Method for activating crop seed by high-voltage electric field cold plasma (HVCP), and use thereof

Abstract

The present disclosure relates to a method for activating a crop seed by high-voltage electric field cold plasma (HVCP), and use thereof, belonging to the technical field of crop planting. The present disclosure provides a method for activating a crop seed by HVCP, including the following steps: (1) mixing a crop seed with water and immersing the crop seed in water for 4 h to 24 h to obtain an immersed crop seed; and (2) conducting discharge activation on the immersed crop seed at a voltage of 80 kV to 130 kV to obtain an activated crop seed. In the present disclosure, the method can improve heat-resistant and disease-resistant properties of crops, and provide a scientific basis for subsequent acquisition of excellent seeds.

Claims

1. A method for mutagenizing a crop seed by high-voltage electric field cold plasma (HVCP), comprising following steps: (1) mixing a crop seed with water and immersing the crop seed in water for 4 h to 8 h to obtain an immersed crop seed; and (2) conducting discharge activation by HVCP on the immersed crop seed at a voltage of 130 kV, wherein the discharge activation is conducted for 2 h to 4 h, and wherein the crop is Brassica campestris ssp. chinensis Makino.

2. The method according to claim 1, wherein the mixing of the crop seed with the water is conducted at a mass-to-volume ratio of 1:4 to 1:6.

3. The method according to claim 2, wherein the mixing of the crop seed with the water is conducted at a mass-to-volume ratio of 1:5.

4. The method according to claim 1, wherein the immersing is conducted in dark; and the immersing is conducted at a water temperature of 18 C. to 22 C.

5. The method according to claim 4, wherein the immersing is conducted at a water temperature of 20 C.

6. The method according to claim 1, wherein the discharge activation by HVCP is conducted at 18 C. to 25 C.

7. The method according to claim 6, wherein the discharge activation by HVCP is conducted at 20 C.

8. A method for mutation breeding by HVCP comprising following steps: (1) mixing a crop seed with water and immersing the crop seed in water for 4 h to 8 h to obtain an immersed crop seed; and (2) conduction discharge activation by HVCP on the immerse crop seed at a voltage of 130 kV, wherein the discharge activation is conducted for 2 h to 4 h, and wherein the crop is Brassica campestris ssp. chinensis Makino.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows an influence of different voltages on seed germination rate and germination potential of Brassica campestris ssp. chinensis Makino;

(2) FIG. 2 shows an influence of different treatment times on seed germination rate and germination potential of Brassica campestris ssp. chinensis Makino:

(3) FIG. 3 shows an influence of different treatment times on seed survival rate of Brassica campestris ssp. chinensis Makino:

(4) FIG. 4 shows the acquisition of forward mutant plants; and

(5) FIG. 5 shows molecular detection results of simple sequence repeat (SSR) markers linked to candidate genes of a flowering time on mutant strains.

DETAILED DESCRIPTION OF THE EMBODIMENTS

(6) The present disclosure provides a method for activating a crop seed by HVCP, including the following steps: (1) a crop seed is mixed with water and immersed in water for 4 h to 24 h to obtain an immersed crop seed; and (2) the immersed crop seed is discharged to activate at a voltage of 80 kV to 130 kV.

(7) In the present disclosure, the crop seed is preferably derived from a crop selected from the group consisting of rice, wheat, corn, rapeseed, soy bean, tomato, chili, eggplant, cucumber, muskmelon, pumpkin, kidney bean, radish, Brassica oleracea, Brassica juncea, Chinese cabbage, and watercress, more preferably Brassica campestris ssp. chinensis Makino. The crop seed is mixed with water at a mass-to-volume ratio of preferably 1:(4-6), more preferably 1:5.

(8) In the present disclosure, the crop seed is immersed preferably in the dark at a water temperature of preferably 18 C. to 22 C., more preferably 20 C. for preferably 8 h to 20 h, more preferably 14 h.

(9) In the present disclosure, the voltage is preferably 105 kV. The immersed crop seed is discharged to activate at preferably 18 C. to 25 C., more preferably 21.5 C. for preferably 0.5 h to 10 h, more preferably 3.5 h to 7 h, and even more preferably 5.25 h.

(10) The present disclosure further provides use of the method in improving a yield and heat resistance of a crop.

(11) The present disclosure further provides use of the method in mutation breeding.

(12) The present disclosure further provides a method for mutagenizing a crop seed by HVCP, including the following steps: (1) a crop seed is mixed with water and immersed in water for 4 h to 24 h to obtain an immersed crop seed; and (2) the immersed crop seed is discharged to activate at a voltage of 80 kV to 130 kV to obtain a mutagenized crop seed.

(13) In the present disclosure, the crop seed is preferably derived from a crop selected from the group consisting of rice, wheat, corn, rapeseed, soy bean, tomato, chili, eggplant, cucumber, muskmelon, pumpkin, kidney bean, radish, Brassica oleracea, Brassica juncea, Chinese cabbage, and watercress, more preferably Brassica campestris ssp. chinensis Makino. In the present disclosure, the crop seed is mixed with water at a mass-to-volume ratio of preferably 1:(4-6), more preferably 1:5: the crop seed is immersed preferably in the dark at a water temperature of preferably 18 C. to 22 C., more preferably 20 C. for preferably 8 h to 20 h, more preferably 14 h.

(14) In the present disclosure, the voltage is preferably 105 kV. The immersed crop seed is discharged to activate at preferably 18 C. to 25 C., more preferably 21.5 C. for preferably 0.5 h to 10 h, more preferably 3.5 h to 7 h, and even more preferably 5.25 h.

(15) The technical solutions provided by the present disclosure will be described in detail below with reference to examples, but the examples should not be construed as limiting the claimed scope of the present disclosure.

(16) In these examples, a Brassica campestris ssp. chinensis Makino cultivar, Suzhouqing, is selected as the material for experiment.

(17) In these examples of the present disclosure, germination rate=(number of germinated seeds/number of tested seeds)100%, which is counted on day 7 of accelerating germination:

(18) germination potential=(number of germinated seeds/number of tested seeds)100%, which is counted on day 3 of accelerating germination; and

(19) survival rate=(number of surviving plants/total number of plants)100%, which is counted on day 30 after planting.

Example 1

(20) A Brassica campestris ssp. chinensis Makino material Suzhouqing was selected to use, and its seeds were added to a petri dish with water at a mass-to-volume ratio of 1:5, and subjected to a water absorption in the dark at 20 C. for 8 h to obtain immersed seeds.

(21) The immersed seeds were randomly divided into 3 groups. The first group was discharged to activate at a voltage of 80 kV. The second group was discharged to activate at a voltage of 100 kV. The third group was discharged to activate at a voltage of 130 kV. The three groups of seeds were all discharged to activate at 20 C. for 4 h, to obtain activated seeds.

(22) In each group. 50 activated seeds were selected and cultured for 3 d at 20 C. in 16 h light/8 h dark. The germination potential of the activated seeds in each group was counted, and the germination rate of the activated seeds in each group was counted on the 7th day. The results were shown in FIG. 1.

(23) As shown in FIG. 1, under different voltage treatments, the germination rate and germination potential of seeds swelled by water absorption for 8 h at 80 kV and 100 kV had no obvious change. As the treatment voltage increased, when the voltage reached 130 kV, the germination rate and the germination potential were significantly inhibited, which were down to 40% and 65%, respectively. The results revealed that the inhibition of germination rate and germination potential of Brassica campestris ssp. chinensis Makino induced by HVCP had the most obvious effectiveness when the treatment voltage was 130 kV.

Example 2

(24) A Brassica campestris ssp. chinensis Makino material Suzhouqing was selected to use, and its seeds were added to a petri dish with water at a mass-to-volume ratio of 1:5, and subjected to a water absorption in the dark at 20 C. for 8 h to obtain immersed seeds.

(25) After germinated seeds were discarded, the immersed seeds were divided into 10 groups. The first group was discharged to activate for 0.5 h at a constant peak voltage of 130 kV. The second group was discharged to activate for 1 h at a constant peak voltage of 130 kV. The third group was discharged to activate for 1.5 h at a constant peak voltage of 130 kV. The fourth group was discharged to activate for 2 h at a constant peak voltage of 130 kV. The fifth group was discharged to activate for 3 h at a constant peak voltage of 130 kV. The sixth group was discharged to activate for 4 h at a constant peak voltage of 130 kV. The seventh group was discharged to activate for 5 h at a constant peak voltage of 130 kV. The eighth group was discharged to activate for 6 h at a constant peak voltage of 130 kV. The ninth group was discharged to activate for 8 h at a constant peak voltage of 130 kV. The tenth group was discharged to activate for 10 h at a constant peak voltage of 130 kV. The ten groups of seeds were all discharged to activate at 20 C. Seeds without activation were used as a control.

(26) In each group, 50 seeds were selected and cultured for 3 d at 20 C. in 16 h light/8 h dark. The germination potential of the seeds in each group was counted, and a germination rate of the seeds in each group was counted on the 7th day. The results are shown in FIG. 2.

(27) As shown in FIG. 2, compared with the control, with the prolongation of treatment time, both germination rate and germination potential were inhibited. The germination rate and germination potential were rapidly inhibited at 2 h to 4 h of treatment, where the germination rate decreased from 82% (2 h) to 60% (4 h), and the germination potential decreased from 64% (2 h) to 32% (4 h), and then tended to be stable. The results revealed that the inhibition of germination rate and germination potential of Brassica campestris ssp. chinensis Makino induced by HVCP were obvious effected when the treatment time was 2 h to 4 h.

Example 3

(28) A Brassica campestris ssp. chinensis Makino material Suzhouqing was selected to use, and its seeds were added to a petri dish with water at a mass-to-volume ratio of 1:4, and subjected to a water absorption in the dark at 20 C. for 8 h to obtain immersed seeds.

(29) After germinated seeds were discarded, the immersed seeds were divided into 10 groups. The first group was discharged to activate for 0.5 h at a constant peak voltage of 130 kV. The second group was discharged to activate for 1 h at a constant peak voltage of 130 kV. The third group was discharged to activate for 1.5 h at a constant peak voltage of 130 kV. The fourth group was discharged to activate for 2 h at a constant peak voltage of 130 kV. The fifth group was discharged to activate for 3 h at a constant peak voltage of 130 kV. The sixth group was discharged to activate for 4 h at a constant peak voltage of 130 kV. The seventh group was discharged to activate for 5 h at a constant peak voltage of 130 kV. The eighth group was discharged to activate for 6 h at a constant peak voltage of 130 kV. The ninth group was discharged to activate for 8 h at a constant peak voltage of 130 kV. The tenth group was discharged to activate for 10 h at a constant peak voltage of 130 kV. The ten groups of seeds were all discharged to activate at 20 C. Seeds without activation were used as a control group.

(30) In each group, 200 seeds were selected to be sown in plug trays, and grown under 16 h light/20 C., and 8 h dark/16 C. After 26 d of cultivation, seedlings were planted in the field and subjected to field management according to conventional methods. A survival rate of the seedlings was counted separately after 30 d, 60 d, and 90 d of planting in the field. The results were shown in FIG. 3.

(31) As shown in FIG. 3, compared with the control, with the prolongation of treatment time, the survival rate showed a downward trend. The decrease in the survival rate was more obvious at 2 h to 4 h of treatment. The survival rate of 30 d of planting decreased from 60% (2 h) to 30% (4 h), the survival rate of 60 d of planting decreased from 50% (2 h) to 34% (4 h), and the survival rate of 90 d of planting decreased from 38% (2 h) to 30% (4 h), and then the survival rate was even lower. The results revealed that HVCP could inhibit the survival rate of Brassica campestris ssp. chinensis Makino. The treatment for 2 h to 4 h was appropriate, which could ensure the survival rate reaching 50% (30 d after planting), creating mutants, producing more forward mutants up to 16.5%.

Example 4

(32) After the plants in each group of Example 3 were planted for 30 d, the growth and development of plants in each group were investigated.

(33) Results: a variety of mutants with positive and negative traits were observed in the plots treated with high-pressure plasma for 2 h and 4 h. There were 33 plants showing better plant type, disease resistance, leaf color (darkness), and heat resistance than those of the control. In addition, there were more reverse mutants in the 2 h treatment group, and the forward mutants did not receive seeds due to weak disease resistance. The results of the obtained forward mutant strains were shown in FIG. 4.

Example 5

(34) 9 SSR markers (including BeVIN3_A02. BeAGL24_A01. BeFRI_A03. BeFLC5_A03. BcFLC1_A10, BeFLC2_A02. BeAGL24_A03. BeFT_A02, and BeFLM_A08) closely linked to candidate genes regulating flowering time in temperature pathway were used. The DNAs of control CK, mutant strain M.sub.0 (generation 0), and mutant strain M.sub.1 (generation 1) were used as templates, and polymorphisms were detected with polyacrylamide. To amplify BeVIN3_A02, a forward primer was: 5-CCACCAGAACTGGCTCTCAT-3 (SEQ ID NO: 1), and a reverse primer was: 5-GAGGGAGGACCTGGAGTTGT-3 (SEQ ID NO: 2). To amplify BcAGL24_A01, a forward primer was: 5-TCTTTGTGATGCCGATGTTG-3 (SEQ ID NO: 3), and a reverse primer was: 5-GATATCTCCAAGCCCGAGAA-3 (SEQ ID NO: 4). To amplify BeFRI_A03, a forward primer was: 5-CTGATACTGCTGCTCCATCG-3 (SEQ ID NO: 5), and a reverse primer was: 5-AGGGAGGGAGTCCTCCATAA-3 (SEQ ID NO: 6). To amplify BeFLC5_A03, a forward primer was: 5-ACGCCGAGATAATGCAGAAG-3 (SEQ ID NO: 7), and a reverse primer was: 5-GTATATTCCGACGCCCTCAA-3 (SEQ ID NO: 8). To amplify BeFLC1_A10, a forward primer was: 5-CTTCCTGCGAATCTTGTGTG-3 (SEQ ID NO: 9), and a reverse primer was: 5-TATGCATCACAGCGTGTCAA-3 (SEQ ID NO: 10). To amplify BcFLC2_A02, a forward primer was: 5-AGGGAAACTAATACAATACGCAA-3 (SEQ ID NO: 11), and a reverse primer was: 5-GTCGACTCCCTCGTTCAGC-3 (SEQ ID NO: 12). To amplify BeAGL.24_A03, a forward primer was: 5-TGGCAAAAATTTGGTAACGA-3 (SEQ ID NO: 13), and a reverse primer was: 5-ATATTGTGCTGCTGCATTGG-3 (SEQ ID NO: 14). To amplify BcFT_A02, a forward primer was: 5-GACGACAGCTTCGAAAGAGA-3 (SEQ ID NO: 15), and a reverse primer was: 5-TGAGCATTGTTTTGGTGATG-3 (SEQ ID NO: 16). To amplify BelIM_A08, a forward primer was: 5-CCAAAAAGCCGAAAAACAGA-3 (SEQ ID NO: 17), and a reverse primer was: 5-TTTGGAACCACCAAGTTGAA-3 (SEQ ID NO: 18). The specific sequences were set forth in the SEQ ID NO: 1 to SEQ ID NO: 18. The genetic diversity results were shown in FIG. 5. The primers were set according to the genome sequence of CK, which was published in website (http://tbir.njau.edu.cn/NhCCDbHubs/). If the M.sub.0 and M.sub.1 generations amplified by the primers were different from CK, it indicated that the M.sub.0 and M.sub.1 generations were mutated.

(35) As shown in FIG. 5, BcAGL24_A01 and BcFLC5_A03 had obvious marks on M.sub.0 and M.sub.1 where the size of the PCR product was, while CK had no such marks. The results revealed that plasma mutagenesis of Brassica campestris ssp. chinensis Makino could obtain excellent mutant strains, which could be stably inherited.

(36) As can be seen from the above examples, the present disclosure provides a method for activating a crop seed by HVCP, and use thereof. In the present disclosure, the method can improve the heat resistance and disease resistance of the seeds, and provide a scientific basis for subsequent acquisition of excellent seeds.

(37) The above are merely preferred embodiments of the present disclosure. It should be noted that several improvements and modifications may further be made by those of ordinary skill in the art without departing from the principle of the present disclosure, and such improvements and modifications should also be deemed as falling within the claimed scope of the present disclosure.