阅读说明：本技术 一种白菜种子引发剂及其研究方法 (Cabbage seed initiator and research method thereof ) 是由 许小勇 张效宁 温艳斌 李小兵 褚润根 李梅兰 张静 于 2021-09-23 设计创作，主要内容包括：本发明公开了一种白菜种子引发剂及其研究方法,白菜种子引发剂包括聚谷氨酸、5-氨基乙酰丙酸、氨基丁酸、复硝酚钠四种成份,研究方法包括以下步骤：S1：设置正常引发实验环境和逆境胁迫环境实验；S2：种子生长指标测定；其中包括种子萌发指标的测定和生理生化指标的测定；S3：分析种子生长指标；引发剂成分为聚谷氨酸、5-氨基乙酰丙酸、氨基丁酸、复硝酚钠；本发明通过以大白菜陈种子为试材,进行种子引发处理试验,可以筛选出合适的引发剂及适合的浓度组合；为提高种子活力,缩短发芽时间,增加种子抗逆性,培育壮苗等生产应用以及研究收集数据,为增加种子抗逆性,培育壮苗、实验研究等生产应用做铺垫。(The invention discloses a cabbage seed initiator and a research method thereof, wherein the cabbage seed initiator comprises four components of polyglutamic acid, 5-aminolevulinic acid, aminobutyric acid and compound sodium nitrophenolate, and the research method comprises the following steps: s1: setting a normal initiation experiment environment and an adversity stress environment experiment; s2: measuring seed growth indexes; the method comprises the steps of measuring germination indexes of seeds and measuring physiological and biochemical indexes of the seeds; s3: analyzing the growth indexes of the seeds; initiator components are polyglutamic acid, 5-aminolevulinic acid, aminobutyric acid and compound sodium nitrophenolate; according to the invention, proper initiators and proper concentration combinations can be screened out by taking aged Chinese cabbage seeds as test materials and carrying out a seed initiation treatment test; the method is used for improving the seed vigor, shortening the germination time, increasing the stress resistance of the seeds, cultivating strong seedlings and other production applications, researching and collecting data, and is used for laying a cushion for increasing the stress resistance of the seeds, cultivating strong seedlings, experimental research and other production applications.)

1. The Chinese cabbage seed initiator is characterized by comprising the components of polyglutamic acid, 5-aminolevulinic acid, aminobutyric acid and compound sodium nitrophenolate.

2. The cabbage seed initiator as claimed in claim 1, wherein the polyglutamic acid is 500mg/L, the 5-aminolevulinic acid is 10mg/L, the molar concentration of aminobutyric acid is 10mmol/L, and the compound sodium nitrophenolate is 10 mg/L.

3. A method of investigating cabbage seed initiators as claimed in claim 1 or 2, characterized in that: comprises the following steps;

s1: setting a normal initiation experiment environment and an adversity stress environment experiment;

s11: the seed treatment process of the normal environment priming experiment comprises the following steps;

s111: selecting seeds with the same size and plumpness and placing the seeds in test tubes containing an initiator, wherein the number of the seeds in each test tube is the same; initiating by an initiator, washing the seeds by distilled water after the initiation is finished, completely washing residual solution on the surface, absorbing the surface moisture, and then drying to the initial moisture content;

s112: after step S111 is completed, sowing the seeds into culture dishes containing a single layer of wet filter paper, wherein the number of seeds in each culture dish is equal; supplementing water every day at regular time, and keeping the filter paper moist;

s12: the seed treatment process of the adversity stress environment initiation experiment comprises the following steps;

s121: setting a plurality of experimental groups, including a clear water control group and an initiation treatment group; drought stress group, drought stress + initiation treatment group; salt stress group, salt stress + priming treatment group;

s122: placing the experimental group in the step S121 in a constant-temperature incubator for germination;

s2: measuring seed growth indexes; the method comprises the steps of measuring germination indexes of seeds and measuring physiological and biochemical indexes of the seeds;

s3: and analyzing the seed growth indexes.

4. The method for researching the cabbage seed initiator as claimed in claim 3, wherein the method comprises the following steps: in step S111, the initiator initiation time is 2 h.

5. The method for researching the cabbage seed initiator as claimed in claim 3, wherein the method comprises the following steps: in step S112, the germination temperature is kept at 22 ℃ in the daytime for 16 hours; the mixture was kept at 18 ℃ overnight for 8 h.

6. The method for researching the cabbage seed initiator as claimed in claim 3, wherein the method comprises the following steps: in step S121, the processing method of each experimental group is as follows; the control group and the drought stress group do not use an initiator, distilled water with the same mass as that of the initiators of other control groups is added into the control group, 80 percent of the distilled water of the control group is added into the drought stress group, NaCl solution with the same mass as that of the control group is added into the salt stress group, and the initiators with different component contents are respectively added into a plurality of standard control groups.

7. The method for researching Chinese cabbage seed initiator according to claim 6, characterized in that: the molar concentration of the NaCl solution is 75 mmol/L.

8. The method for researching the cabbage seed initiator as claimed in claim 3, wherein the method comprises the following steps: in step S122, the germination temperature is kept at 22 ℃ in the daytime for 16 hours; at 18 ℃ in the dark, the temperature is kept for 8 hours; distilled water was added to the control group and the drought-stressed group to the initial state every 8h, the NaCl solution was added to the salt-stressed group to the initial state every 8h, and the initiator was added to each standard to the initial state every 8 h.

9. The method for researching the cabbage seed initiator as claimed in claim 3, wherein the method comprises the following steps: in step S2, the method for measuring the seed germination index is as follows; selecting a plurality of seedlings from each culture dish, cleaning the seedlings by using distilled water, and measuring the hypocotyl length and the root length after different initiation treatments after the seedlings are dried by using filter paper; the germination standard is 1/2 with the hypocotyl length being the length of the seeds, the germination number of the seeds is recorded by regularly observing the seeds in the morning and evening every day, the germination vigor is counted on the 3 rd day, the germination rate is counted on the 7 th day, and the hypocotyl length of the germinated seeds is measured; calculating the following indexes according to the germination number and hypocotyl length of the seeds;

the germination potential is (Gt/T). times.100%

The germination rate is (Gt/T). times.100%

Germination index ═ Σ Gt/Dt

Vitality index is germination index × S

Wherein Gt is the germination number at the t day after the germination begins; dt is the corresponding germination days (d); s is the root length (cm) of the seedling; t is the number of test seeds in each dish.

10. The method for researching the cabbage seed initiator as claimed in claim 3, wherein the method comprises the following steps: in step S2, the physiological and biochemical indexes are measured by the method of; measuring physiological indexes of seedlings growing on the 7 th day; the physiological index includes; measuring the activity of superoxide dismutase (SOD), Peroxidase (POD), Catalase (CAT), Malondialdehyde (MDA) and soluble protein, wherein the method for measuring the activity of the superoxide dismutase (SOD) adopts a Nitrogen Blue Tetrazole (NBT) photoreduction method; peroxidase (POD) activity assay guaiacol method was used; determination of Catalase (CAT) Activity Using UV absorption; the content of Malondialdehyde (MDA) is measured by a thiobarbituric acid method; soluble protein assay Coomassie Brilliant blue G-250 method was used.

Technical Field

The invention relates to the technical field of seed initiation, in particular to a cabbage seed initiator and a research method thereof.

Background

The seed priming technology can shorten the seed germination time and improve the germination rate, is a seed treatment technology capable of effectively improving the seed quality, and has wide application prospect. After the initiation, a series of complex physiological processes of hormone, enzyme activity and nutrient substances are changed, the seed activity is improved, so that the seed activity, the seed germination rate and the seed uniformity are improved, and meanwhile, the germination time is also obviously shortened. However, the conventional technology is mainly used for researching seed initiation at present, the research is not deep enough, and the research on the initiation mechanism of different initiators is weak. Different types of seeds have different requirements on an initiation method, initiation time and temperature, initiator concentration and the like, and can be widely popularized in production practice only through a series of tests and verifications. Therefore, further research and development of seed priming technologies suitable for different species of plants and different physiological states are important tasks for seed priming research in the future.

The prior initiating technology has less research on the restoration of the vitality of the aged seeds. Although the seeds can maintain activity for a longer period of time after physiological maturation, as the storage time is prolonged, the membrane system is damaged, the enzyme activity is reduced, the integrity of genetic materials is reduced, and the germination vigor, the germination rate, the germination index and the vitality index are reduced, and the growth of buds and roots is inhibited. This directly causes huge economic losses in agricultural production and also poses certain difficulties in the screening and improvement of breeding materials. Chinese cabbage (Brassica rapa L. ssp. pekinensis) originates from China, belongs to cruciferae plants, is also called as Chinese cabbage and white cabbage, is one of special vegetables in China, is planted in all regions of China in planting areas, and is one of the vegetable crops with the largest planting area in China at present. The vegetable is tender in quality, is transport-resistant and storage-resistant, is rich in a large number of nutrient elements and crude fibers in the body, can promote digestion and invigorate the stomach, has medicinal effects of preventing cardiovascular diseases and the like, and becomes a main vegetable for eating in winter and spring in most areas of China. Therefore, the initiation research of the aged seeds of the Chinese cabbage and other vegetables is particularly important.

Disclosure of Invention

The invention aims to provide a cabbage seed initiator and a research method thereof.

In order to achieve the purpose, the invention provides the following technical scheme:

a Chinese cabbage seed initiator comprises polyglutamic acid, 5-aminolevulinic acid, aminobutyric acid and compound sodium nitrophenolate.

Further, the content of the polyglutamic acid is 500mg/L, the content of the 5-aminolevulinic acid is 10mg/L, the molar concentration of the aminobutyric acid is 10mmol/L, and the content of the compound sodium nitrophenolate is 10 mg/L.

Further, the invention provides a research method of the Chinese cabbage seed initiator, which comprises the following steps:

s1: setting a normal initiation experiment environment and an adversity stress environment experiment;

s11: the seed treatment process of the normal environment priming experiment comprises the following steps;

s111: selecting seeds with the same size and plumpness and placing the seeds in test tubes containing an initiator, wherein the number of the seeds in each test tube is the same; initiating by an initiator, washing the seeds by distilled water after the initiation is finished, completely washing residual solution on the surface, absorbing the surface moisture, and then drying to the initial moisture content;

s112: after step S111 is completed, sowing the seeds into culture dishes containing a single layer of wet filter paper, wherein the number of seeds in each culture dish is equal; supplementing water every day at regular time, and keeping the filter paper moist;

s12: the seed treatment process of the adversity stress environment initiation experiment comprises the following steps;

s121: setting a plurality of experimental groups, including a clear water control group and an initiation treatment group; drought stress group, drought stress + initiation treatment group; salt stress group, salt stress + priming treatment group;

s122: placing the experimental group in the step S121 in a constant-temperature incubator for germination;

s2: measuring seed growth indexes; the method comprises the steps of measuring germination indexes of seeds and measuring physiological and biochemical indexes of the seeds;

s3: and analyzing the seed growth indexes.

Further, in step S111, the initiator initiation time is 2 h.

Further, in step S112, the germination temperature is maintained at 22 ℃ during the day for 16 hours; the mixture was kept at 18 ℃ overnight for 8 h.

Further, the molar concentration of the NaCl solution is 75 mmol/L.

Further, in step S122, the germination temperature is maintained at 22 ℃ during the day for 16 hours; at 18 ℃ in the dark, the temperature is kept for 8 hours; distilled water was added to the control group and the drought-stressed group to the initial state every 8h, the NaCl solution was added to the salt-stressed group to the initial state every 8h, and the initiator was added to each standard to the initial state every 8 h.

Further, in step S2, the method for measuring the seed germination index is as follows; selecting a plurality of seedlings from each culture dish, cleaning the seedlings by using distilled water, and measuring the hypocotyl length and the root length after different initiation treatments after the seedlings are dried by using filter paper; the germination standard is 1/2 with the hypocotyl length being the length of the seeds, the germination number of the seeds is recorded by regularly observing the seeds in the morning and evening every day, the germination vigor is counted on the 3 rd day, the germination rate is counted on the 7 th day, and the hypocotyl length of the germinated seeds is measured; calculating the following indexes according to the germination number and hypocotyl length of the seeds;

the germination potential is (Gt/T). times.100%

The germination rate is (Gt/T). times.100%

Germination index ═ Σ Gt/Dt

Vitality index is germination index × S

Wherein Gt is the germination number at the t day after the germination begins; dt is the corresponding germination days (d); s is the root length (cm) of the seedling; t is the number of test seeds in each dish.

Further, in step S2, the physiological and biochemical indexes are measured by the method of; measuring physiological indexes of seedlings growing on the 7 th day; the physiological index includes; measuring the activity of superoxide dismutase (SOD), measuring the activity of Peroxidase (POD), measuring the activity of Catalase (CAT), measuring the content of Malondialdehyde (MDA) and measuring soluble protein.

Further, the superoxide dismutase (SOD) activity is measured by using a Nitrogen Blue Tetrazolium (NBT) photoreduction method; peroxidase (POD) activity assay guaiacol method was used; determination of Catalase (CAT) Activity Using UV absorption; the content of Malondialdehyde (MDA) is measured by a thiobarbituric acid method; soluble protein assay Coomassie Brilliant blue G-250 method was used.

Compared with the prior art, the invention has the beneficial effects that:

according to the invention, proper initiators and proper concentration combinations can be screened out by taking aged Chinese cabbage seeds as test materials and carrying out a seed initiation treatment test; the method is used for improving the seed vigor, shortening the germination time, increasing the stress resistance of the seeds, cultivating strong seedlings and other production applications, researching and collecting data, and is used for laying a cushion for increasing the stress resistance of the seeds, cultivating strong seedlings, experimental research and other production applications.

Description of the drawings:

in order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a schematic diagram of germination and growth of Chinese cabbage seeds under stress of an orthogonal optimal combination of stresses.

The specific implementation mode is as follows:

in order to make the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments.

Thus, the following detailed description of embodiments of the invention is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.

In addition, the embodiments of the present invention and the features of the embodiments may be combined with each other without conflict.

As shown in figure 1, the Chinese cabbage seed initiator comprises polyglutamic acid, 5-aminolevulinic acid, aminobutyric acid and compound sodium nitrophenolate.

Further, the content of the polyglutamic acid is 500mg/L, the content of the 5-aminolevulinic acid is 10mg/L, the molar concentration of the aminobutyric acid is 10mmol/L, and the content of the compound sodium nitrophenolate is 10 mg/L.

The research method of the Chinese cabbage seed initiator comprises the following steps:

s1: setting a normal initiation experiment environment and an adversity stress environment experiment;

s11: the seed treatment process of the normal environment priming experiment comprises the following steps;

s111: selecting seeds with the same size and plumpness and placing the seeds in test tubes containing an initiator, wherein the number of the seeds in each test tube is the same; initiating by an initiator, washing the seeds by distilled water after the initiation is finished, completely washing residual solution on the surface, absorbing the surface moisture, and then drying to the initial moisture content;

s112: after step S111 is completed, sowing the seeds into culture dishes containing a single layer of wet filter paper, wherein the number of seeds in each culture dish is equal; supplementing water every day at regular time, and keeping the filter paper moist;

s12: the seed treatment process of the adversity stress environment initiation experiment comprises the following steps;

s121: setting a plurality of experimental groups, including a clear water control group and an initiation treatment group; drought stress group, drought stress + initiation treatment group; salt stress group, salt stress + priming treatment group;

s122: placing the experimental group in the step S121 in a constant-temperature incubator for germination;

s2: measuring seed growth indexes; the method comprises the steps of measuring germination indexes of seeds and measuring physiological and biochemical indexes of the seeds;

s3: and analyzing the seed growth indexes.

In this embodiment, in step S111, the initiation time of the initiator is 2 hours.

In this embodiment, in step S112, the germination temperature is maintained at 22 ℃ during the day for 16 hours; the mixture was kept at 18 ℃ overnight for 8 h.

In this embodiment, in step S121, the processing method of each experimental group is as follows; the control group and the drought stress group do not use an initiator, distilled water with the same mass as that of the initiators of other control groups is added into the control group, 80 percent of the distilled water of the control group is added into the drought stress group, NaCl solution with the same mass as that of the control group is added into the salt stress group, and the initiators with different component contents are respectively added into a plurality of standard control groups.

In this example, the molar concentration of the NaCl solution was 75 mmol/L.

In this embodiment, in step S122, the germination temperature is maintained at 22 ℃ in the daytime for 16 hours; at 18 ℃ in the dark, the temperature is kept for 8 hours; distilled water was added to the control group and the drought-stressed group to the initial state every 8h, the NaCl solution was added to the salt-stressed group to the initial state every 8h, and the initiator was added to each standard to the initial state every 8 h.

In this embodiment, in step S2, the method for determining the seed germination index is as follows; selecting a plurality of seedlings from each culture dish, cleaning the seedlings by using distilled water, and measuring the hypocotyl length and the root length after different initiation treatments after the seedlings are dried by using filter paper; the germination standard is 1/2 with the hypocotyl length being the length of the seeds, the germination number of the seeds is recorded by regularly observing the seeds in the morning and evening every day, the germination vigor is counted on the 3 rd day, the germination rate is counted on the 7 th day, and the hypocotyl length of the germinated seeds is measured; calculating the following indexes according to the germination number and hypocotyl length of the seeds;

the germination potential is (Gt/T). times.100%

The germination rate is (Gt/T). times.100%

Germination index ═ Σ Gt/Dt

Vitality index is germination index × S

Wherein Gt is the germination number at the t day after the germination begins; dt is the corresponding germination days (d); s is the root length (cm) of the seedling; t is the number of test seeds in each dish.

In this embodiment, in step S2, the physiological and biochemical indexes are measured by the following method; measuring physiological indexes of seedlings growing on the 7 th day; the physiological index includes; measuring the activity of superoxide dismutase (SOD), measuring the activity of Peroxidase (POD), measuring the activity of Catalase (CAT), measuring the content of Malondialdehyde (MDA) and measuring soluble protein.

In this example, the superoxide dismutase (SOD) activity was measured by a Nitrobluetetrazolium (NBT) photoreduction method; peroxidase (POD) activity assay guaiacol method was used; determination of Catalase (CAT) Activity Using UV absorption; the content of Malondialdehyde (MDA) is measured by a thiobarbituric acid method; soluble protein assay Coomassie Brilliant blue G-250 method was used.

The specific embodiment of the invention is as follows:

1. selection of materials and reagents:

the test material is aged Chinese cabbage seed naturally stored at room temperature for 5 years in laboratory, and the variety is Taiyuan Erqing (selected from Shanxi Jinnong Co., Ltd.); the test reagent, polyglutamic acid (γ -PGA), was purchased from Reilin Biotech, Inc.; 5-Aminolevulinic acid (5-ALA) was purchased from Seisan Tianfeng Biotech, Inc.; aminobutyric acid (GABA) was purchased from SIGMA; the compound sodium nitrophenolate is purchased from Shandong Hangao bioengineering Co.

2. Seed priming treatment:

2.1 seed priming treatment method under normal treatment:

selecting seeds with consistent size and plumpness, placing the seeds in test tubes containing an initiator, and treating 100 seeds in each test tube; the initiation time is 2h, the seeds are washed by distilled water after initiation is finished, the residual solution on the surface is washed out, the surface moisture is absorbed, and then the seeds are dried back to the initial moisture content; after the above operations are completed, sowing the seeds into culture dishes containing single-layer wet filter paper, wherein each culture dish contains 30 seeds, the number of the seeds is repeated for 3 times, the germination temperature of the Chinese cabbage is kept at 22 ℃ in the daytime and lasts for 16 hours, and the germination temperature of the Chinese cabbage is kept at 18 ℃ in the dark and lasts for 8 hours; periodically, water was replenished every day, keeping the filter paper wet.

2.2, seed priming treatment method under adversity stress treatment:

according to the previous test results, the cabbage seeds are subjected to initiation treatment by adopting the treatment of reagent ratio T9 (namely 500mg/L gamma-PGA, 10mg/L5-ALA, 10mmol/L GABA and 10mg/L compound sodium nitrophenolate reagent), and distilled water treatment is set as a control T0, and the rest is the same as above.

The germination test is carried out in a constant-temperature incubator, and three germination conditions are set through pre-experimental screening: (1) drought stress D, relative water content 80% of control treatment; (2) salt stress S, setting the salt concentration to be 75 mmol/L; (3) standard germination conditions CK; the seeds treated with T9 and T0 were placed under the three germination conditions described above, and the dishes were replenished with the required water every 8h (drought stress D with 80% water CK, salt stress S with the same amount of NaCl solution at the corresponding concentration).

3. Determination of growth indicators

3.1 measurement of seed germination indexes:

the germination standard is 1/2 with the hypocotyl length being the length of the seeds, the germination number of the seeds is recorded by regularly observing the seeds in the morning and evening every day, the germination vigor is counted on the 3 rd day, the germination rate is counted on the 7 th day, and the hypocotyl length of the germinated seeds is measured. The following indices were calculated from the number of seed germination and hypocotyl length.

The germination potential is (Gt/T). times.100%

The germination rate is (Gt/T). times.100%

Germination index ═ Σ Gt/Dt

Vitality index is germination index × S

Wherein Gt is the germination number at the t day after the germination begins; dt is the corresponding germination days (d); s is the root length (cm) of the seedling; t is the number of test seeds in each dish.

3.2 measurement of hypocotyl length and root length of Chinese cabbage seedlings:

after the test was completed, 5 seedlings of Chinese cabbage were selected from each petri dish, washed with distilled water, and blotted with filter paper, and then hypocotyl length and root length after different initiation treatments were measured.

3.3, measurement of physiological and biochemical indexes:

measuring physiological index of Chinese cabbage seedling growing on day 7. Wherein, the activity of superoxide dismutase (SOD) is measured by Nitrogen Blue Tetrazole (NBT) photoreduction method such as Gaojunfeng; peroxidase (POD) activity measurement referring to guaiacol method of Junfeng et al; determination of Catalase (CAT) Activity with reference to the ultraviolet absorption method by Haebin, et al; the content of Malondialdehyde (MDA) is measured by a thiobarbituric acid method; soluble protein assay is performed by Coomassie brilliant blue G-250 method of Zhangiang et al.

4. Results and analysis

4.1 Effect of orthogonal combination treatment on cabbage seed Germination

TABLE 1 test factors and levels

On the basis of improving the germination of old Chinese cabbage seeds and the growth of seedlings by a single initiator test, selecting 3 concentrations with the best initiating effect of each reagent for mixing, and designing a four-factor three-level test (table 1) according to an L9(34) orthogonal table; determining the optimal concentration ratio of gamma-PGA (A), 5-ALA (B), GABA (C) and compound sodium nitrophenolate (D) for improving the germination of the Chinese cabbage seeds by measuring the influence of each treatment on the germination characteristics of the Chinese cabbage seeds, and on one hand, screening the combination ratio of reagents with obvious synergistic effect on the germination of the Chinese cabbage seeds; on the other hand, a foundation is laid for the interaction among the reagents.

TABLE 2 Effect of different combinations of initiators on cabbage Germination and growth

The gamma-PGA, 5-ALA, GABA and compound sodium nitrophenolate with different concentrations are combined for initiation, and all germination indexes of the Chinese cabbage seeds show obvious difference (table 2); the germination potential of T6, T7 and T9 is obviously higher than that of the control treatment, and is respectively increased by 32.13%, 35.67% and 46.40% compared with the control treatment; the vitality indexes of T6, T7 and T9 are obviously higher than those of control treatment, and are respectively increased by 22.35%, 17.35% and 45.68% compared with the control treatment; except for T5 and T8, the germination rates of other treatments are obviously higher than those of a control, wherein T6, T7 and T9 have the most obvious effect of improving the germination rates and respectively improve the germination rates by 23.84%, 19.23% and 24.62% compared with the control treatment; only the germination indexes of T6 and T9 are obviously higher than those of a control, the germination indexes are improved by 19.49 percent and 31.51 percent compared with the control, and the promotion effect of the rest treatments on the germination indexes is not obvious; in the aspect of hypocotyl length, except the T3, T5 and T8 treatments, the other treatments are all obviously higher than the control, and the treatment is most obvious in T1 and T2 and is increased by 16.19 percent compared with the control; in the aspect of root length, the treatment difference is large, the inhibition effect of T1, T2 and T3 on the root length is obvious, and the promotion effect of T4, T6, T7, T8 and T9 on the root length is obvious; in conclusion, the T9 treatment, namely the combination of 500mg/L gamma-PGA, 10mg/L5-ALA, 10mmol/L GABA and 10mg/L compound sodium nitrophenolate has obviously better inducing effect than other combinations, and the germination vigor, the germination rate, the germination index, the vitality index and the root length are respectively improved by 46.40%, 24.62%, 31.51%, 45.68% and 10.77% compared with the T0 treatment.

TABLE 3 Effect of different combinations of initiators on the antioxidase Activity of Chinese cabbage seedlings

As can be seen from Table 3, the combination of gamma-PGA, 5-ALA, GABA and sodium nitrophenolate with different concentrations is used for initiation, so that the activities of soluble protein, MDA and antioxidant enzyme of the Chinese cabbage seedlings are greatly different; the content of the T9 soluble protein is obviously improved by about 70 percent compared with that of a control, and the content of the soluble protein is reduced by other treatments; other treatments besides T5, T7, and T8 significantly reduced MDA content, indicating that reagent treatment can effectively repair membrane damage; other treatments except T2 and T4 had significantly increased SOD activity; POD activity was significantly increased by treatment with T2, T6, T7 and T9; the CAT activity of T2, T6 and T9 treatment is obviously improved; POD, SOD and CAT activities are used as 3 important enzyme structures of an antioxidant enzyme system, the enzyme activity is improved, the peroxidation degree can be effectively reduced, the metabolic process is promoted, and the seed activity is further improved; in conclusion, after the treatment of T9, namely the treatment of the combination of 500mg/L gamma-PGA, 10mg/L ALA, 10mmol/L GABA and 10mg/L compound sodium nitrophenolate reagent, the activities of soluble protein and antioxidant enzyme in the seedlings of the Chinese cabbage are obviously improved.

In conclusion, by comparing the influences of different reagent combination treatments on the germination of the Chinese cabbage seeds and the growth of seedlings, the germination vigor, the germination rate, the germination index, the vigor index and the root length of the T9 treatment are respectively improved by 46.40%, 24.62%, 31.51%, 45.68% and 10.77% compared with the T0 treatment; in addition, the content of soluble protein, POD, SOD and CAT activities of T9 treated by the method are also obviously higher than those of T0 treated by the method; in conclusion, T9 treatment has obvious promotion effect on the germination index of seeds and the growth of seedlings.

4.2 influence of the optimal combination of orthogonal treatment on the germination of the Chinese cabbage seeds under adversity stress:

the optimal combined T9 treatment obtained by orthogonal treatment has certain promotion effect on seed germination and seedling growth under drought stress and salt stress (figure 1); as can be seen in fig. 1A, the germination potential of the T9-treated seeds was improved by 36.65%, 15.44% and 16.73% compared to the T0-treated seeds under CK, D and S conditions, respectively, but the difference was not significant; as can be seen in FIG. 1B, the germination rates of the T9-treated seeds are respectively improved by 8.62%, 6.28% and 6.91% compared with the germination rate of the T0-treated seeds; the germination index and the vitality index of the seeds treated by the T9 are obviously changed (figures 1C and 1D), and under the conditions of CK, D and S, the germination index is obviously improved after the treatment of the T9 and is respectively increased by 15.19%, 20.81% and 27.86% compared with the treatment of the T0; viability index after T9 treatment was significantly higher than T0 treatment under CK and D conditions, but the difference was not significant under S conditions; in terms of root length (fig. 1E), treatment with T9 and T0 did not differ significantly, nor did the difference between D and S conditions; in terms of root length (fig. 1F), the difference between treatment with T9 and treatment with T0 was not significant under each condition, and the root length was significantly reduced with T0 under D condition, but the change in root length was not significant with T9 treatment.

TABLE 4 influence of antioxidant enzyme activity of Chinese cabbage seedlings under stress of orthogonal optimal combination

Seed treatment is carried out by adopting an orthogonal optimal combination of reagents, the soluble protein content, MDA content and antioxidant enzyme activity of the Chinese cabbage seedlings treated at different time T9 and T0 are compared, and the difference among treatments and the change trend at 6d are found (table 4); under CK conditions, the change trend of the T9 and the T0 on the content of the soluble protein is firstly increased and then reduced, wherein the content of the soluble protein is not greatly influenced by the T9 and the T0 on 0d and 6d, and the content of the soluble protein is increased by 14.61 percent after the T9 is treated compared with the T0 on 3 d; under the conditions of D and S, the content of soluble protein of T9 and T0 is obviously increased, the change trend is consistent with that of CK conditions, and the content of the soluble protein is increased but is not obvious after T9 treatment compared with T0 treatment at 3D and 6D; the MDA content of different treatments shows a trend of increasing firstly and then decreasing along with the change of time, under the CK condition, the MDA content of 0d and 3d treated by T9 is increased by 23.33 percent and 23.33 percent respectively compared with that of T0 treated, and no obvious difference exists in 6 d; the MDA content after T9 treatment is increased by 10.81% compared with that of T0 treatment at 3D under D condition, but the MDA content of T9 and that of T0 treatment are at the same level at 6D; under S conditions, T9 treatment had no significant effect on MDA content over time; the SOD activity and the time change are in positive correlation in different treatments, and the activity is obviously enhanced along with the increase of time; the SOD activity was not much affected by treatment with T9 and T0 at 0d and 6d under CK conditions, but increased by 17.52% after T9 treatment at 3d compared with T0 treatment; SOD activity of T9 treated at 3D and 6D under D condition was increased by 20.12% and 10.73% respectively compared with T0 treated; the activity of T9 treated SOD at 6d is not obviously different from that of 3d, but the activity of T0 treated SOD at 6d is increased by 8.59 percent compared with that of 3 d; the SOD activity of T9 treated by the method is increased by 7.75 percent compared with that of T0 treated by the method at 3d under the S condition, and the SOD activity of T9 treated by the method at 3d is not greatly different from that of T0 treated by the method at 3 d; the POD activity of different treatments firstly increases and then decreases along with the change of time, and the POD activity of other treatments is not obviously changed from 3d to 6d except the condition of the T9 treatment S; treatment with T9 and T0 at 0d and 6d under CK had little effect on POD activity, but at 3d T9 increased POD activity by 5.58% over T0; POD activity was increased by 4.48% and 6.31% in T9 treatment compared to T0 treatment at 3D and 6D, respectively, under D conditions; POD activity is increased by 4.83% when the POD is treated by T9 at 3d under the S condition compared with that of the POD treated by T0, and the POD activity is not greatly influenced by T9 and T0 at 6 d; the CAT activity is not greatly influenced by the T9 treatment and the T0 treatment at 0d under the CK condition, but the CAT activity of the T9 treatment is respectively increased by 9.74 percent and 3.61 percent compared with the T0 treatment at 3d and 6 d; CAT activity of T9 treatment was increased by 5.66% and 2.74% compared with that of T0 treatment at 3D and 6D under D condition, respectively, and the treatment change was not obvious from 3D to 6D; CAT activities treated by T9 and T0 at 3d and 6d under S condition were not much different.

In conclusion, under the conditions of D and S, the germination indexes of the T0-treated seeds are reduced compared with those of the CK-treated seeds; the soluble protein, MDA content and antioxidant enzyme activity in the seedlings are increased, and gradually increased along with the severe stress degree; in addition, as the stress time increases and the stress degree deepens, the soluble protein, MDA content and antioxidant enzyme activity in seedlings approximately show the trend of increasing and then decreasing; the germination index of the seeds is improved to a certain extent by the treatment of T9 under the stress condition, but the germination index is still lower than that of the seeds treated by T9 under the CK condition; at each stage, soluble protein, MDA content and antioxidant enzyme activity in T9-treated seedlings were increased compared to the T0-treated level; the T9 treatment can relieve the damage of the adversity stress to the seeds and improve the stress resistance of the seedlings.

5. Conclusion

(1) The treatment of different combination ratios T9, namely the combination initiation effect of 500mg/L gamma-PGA, 10mg/L5-ALA, 10mmol/L GABA and 10mg/L compound sodium nitrophenolate reagent is obviously better than that of other combinations, the content of soluble protein and the activities of POD, SOD and CAT are also obviously higher than that of the treatment of T0, and the promotion effect on seed germination and seedling growth exists.

(2) Under drought stress and salt stress, the germination index of the untreated Chinese cabbage seeds is reduced compared with that under CK conditions; the soluble protein, MDA content and antioxidant enzyme activity in the seedlings are increased, and gradually increased along with the severe stress degree; in addition, the soluble protein, MDA content and antioxidant enzyme activity in seedlings also show a general tendency to increase and then decrease as the stress time increases; under drought and salt stress treatment, although the germination index of the seeds treated by the T9 is still lower than that of the seeds after initiation treatment under non-stress treatment, the germination vigor, the germination rate, the germination index and the vitality index of the seeds are improved to a certain extent, and each germination index is higher than that of the seeds which are not treated under the non-stress condition; the content of soluble protein and MDA in seedlings and the activity of antioxidant enzyme are increased compared with the untreated level; the T9 treatment can relieve the damage of the adversity stress to the seeds and improve the stress resistance of the seedlings.

It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.