A priming method for improving the germination ability of Xinjiang Lithospermum officinale seeds

Abstract

The invention discloses a priming method for improving the germination ability of Xinjiang lithospermum seeds. The invention belongs to the field of biotechnology, and specifically relates to a priming method for improving the germination ability of Xinjiang lithospermum seeds. The method for improving the germination ability of Xinjiang lithospermum seeds of the present invention comprises the following steps: soaking Xinjiang lithospermum seeds in an initiator to obtain seeds after initiation treatment by the initiator. Specifically, Xinjiang lithospermum seeds can be soaked in 100-200 μmol / L melatonin solution or 0.01-0.1 μmol / L brassinolide solution under dark conditions at 25°C for 24 hours, and then dried to the original weight. The above treatment can improve the germination ability of Xinjiang lithospermum seeds, and has certain application prospects.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for improving the germination ability of Xinjiang lithospermum officinale seeds. Background Art

[0002] Xinjiang lithospermum [Arnebia euchroma (Royle) Johnst] is a perennial herbaceous plant of the Boraginaceae family. Its dried root is a traditional Chinese medicinal material with extensive medicinal value, which has pharmacological effects such as antibacterial, anti-inflammatory, and inhibition of tumor cell growth. The wild population of Xinjiang lithospermum is mainly distributed in Xinjiang, with a small amount distributed in western Tibet, and the distribution range is narrow. In recent years, wild resources have shown a sharp decline year by year. In 2022, Xinjiang lithospermum was listed as a national second-level endangered protected plant. As lithospermum medicinal materials mainly rely on imports, artificial breeding is particularly urgent. Xinjiang lithospermum is mainly propagated by seeds. Studies have shown that Xinjiang lithospermum has a low fruiting rate under natural conditions, resulting in a small number of seeds and high prices. In addition, the cultivation of Xinjiang lithospermum also faces the problem of low germination rate. Under suitable experimental conditions, the germination rate is as high as 67.33%. There are many factors that affect the germination ability of seeds, including intrinsic factors such as the quality of the seeds themselves, such as embryo vitality, and external factors such as moisture, light, temperature and humidity during the germination process. There is no report on the research of Xinjiang Lithospermum officinale seed initiator.

[0003] Seed priming technology is widely used in agricultural production. It is an effective seed pre-sowing treatment technology in agricultural production, which can promote seed germination and solve problems such as slow and uneven seed emergence. The development of priming technology can greatly save seed usage, reduce costs, and contribute to the development and popularization of precision seeding. However, there is little research on the priming technology of Xinjiang Lithospermum officinale seeds. Summary of the invention

[0004] The main problem to be solved by the invention is how to improve the germination ability of Xinjiang Lithospermum officinale seeds.

[0005] In order to solve the above problems, the present invention provides a method for improving the germination ability of Xinjiang Lithospermum officinale seeds.

[0006] The method for improving the germination ability of Xinjiang lithospermum seeds provided by the invention comprises the following steps: soaking the Xinjiang lithospermum seeds in an initiator to obtain seeds treated with the initiator.

[0007] In the above method, the initiator may be melatonin.

[0008] Furthermore, the melatonin may be used at a concentration of 100-200 μmol / L.

[0009] In the above method, the initiator may be brassinolide.

[0010] Furthermore, the brassinolide may be used at a concentration of 0.01-0.1 μmol / L.

[0011] In the above method, the soaking time is 24 hours and the soaking temperature is 25°C.

[0012] In the above method, the immersion condition is a dark condition.

[0013] In the above method, the germination ability of the Xinjiang Lithospermum officinale seeds is expressed as germination rate and / or germination potential.

[0014] The evaluation standard of the germination rate is germination standard 1 or germination standard 2:

[0015] The germination standard 1 is that the seeds germinate from the first day after being placed on the paper bed, and the whitening ratio of seeds is 1-2 mm as the germination standard 1. The observation and record are made once every 24 hours, and the number of seeds germinated on that day is recorded. The whitening ratio of seeds is used as the statistical standard 1, and the recording period is 15 days.

[0016] The germination standard 2 is to observe and record once every 24 hours from the 8th day after being placed on the paper bed, and record the number of normal seedlings, abnormal seedlings, hard and solid, fresh non-germinated seeds and dead seeds. The recording standards for normal seedlings, abnormal seedlings, hard and solid, fresh non-germinated seeds and dead seeds are the same as Appendix A of GB / T 3543.4-1995 "Germination Test of Crop Seed Inspection Procedure". The proportion of seeds growing into normal seedlings is the germination standard 2, and the number of seeds germinated on the day is recorded, and the recording period is 11 days.

[0017] Under germination standard 1 and germination standard 2, record the germination of seeds every day, calculate the daily germination rate, and record the germination rate with the largest number of germinated seeds as the germination potential 1 and 2 of the seeds under the treatment. Compare the germination potentials that can be achieved in each group of treatments to evaluate the uniformity of seed germination.

[0018] The calculation formulas for the germination rate and germination potential are as follows:

[0019] Germination rate 1 = S 1 / S T ×100%

[0020] Germination potential 1 = S n1 / S T ×100%

[0021] Germination rate 2 = S 2 / S T ×100%

[0022] Germination potential 2 = S n2 / ST ×100%

[0023] —S 1 The number of seeds germinated at the end of the experiment was recorded with germination standard 1;

[0024] —S n1 It is the number of seeds that germinated when the number of seeds germinated daily recorded by germination standard 1 reached the peak;

[0025] —S 2 The number of seeds germinated at the end of the experiment was recorded with germination standard 2;

[0026] —S n2 It is the number of seeds that germinated when the number of seeds germinated daily recorded by germination standard 2 reached the peak;

[0027] —S T The number of seeds for the test.

[0028] The application of the above method in improving the germination ability of Xinjiang Lithospermum officinale seeds also belongs to the scope of protection required by the present invention.

[0029] The invention also provides the use of melatonin in improving the germination ability of Xinjiang lithospermum seeds.

[0030] The invention also provides application of brassinolide in improving germination ability of Xinjiang lithospermum officinale seeds.

[0031] The present invention uses a specific initiator to pre-treat Xinjiang lithospermum seeds, and proposes an optimal initiation method that can significantly improve the germination ability of Xinjiang lithospermum seeds by optimizing the initiation conditions. The optimal initiation treatment conditions are: soaking Xinjiang lithospermum seeds in 100-200 μmol / L melatonin solution or 0.01-0.1 μmol / L brassinolide solution for 24 hours under 25°C dark conditions, drying to the original weight, and then germinating the seeds. The method has the advantages of simple operation, strong controllability of initiation conditions, low cost, etc., and is of great significance to the cultivation of Xinjiang lithospermum. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The germination of Xinjiang Lithospermum officinale seeds under different statistical standards. A represents seeds with 1-2mm white germination under statistical standard 1, and B represents normal seedlings under statistical standard 2.

[0033] Figure 2 Seed germination rate of Lithospermum chinense seeds after different priming treatments.

[0034] Figure 3The germination of Xinjiang Lithospermum officinale seeds after different priming treatments. A, CK group; B, polyethylene glycol PEG-6000 (PEG-6000) treatment group; C, chitosan (CTS) treatment group; D, melatonin (MT) treatment group; E, brassinolide (BR) treatment group.

[0035] Figure 4 The germination potential of Xinjiang Lithospermum officinale seeds treated with different initiators at different concentrations.

[0036] Figure 5 The root length of normal seedlings of Lithospermum erythrorhizon treated with different initiators at different concentrations.

[0037] Figure 6 The root length traits of normal Xinjiang Lithospermum seedlings after being treated with different initiators at different concentrations, including A, treatment 3 (3); B, CK; C, treatment 2 (5). The most representative root length traits were selected from the three treatment groups A, B, and C and arranged in order from long to short. DETAILED DESCRIPTION

[0038] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0039] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0040] The quantitative experiments in the following examples were performed in triplicate unless otherwise specified.

[0041] The seeds of Xinjiang Lithospermum in the following examples were donated by the laboratory of Zhu Jun, a teacher of the College of Agriculture of Xinjiang Agricultural University, and have been recorded in: Li Xiaojun, Wang Fang, Li Xiaojin, et al. Effects of different treatments on germination of Xinjiang Lithospermum seeds [J]. Seeds, 2005(09):16-18.DOI:10.16590 / j.cnki.1001-4705.2005.09.053. The public can obtain the biological material from the applicant, and the biological material is only used for repeating the experiment of the present invention and cannot be used for other purposes.

[0042] Embodiment 1, the method for improving the germination ability of Xinjiang Lithospermum officinale seeds

[0043] Screening of initiators for germination of Lithospermum officinale seeds

[0044] Initiator type: polyethylene glycol PEG-6000 (Beijing Biotechnology Co., Ltd., BS902-500g), chitosan (Beijing Biotechnology Co., Ltd., BY-C8320), melatonin (Beijing Biotechnology Co., Ltd., BY-M8600D), brassinolide (Shanghai Yuanye Biotechnology Co., Ltd., B21633-20mg-1).

[0045] 1. Triggering treatment

[0046] Select Xinjiang lithospermum seeds with good development, full grains, regular appearance, no damage, pests and diseases, and uniform grain size, peel off the shell and set aside. The selected lithospermum seeds were soaked for 24 hours in the dark at 25°C using four initiators, polyethylene glycol PEG-6000 (referred to as PEG-6000), chitosan (referred to as CTS), melatonin (referred to as MT), and brassinolide (referred to as BR) and their 5 concentration gradients (specific concentrations are shown in Table 1). The control group was treated without any initiator treatment, with a total of 21 treatments, 3 replicates per treatment, and 50 seeds per replicate. After the treatment, dry them back to the original weight.

[0047] Table 1. Different triggering treatment concentration settings

[0048] Group deal with Group deal with CK No treatment 3(1) 10μmol / L MT 1(1) 5% PEG-6000 3(2) 50μmol / L MT 1(2) 10% PEG-6000 3(3) 100μmol / L MT 1(3) 15% PEG-6000 3(4) 150μmol / L MT 1(4) 20% PEG-6000 3(5) 200μmol / L MT 1(5) 25% PEG-6000 4(1) 0.01μmol / L BR 2(1) 50mg / L CTS 4(2) 0.05μmol / L BR 2(2) 100mg / L CTS 4(3) 0.1μmol / L BR 2(3) 150mg / L CTS 4(4) 0.5μmol / L BR 2(4) 200mg / L CTS 4(5) 1μmol / L BR 2(5) 250mg / L CTS

[0049] 2. Germination test

[0050] According to GB / T 3543.4-1995 "Germination Test for Inspection Procedures of Crop Seeds", a standard germination test was conducted. After soaking, the seeds of Xinjiang Lithospermum officinale were placed in a culture dish with two layers of filter paper, pure water was added, and the culture dish was covered. The number was marked on the surface of the culture dish, and the seeds were placed in an artificial climate box (25℃ light for 8h; 15℃ dark for 16h) for cultivation. Pure water was added regularly during the period to keep the filter paper moist. The seed placement time was set to the 0th day. The number of seeds exposed to white (seeds exposed to white 1-2mm) was recorded from the first day after placement, and the number was recorded once every 24h. The proportion of seeds exposed to white was used as the statistical standard 1, and the record was recorded until the 15th day. From the 8th day, the number of normal seedlings, abnormal seedlings, fresh non-germinated seeds, hard seeds, and dead seeds was recorded respectively. The recording standard refers to Appendix A of GB / T 3543.4-1995 "Germination Test for Inspection Procedures of Crop Seeds", and the proportion of normal seedlings was used as the statistical standard 2, and the record was recorded until the 18th day. After the germination test, the germination rate and germination potential were calculated. Statistical Standards 1 and 2 are shown in Figure 1 A and B.

[0051] 3. Germination index

[0052] Germination rate 1: S 1 / S T ×100%

[0053] Germination rate 2: S 2 / S T ×100%

[0054] Germination potential 1: S n1 / S T ×100%

[0055] Germination potential 2: S n2 / S T ×100%

[0056] ——S 1 is the number of white seeds recorded according to statistical standard 1;

[0057] ——S 2 is the number of seeds germinated recorded by statistical standard 2;

[0058] ——S n1 It is the number of seeds that germinated when the number of seeds that germinated daily recorded by statistical standard 1 reached the peak;

[0059] ——S n2 It is the number of seeds that germinated when the number of seeds that germinated daily recorded by statistical standard 2 reached the peak;

[0060] ——S T is the number of seeds tested.

[0061] Root length: Use a vernier caliper with a scale of 0-150mm to measure the root length of normal seedlings. The length from the root base attachment point to the root tip is used as the measurement standard. The unit is expressed in cm, and 3 significant digits are retained after the decimal point.

[0062] 4. Statistics

[0063] The germination rate data were analyzed by one-way analysis of variance (ANOVA) and the values ​​were expressed as the mean ± SD of three replicates, with two significant figures after the decimal point.

[0064] The germination data were recorded, sorted, and plotted using Microsoft Excel 2019. The values ​​were expressed as the average of three replicates, with two significant digits after the decimal point.

[0065] The root length data were analyzed by one-way analysis of variance (ANOVA) and analyzed using IBM SPSS Statistics 27.0 software. The values ​​were expressed as the mean ± SD of three replicates, with three significant figures after the decimal point.

[0066] The results of the initiator screening experiment are as follows:

[0067] 1) Germination rate results

[0068] The seeds of Xinjiang Lithospermum were treated with 21 groups of different initiators and concentrations. The results of seed germination rates under two statistical standards are shown in Figure 2 , Figure 3 and Table 2. Under standard 1 statistics, each group had significant differences from the CK group. Among them, the germination rates of treatment 3 (MT) and treatment 4 (BR) at all concentrations were higher than those of the CK group, and the germination rate of treatment 3 (3) (100μmol / L MT) was the highest, reaching 95.33%. Under standard 2 statistics, except for treatment 4 (4) (0.5μmol / L BR) and treatment 4 (5) (1μmol / L BR), which had no significant difference from the CK group, the other groups had significant differences. Among them, the germination rates of treatment 3 (3), treatment 3 (5), treatment 4 (3), treatment 3 (1), and treatment 4 (1) were 94.67%, 90.67%, 89.33%, 86.00%, and 85.33%, respectively. Compared with the CK group, the germination rates of treatment 3 (3) (100μmol / L MT) and treatment 4 (3) (0.1μmol / L BR) increased by 30.27% and 22.93%, respectively. The results showed that 100-200μmol / LMT and 0.01-0.1μmol / L BR priming treatments could significantly increase the germination rate of Xinjiang Lithospermum officinale seeds.

[0069] Table 2. Germination rate of Lithospermum officinale seeds after different priming treatments

[0070]

[0071]

[0072] 2) Germination and fruiting

[0073] The seeds of Xinjiang Lithospermum were subjected to 21 different priming treatments. The germination potential results under two statistical standards are shown in Tables 3, 4 and Figure 4 . Under standard 1 statistics, both treatment 3 (MT) and treatment 4 (BR) advanced the time of germination and increased the value of germination potential. Among them, treatment 4 (3) (0.1μmol / L BR) had the highest germination potential, reaching 37.33% on the 4th day; under standard 2 statistics, the germination potential values ​​of treatments 3 (MT) and 4 (BR) were improved, and the 100-200μmol / L MT and 0.01-0.1μmol / L BR priming treatments performed best, and treatment 3 (3) (100μmol / L MT) had the highest germination potential, reaching 32.00%. The results showed that the 100-200μmol / L MT and 0.01-0.1μmol / L BR priming treatments could improve the germination potential of Xinjiang Lithospermum officinale seeds.

[0074] Table 3. Daily germination rate of Xinjiang Lithospermum officinale seeds after different priming treatments (standard 1)

[0075]

[0076]

[0077]

[0078] Table 4. Daily germination rate of Xinjiang Lithospermum officinale seeds after different priming treatments (standard 2)

[0079]

[0080] 3) Root growth and fruiting

[0081] The root length of 21 groups of normal seedlings of Lithospermum erythrorhizon with different priming treatments was measured. Figure 5 , Figure 6 As shown in Table 5, there were significant differences among the groups. Among them, treatment 3 (3) (100 μmol / L MT) had the longest root length, reaching 3.472 cm, and treatment 2 (5) (250 mg / L CTS) had the shortest root length, only 0.553 cm.

[0082] Comprehensive germination rate, germination potential and root length results show that 100-200 μmol / L melatonin and 0.01-0.1 μmol / L brassinolide for 24 hours can be used as the best method to improve the germination ability of Xinjiang lithospermum seeds. The present invention obtains the optimal initiator treatment conditions by screening the type and concentration of initiators: soaking Xinjiang lithospermum seeds in 100-200 μmol / L melatonin solution or 0.01-0.1 μmol / L brassinolide solution for 24 hours under dark conditions at 25°C, and drying to the original weight.

[0083] Table 5. Root length of normal seedlings of Lithospermum chinense after different priming treatments

[0084] deal with Root length (cm) deal with Root length (cm) CK 2.682±0.171b 3(1) 2.027±0.152de 1(1) 2.557±0.122bc 3(2) 1.934±0.090e 1(2) 2.529±0.165cf 3(3) 3.472±0.047a 1(3) 1.721±0.111g 3(4) 2.148±0.120d 1(4) 1.809±0.040fg 3(5) 2.054±0.101de 1(5) 1.745±0.263g 4(1) 1.909±0.090ef 2(1) 0.900±0.077j 4(2) 2.055±0.076de 2(2) 0.921±0.039j 4(3) 2.534±0.132bc 2(3) 0.653±0.056k 4(4) 1.560±0.098h 2(4) 0.581±0.020k 4(5) 1.108±0.075i 2(5) 0.553±0.027k

[0085] Note: Different lowercase letters after the data in the same column indicate significant differences (P<0.05)

[0086] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A method for improving the germination ability of Xinjiang Lithospermum officinale seeds, characterized in that: The method comprises the following steps: soaking Xinjiang lithospermum seeds in an initiator to obtain seeds treated with the initiator; The initiator is melatonin or brassinolide; The melatonin is used at a concentration of 100-200 μmol / L; The brassinolide is used at a concentration of 0.01-0.1 μmol / L.

2. The method according to claim 1, characterized in that The soaking time is 24 hours and the soaking temperature is 25°C.

3. The method according to claim 1 or 2, characterized in that: The germination ability of Xinjiang Lithospermum officinale seeds is manifested in germination rate and / or germination potential.

4. Application of melatonin in improving the germination ability of Xinjiang Lithospermum officinale seeds; the use concentration of the melatonin is 100-200 μmol / L.

5. Application of brassinolide in improving the germination ability of Xinjiang Lithospermum officinale seeds; the use concentration of brassinolide is 0.01-0.1 μmol / L.

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