancy: Mix perilla seeds thoroughly with fine sand, knead and vortex to break the waxy layer on the outer layer of the seed coat; bury perilla seeds in dry fine sand, add an appropriate amount of water to keep the sand moist, the amount of water should be such that there is no flowing water when tilted to the side; and store in a refrigerator at 4℃. Step 2: Identification of dormancy level of perilla seeds: A germination test was conducted on a small number of perilla seeds that had been stored in a 4℃ refrigerator for 3 days after step 1. After 1-2 days, the germination rate was calculated based on the degree of white sprouting of the perilla seeds. If the germination rate was greater than 85%, the seeds were removed without further treatment. If the germination rate was less than 85%, further tests were conducted. Step 3: Further treatment of deeply dormant seeds: If the germination rate in Step 2 is less than 85%, take out the perilla seeds that have been stored in a 4℃ refrigerator for 5 days, wash them, and treat them with 40kHz-60kHz ultrasound for 60min-120min; then soak the perilla seeds in 50-100mg / L gibberellin for 6-10 hours. Step 4: Germination of Deep Dormant Seeds: Germinate the perilla seeds from Step 3 by placing them in a breathable mesh bag and keeping them at a temperature of 20-25℃, humidity of 50-75%, and 8-10 hours of light / 14-16 hours of darkness.

2. The method as described in claim 1, characterized in that, The breathable mesh bag is made of double-layered filter paper or gauze.

3. The method as described in claim 2, characterized in that, The gibberellin soaking step is followed by a PEG treatment step, which includes soaking perilla seeds in a 30-120 mg / L PEG2000 solution for 6-8 hours.

4. The method as described in claim 3, characterized in that, The gibberellin solution contains a small amount of ethanol.

5. The method as described in claim 4, characterized in that, The method for preparing the gibberellin solution is as follows: dissolve the gibberellin in a small amount of high-concentration ethanol, and then add purified water to make up to the required volume.

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