CN108055893A - A kind of processing method for improving seed vitality - Google Patents

Abstract

The invention discloses a treatment method for improving seed vigor, comprising the following steps: 1) artificially synthesizing miR168a, the nucleotide sequence of which is shown in SEQ ID 1; then dissolving the synthesized miR168a in sterile purified water to obtain exogenous miR168a solution; 2) Soak the seeds in the miR168a solution to improve the vigor of the seeds. The present invention mainly uses exogenous miR168a to pre-germinate seeds to improve seed vigor, promote germination of low vigor seeds, increase seed germination rate, and promote growth of seeds' roots and buds.

Description

A treatment method for improving seed vigor

technical field

The invention belongs to the technical field of agriculture, and in particular relates to a treatment method for improving seed vigor.

Background technique

At present, the germination treatment of most seeds is to sterilize the seeds, soak the seeds in water at a suitable temperature, and then germinate them. However, during the storage of seeds, due to the influence of internal and external factors, the vitality of the seeds will be reduced or even inactivated, so there will always be some seeds that still have certain vitality but cannot germinate in the seed batch. Therefore, how to promote the germination of low vigor seeds is the key to improving the germination rate of seeds.

Contents of the invention

The purpose of the invention is to provide a treatment method for improving seed vigor to solve the defect that low vigor seeds are difficult to germinate.

The treatment method for improving seed vigor of the present invention comprises the following steps:

1) Artificially synthesizing miR168a, the nucleotide sequence of which is shown in SEQ ID 1; then dissolving the synthesized miR168a in sterile purified water to obtain an exogenous miR168a solution;

2) The seeds are soaked in the miR168a solution to improve the vigor of the seeds.

In the step 1), the concentration of the exogenous miR168a solution is 0.5-1.5 μg/ml.

In the step 2), the specific steps of soaking the seeds in the miR168a solution are as follows: after sterilizing and cleaning the seeds to be germinated, soak them in the exogenous miR168a solution for 1 day, then take out the seeds, and then use water as the germination matrix , and germinate the seeds at a set temperature.

In the step 2), the specific steps of soaking the seeds in the miR168a solution are as follows: completely immerse the seeds in the exogenous miR168a solution for 2 to 3 hours, air-dry the seeds, and store them in sealed packaging; store until the seeds are to be germinated, After the seeds were sterilized, cleaned and soaked in exogenous miR168a solution for 1 day, the seeds were taken out, and then the seeds were germinated at a set temperature using water as the germination substrate.

During the germination, the mass volume ratio (W:V=g/ml) of the seed to the exogenous miR168a solution is 1:(1.5-2); the set temperature is 27-30° C.; the germination time is 3-7 days.

miR168a belongs to one kind of miRNA. miRNA is a kind of single-stranded short RNA sequence of non-coding protein; plant miRNA can regulate the whole life process of plant growth and development. Studies have shown that seed vigor is related to the expression of miR168a, and the higher the expression of miR168a, the higher the seed vigor. Therefore, it is speculated that exogenous miR168a can also promote the improvement of seed vigor.

Beneficial effects of the present invention: the present invention mainly adopts the exogenous miR168a to treat the seeds, improve the vigor of the seeds, promote the germination of low vigor seeds, increase the germination rate of the seeds, and promote the growth of the roots and buds of the seeds.

Description of drawings

Figure 1 Comparison of seedling morphology after germination of rice Wuyou 308 seeds (harvested in 2015, stored for 2 years) in Example 6 (A, miR168a treatment) and Comparative Example 6 (B, water treatment).

Fig. 2 Comparison of seedling morphology after germination of rice Wuyou 308 seeds (harvested in 2016, stored for 1 year) in Example 7 (A, miR168a treatment) and Comparative Example 7 (B, water treatment).

Fig. 3 Comparison of seedling morphology after germination of rice Fengliangyousi seedlings (harvested in 2015, stored for 2 years) in Example 8 (A, miR168a treatment) and Comparative Example 8 (B, water treatment).

After seed germination in Fig. 4 embodiment 9-13 and comparative example 9-13, take the DNA of seedling as template, take the comparison of the PCR product of primer with ISSR UBC-808 sequence (5'-AGAGAGAGAGAGAGAGC-3'); 1: H ₂ O treatment; 2: exogenous miR168a solution treatment.

Detailed ways

Example 1

The nucleotide sequence of miR168a (exogenous miR168a) was artificially synthesized, and then the exogenous miR168a was dissolved in sterile purified water to prepare a 1.0 μg/ml aqueous solution.

Wash the rice phoenix seedling seeds harvested in 2013 and stored for 4 years with sterile pure water for 3 to 4 times, and then soak in 75% alcohol for 1 min; after draining the alcohol, soak in 3% sodium hypochlorite , shake at 180rpm for 30min, discard the solution, wash the seeds with sterile pure water 4 to 5 times, put them on filter paper to absorb the surface moisture. Put the sterilized seeds in a Φ11.0cm petri dish, soak the seeds in exogenous miR168a solution for 1d [the mass-volume ratio of seeds to exogenous miR168a solution (W:V=g/ml) is 1:1.5], after soaking, pour Remove the residual exogenous miR168a solution, then use pure water as the germination substrate, and place it in a constant temperature incubator at 28±1°C for germination experiments. After 7 days of germination treatment, the seed germination rate, seedling root and shoot length, and seed vigor index were calculated, and the results are shown in Table 1.

Germination rate=(number of germinated seeds/total number of test seeds)×100%;

Vitality index = germination rate (%) × root length (cm).

Comparative example 1

In Example 1, the miR168a solution in the soaking treatment of rice phoenix seedling seeds was replaced with water, and other operations and conditions remained unchanged. The seed germination rate, seedling root length and bud length, and seed vigor index were calculated, and the results are shown in Table 1.

Table 1 Effect of exogenous miR168a on seed germination of rice Fengliangyousi seedlings stored for 4 years

*: The number in brackets is the year of seed harvesting, the same below.

As can be seen from Table 1, compared with Comparative Example 1, the germination rate of Fengliangyou silk seedlings in Example 1 was increased from 74% to 80%, and its root length, bud length and vitality index were also greater than Comparative Example 1, indicating that miR168a solution Soaking increases the vigor level of the seeds, promotes the germination of the seeds, improves the germination rate of the seeds, and accelerates the growth of their roots and shoots.

Example 2

The nucleotide sequence of miR168a (exogenous miR168a) was artificially synthesized, and then the exogenous miR168a was dissolved in sterile purified water to prepare a 1.0 μg/ml aqueous solution.

The rice Fengliangyousi seedlings harvested in 2013 and stored for 4 years were completely soaked in the exogenous miR168a solution. After soaking for 2 hours, the seeds were air-dried to reduce the water content of the seeds to a safe storage water content of 10-12 %, put it in a kraft paper bag; keep it sealed for 3 months.

Wash the seeds stored for 3 months with sterile pure water for 3 to 4 times, and then soak them in 75% alcohol for 1 minute; drain the alcohol, soak them in 3% sodium hypochlorite, shake at 180rpm for 30 minutes, discard the solution, The seeds were washed 4 to 5 times with sterile pure water, and put on filter paper to absorb the surface moisture. Put the sterilized seeds in a Φ11.0cm petri dish, soak the seeds in exogenous miR168a solution for 1d [the mass-volume ratio of seeds to exogenous miR168a solution (W:V=g/ml) is 1:1.5], after soaking, pour Remove the residual exogenous miR168a solution, then use pure water as the germination substrate, and place it in a constant temperature incubator at 28±1°C for germination experiments. After 7 days of germination treatment, the seed germination rate, seedling root length and shoot length, and seed vigor index were calculated, and the results are shown in Table 2.

Comparative example 2

Soak the rice Fengliangyousi seedlings harvested in 2013 and stored for 4 years in water for 2-3 hours, then air-dry to reduce the water content of the seeds to 10-12% of the safe storage water content, and then pack them into kraft paper bag; sealed for 3 months.

The germination test of the seeds preserved for 3 months was carried out according to the method of Comparative Example 1, and the results of seed germination are shown in Table 2.

Table 2 Effects of exogenous miR168a soaking storage and soaking before germination on seed germination of rice Fengliangyousi seedlings

As can be seen from Table 2, compared with Comparative Example 2, the germination rate of Fengliangyou silk seedlings in Example 2 is increased from 82% to 97%, and its root length and shoot length are also longer than Comparative Example 2, indicating that the seeds are soaked in miR168a solution Post-storage and soaking treatment with miR168a solution before germination can further promote the germination of low vigor seeds (compared with Example 1), increase the germination rate of seeds, and also accelerate the growth of their roots and shoots.

Example 3

The nucleotide sequence of miR168a (exogenous miR168a) was artificially synthesized, and then the exogenous miR168a was dissolved in sterile purified water to prepare a 1.5 μg/ml aqueous solution.

Wash the seeds of rice Fengliangyousi seedlings harvested in 2015 and stored for 2 years with sterile pure water for 3 to 4 times, then soak them in 75% alcohol for 1 minute, drain the alcohol, and wash them with 3% sodium hypochlorite Soak, shake at 180rpm for 30min, discard the solution, wash the seeds with sterile pure water 4 to 5 times, put them on filter paper to absorb the surface moisture. Put the sterilized seeds in a Φ11.0cm petri dish, soak the seeds in exogenous miR168a solution for 1d [the mass-volume ratio of seeds to exogenous miR168a solution (W:V=g/ml) is 1:2], after soaking, pour The remaining exogenous miR168a solution was removed, and then pure water was used as the germination matrix, and it was placed in a constant temperature incubator at 28±1°C for germination test. After 6 days of germination treatment, the seed germination rate, seedling root length and shoot length, Seed vigor index, the results are shown in Table 3.

Comparative example 3

In Example 3, the miR168a solution in the soaking treatment of rice phoenix seedling seeds was replaced with water, and other operations and conditions remained unchanged. Calculate seed germination rate, seedling root length and bud length, and seed vigor index, and the results are shown in Table 3.

Table 3 Effects of exogenous miR168a on seed germination of rice Fengliangyousi seedlings stored naturally for 2 years

From comparative example 1 and comparative example 3, it can be known that the seed vigor of rice Fengliangyousi seedlings stored for 2 years after harvest in 2015 is higher than that of 2013 after harvest and stored for 4 years, indicating that the longer the storage time of rice seeds , the lower the seed vigor. As can be seen from Table 3, compared with Comparative Example 3, the germination rate of Fengliangyousi seedlings in Example 3 is increased to 95.0% from 93.0%, and its root length and bud length are also better than Comparative Example 3, indicating that for storage for 2 years The seeds of the high-viability Fenniferus seedlings were soaked in miR168a solution (Example 3), and the level of seed vigor could still be further improved.

Example 4

The nucleotide sequence of miR168a (exogenous miR168a) was artificially synthesized, and then the exogenous miR168a was dissolved in sterile purified water to prepare a 1.0 μg/ml aqueous solution.

The rice Fengliangyousi seedlings harvested in 2015 and stored for 2 years were completely soaked in the exogenous miR168a solution. After soaking for 2-3 hours, the seeds were air-dried to reduce the water content of the seeds to 10% of the safe storage water content. After ~12%, put it into a kraft paper bag; keep it sealed for 4 months.

Wash the seeds of rice phoenix seedlings that have been stored for 4 months with sterile pure water for 3 to 4 times, then soak them in 75% alcohol for 1 minute, drain the alcohol, soak them in 3% sodium hypochlorite, and vibrate at 180rpm. Shake for 30 minutes, discard the solution, wash the seeds with sterile pure water 4 to 5 times, and put them on filter paper to absorb the surface moisture. Put the sterilized seeds in a Φ11.0cm petri dish, soak the seeds in exogenous miR168a solution for 1d [the mass-volume ratio of seeds to exogenous miR168a solution (W:V=g/ml) is 1:2], after soaking, pour The remaining exogenous miR168a solution was removed, and then pure water was used as the germination matrix, and it was placed in a constant temperature incubator at 28±1°C for germination test. After 7 days of germination treatment, the seed germination rate, seedling root length and shoot length, Seed vigor index, the results are shown in Table 4.

Comparative example 4

Soak the rice Fengliangyousi seedlings harvested in 2015 and stored for 2 years in water completely. After soaking for 3 hours, air dry the seeds to reduce the water content of the seeds to 10-12% of the safe storage water content. Put into a kraft paper bag; keep sealed for 4 months.

The seed germination treatment of rice phoenix seedlings stored for 4 months was carried out according to the method of Comparative Example 3, and the results of seed germination are shown in Table 4.

Table 4 Effects of exogenous miR168a soaking storage and soaking before germination on seed germination of rice Fengliangyousi seedlings

As can be seen from Table 4, compared with Comparative Example 4, the germination rate of rice phoenix seedling seedlings processed in Example 4 was harvested in 2015 and stored for 2 years by 93.5% to 97.5%. The growth rate was also significantly better than that of Comparative Example 4, indicating that exogenous miR168a soaked and stored, and then soaked in miR168a solution before germination can maintain the vigor level of rice seeds with high vigor, ensure a high germination rate of seeds and seedlings after germination. Normal growth of shoots and roots.

Example 5

The nucleotide sequence of miR168a (exogenous miR168a) was artificially synthesized, and then the exogenous miR168a was dissolved in sterile purified water to prepare a 1.0 μg/ml aqueous solution.

The rice Zhongliangyou 2727 seeds harvested in 2016 and stored for one year were first washed 3 to 4 times with sterile pure water, then soaked in 75% alcohol for 1 min, drained the alcohol, and soaked in 3% sodium hypochlorite. Shake at 180rpm for 30min, discard the solution, wash the seeds with sterile pure water 4 to 5 times, and put them on filter paper to absorb the surface moisture. Put the sterilized seeds in a Φ11.0cm petri dish, soak the seeds in the exogenous miR168a solution for 1d [the mass-volume ratio of the seeds to the exogenous miR168a solution (W:V=g/ml) is 1:2], after soaking, Pour off the remaining exogenous miR168a solution, then use pure water as the germination matrix, and place it in a constant temperature incubator at 27±1°C for germination test. After 6 days of germination treatment, calculate the seed germination rate, seedling root length and shoot length , Seed vigor index, the results are shown in Table 5.

Comparative example 5

The miR168a solution when soaking the seeds in Example 5 was replaced with water, and other operations and conditions remained unchanged. Calculate seed germination rate, seedling root length and bud length, seed vigor index, and the results are shown in Table 5.

Table 5 Effects of exogenous miR168a on seed germination of Liangyou 2727 rice stored for 1 year

The results in Table 5 show that the seeds of Liangyou 2727 in rice are typical seeds that are not resistant to storage. After being harvested in 2016 and stored for one year, the germination rate of the seeds was reduced to 68%; but using miR168a solution (Example 5) for The soaking treatment of the seeds can increase the germination rate of the seeds to 80%, the root length and the shoot length of the seeds are also obviously increased, and the vigor index of the seeds is obviously improved.

Example 6

The nucleotide sequence of miR168a (exogenous miR168a) was artificially synthesized, and then the exogenous miR168a was dissolved in sterile purified water to prepare a 0.5 μg/ml aqueous solution.

The rice Wuyou 308 seeds harvested in 2015 and stored for 2 years were artificially aged for 8 days at 42°C and 100% RH. Wash the aged seeds with sterile pure water for 3 to 4 times, then soak in 75% alcohol for 1 min, drain the alcohol, soak in 3% sodium hypochlorite, shake at 180rpm for 30 min, discard the solution, and use sterile water for the seeds. Wash with pure water 4 to 5 times, put on filter paper to absorb the surface moisture. Place the sterilized seeds in a Φ11.0cm petri dish, soak the seeds in the miR168a solution for 1d [the mass-volume ratio of the seeds to the exogenous miR168a solution (W:V=g/ml) is 1:1.5], pour it out after soaking The residual exogenous miR168a solution was then used as the germination matrix in purified water, and placed in a constant temperature incubator at 28±1°C for germination test. After 6 days of germination treatment, the seed germination rate, seedling root length and shoot length, and seed germination rate were calculated. The vigor index, the results are shown in Table 6; the seedling morphology after germination is shown in Figure 1-A.

Comparative example 6

The miR168a solution in Example 6 when the seeds were soaked was replaced with water, and other conditions and operations remained unchanged. Calculate the seed germination rate, seedling root length and bud length, and seed vigor index, and the results are shown in Table 6; the seedling morphology after germination is shown in Figure 1-B.

Table 6 Effect of exogenous miR168a on germination of artificially aged rice Wuyou 308 seeds

After the artificial aging treatment, the vigor of rice Wuyou 308 seeds harvested in 2015 and stored for 2 years was greatly reduced, and the germination rate was only 10% during the conventional germination treatment (comparative example 6); soaking treatment with miR168a solution (implementation Example 6), the germination rate can be significantly increased to 40%, and the shoot length, root length and vigor index are significantly higher than those of Comparative Example 6 (Table 6, Figure 1).

Example 7

The nucleotide sequence of miR168a (exogenous miR168a) was artificially synthesized, and then the exogenous miR168a was dissolved in sterile purified water to prepare a 1.0 μg/ml aqueous solution.

The rice Wuyou 308 seeds harvested in 2016 and stored for one year were artificially aged for 8 days at 42°C and 100% RH. Wash the aged seeds with sterile pure water for 3 to 4 times, then soak in 75% alcohol for 1 min, drain the alcohol, soak in 3% sodium hypochlorite, shake at 180rpm for 30 min, discard the solution, and use sterile water for the seeds. Wash with pure water 4 to 5 times, put on filter paper to absorb the surface moisture. Place the sterilized seeds in a Φ11.0cm petri dish, soak the seeds in the miR168a solution for 1d [the mass-volume ratio of the seeds to the exogenous miR168a solution (W:V=g/ml) is 1:2], pour it out after soaking The residual exogenous miR168a solution was then placed in a constant temperature incubator at 28±1°C for germination experiments using pure water as the germination substrate. After 7 days of germination treatment, the seed germination rate, seedling root length and bud length, and seed vigor index were calculated, and the results are shown in Table 7; the morphology of the germinated seedlings is shown in Figure 2-A.

Comparative example 7

The miR168a solution in which the seeds were soaked in Example 7 was replaced with water, and other conditions and operations remained unchanged. The seed germination rate, seedling root length and bud length, and seed vigor index were calculated, and the results are shown in Table 7; the morphology of the seedling after germination is shown in Figure 2-B.

Table 7 Effect of exogenous miR168a on germination of artificially aged rice Wuyou 308 seeds

After the aging treatment, the vigor of rice Wuyou 308 seeds harvested in 2016 and stored for 1 year was greatly reduced, and the germination rate was only 22% during the conventional germination treatment (comparative example 7); soaking treatment with miR168a solution (Example 7) 7), the seed germination rate can be increased to 32%; root length, shoot length and vigor index are also improved (Table 7, Fig. 2).

Example 8

The nucleotide sequence of miR168a (exogenous miR168a) was artificially synthesized, and then the exogenous miR168a was dissolved in sterile purified water to prepare a 1.0 μg/ml aqueous solution.

The seeds of rice Fengliangyousi seedlings harvested in 2015 and stored for 2 years were artificially aged for 8 days under the conditions of 42°C and 100% RH. Wash the aged seeds with sterile pure water for 3 to 4 times, then soak in 75% alcohol for 1 min, drain the alcohol, soak in 3% sodium hypochlorite, shake at 180rpm for 30 min, discard the solution, and use sterile water for the seeds. Wash with pure water 4 to 5 times, put on filter paper to absorb the surface moisture. Place the seeds in a Φ11.0cm petri dish, soak the seeds in the miR168a solution for 1d [the mass-to-volume ratio of the seeds to the exogenous miR168a solution (W:V=g/ml) is 1:1.5], and pour off the remaining The exogenous miR168a solution was then used as the germination substrate in purified water, and placed in a constant temperature incubator at 28±1°C for germination experiments. After 7 days of germination treatment, the seed germination rate, seedling root length and bud length, and seed vigor index were calculated. The results are shown in Table 8, and the morphology of the seedling after seed germination is shown in Figure 3-A.

Comparative example 8

The miR168a solution in which the seeds were soaked in Example 8 was replaced with water, and other conditions and operations remained unchanged. The seed germination rate, seedling root length and bud length, and seed vigor index were calculated, and the results are shown in Table 8; the morphology of the seedling after seed germination is shown in Figure 3-B.

Table 8 Effects of exogenous miR168a on seed germination of artificially aged rice Fengliangyousi seedlings

After the seed aging treatment, the vitality of the rice Fengliangyousi seedlings harvested in 2015 and stored for 2 years was greatly reduced, and the germination rate was only 18% in the conventional germination treatment (comparative example 8); soaking treatment with miR168a solution (Example 8) can make its germination rate be increased to 34%, and bud length, root length and vigor index also obviously improve (table 8, Fig. 3).

Example 9

The nucleotide sequence of miR168a (exogenous miR168a) was artificially synthesized, and then the exogenous miR168a was dissolved in sterile purified water to prepare a 1.0 μg/ml aqueous solution.

Wash the tomato (thick skin and early abundance) seeds purchased from the seed company 3 to 4 times with sterile pure water, then soak for 1 min with 75% alcohol, drain the alcohol, soak with 3% sodium hypochlorite, and shake at 180rpm After 30 minutes, the solution was discarded, the seeds were washed 4 to 5 times with sterile pure water, and the surface moisture was dried on filter paper. Place the sterilized seeds in a Φ11.0cm petri dish, soak the seeds in the miR168a solution for 1d [the mass-volume ratio of the seeds to the exogenous miR168a solution (W:V=g/ml) is 1:1.5], pour it out after soaking The remaining exogenous miR168a solution was then used as the germination matrix in purified water, and placed in a constant temperature incubator at 28±1°C for germination test. After germination treatment for 7 days, the seed germination rate, seedling root length and shoot length, Seed vigor index, the results are shown in Table 9.

Comparative example 9

In Example 9, the exogenous miR168a solution used for soaking the seeds of tomato (Pachyderma early abundance) was replaced with water, and other conditions and operations remained unchanged. Calculate seed germination rate, seedling root length and bud length, seed vigor index, and the results are shown in Table 9.

Table 9 Effects of exogenous miR168a on seed germination of tomato (thick skin and early abundance)

As can be seen from Table 9, compared with Comparative Example 9, the germination rate of tomato seeds in Example 9 is increased from 40% to 46%, and the vitality index of the seeds is increased from 0.95 to 1.50, indicating that soaking in miR168a solution can improve the vitality level of tomato seeds .

Example 10

The nucleotide sequence of miR168a (exogenous miR168a) was artificially synthesized, and then the exogenous miR168a was dissolved in sterile purified water to prepare a 1.0 μg/ml aqueous solution.

Wash the cucumber (Jinyou) seeds purchased from the seed company 3 to 4 times with sterile pure water, then soak them in 75% alcohol for 1 minute, drain the alcohol, soak them in 3% sodium hypochlorite, and shake them at 180 rpm for 30 minutes. Discard the solution, wash the seeds with sterile pure water 4 to 5 times, and put them on filter paper to absorb the surface moisture. Place the sterilized seeds in a Φ11.0cm petri dish, soak the seeds in the miR168a solution for 1d [the mass-volume ratio of the seeds to the exogenous miR168a solution (W:V=g/ml) is 1:1.5], pour it out after soaking The residual exogenous miR168a solution was then used as the germination matrix in purified water, and placed in a constant temperature incubator at 27±1°C for germination test. After 7 days of germination treatment, the seed germination rate, seedling root length and shoot length, and seed germination rate were calculated. The results are shown in Table 10.

Comparative example 10

The exogenous miR168a solution soaked in cucumber (Jinyou) seeds in Example 10 was replaced with water, and other conditions and operations remained unchanged. Calculate the vigor index of seed germination rate, seedling root length and bud length, seed, and the results are shown in Table 10.

Table 10 Effect of exogenous miR168a on germination of cucumber (Jinyou) seeds

The germination rate of cucumber (Jinyou) seeds in this test is relatively high, indicating that its seed vigor is already high; however, soaking treatment with the miR168a solution of the present invention (Example 10) still has the effect of improving its seed vigor.

Example 11

The nucleotide sequence of miR168a (exogenous miR168a) was artificially synthesized, and then the exogenous miR168a was dissolved in sterile purified water to prepare a 1.0 μg/ml aqueous solution.

The mung bean (Lvbao) seeds purchased from the seed company were first washed 3 to 4 times with sterile pure water, then soaked in 75% alcohol for 1 min, drained the alcohol, soaked in 3% sodium hypochlorite, and shaken at 180 rpm for 30 min. Discard the solution, wash the seeds with sterile pure water 4 to 5 times, and put them on filter paper to absorb the surface moisture. Place the sterilized seeds in a Φ11.0cm petri dish, soak the seeds in the miR168a solution for 1d [the mass-volume ratio of the seeds to the exogenous miR168a solution (W:V=g/ml) is 1:1.5], pour it out after soaking The remaining exogenous miR168a solution was then used as the germination matrix in pure water, and placed in a constant temperature incubator at 28±1°C for germination test. After 7 days of germination treatment, the seed germination rate, seedling root length and shoot length, and seed germination rate were calculated. The results are shown in Table 11.

Comparative example 11

The exogenous miR168a solution used to soak mung bean (Lvbao) seeds in Example 11 was replaced with water, and other conditions and operations remained unchanged. Calculate seed germination rate, seedling root length and bud length, seed vigor index, and the results are shown in Table 11.

Table 11 Effect of exogenous miR168a on seed germination of mung bean (Lvbao)

The germination rate of mung bean (Lvbao) seeds in this test is relatively high, indicating that the seed vigor is already high; but soaking treatment with miR168a solution of the present invention (Example 11) can still improve the vigor level of mung bean seeds.

Example 12

The nucleotide sequence of miR168a (exogenous miR168a) was artificially synthesized, and then the exogenous miR168a was dissolved in sterile purified water to prepare a 1.0 μg/ml aqueous solution.

The pakchoi (536) seeds purchased from the seed company were first washed with sterile pure water for 3 to 4 times, then soaked in 75% alcohol for 1 min, drained the alcohol, soaked in 3% sodium hypochlorite, and shaken at 180 rpm for 30 min. Discard the solution, wash the seeds with sterile pure water 4 to 5 times, and put them on filter paper to absorb the surface moisture. Put the sterilized seeds in a Φ11.0cm petri dish, soak the seeds with miR168a solution for 1d [the mass-volume ratio of seeds to exogenous miR168a solution (W:V=g/ml) is 1:1.5], after soaking, pour The remaining exogenous miR168a solution was removed, and then pure water was used as the germination matrix, and it was placed in a constant temperature incubator at 28±1°C for germination test. After 7 days of germination treatment, the seed germination rate, seedling root length and shoot length, Seed vigor index, the results are shown in Table 12.

Comparative example 12

In Example 12, the exogenous miR168a solution soaking the pakchoi (536) seeds was replaced with water, and other conditions and operations remained unchanged. The results of seed germination rate, seedling root length and shoot length, and seed vigor index are shown in Table 12.

Table 12 Effect of exogenous miR168a on seed germination of pakchoi (536)

The germination rate of pakchoi (536) seeds in this test is relatively high, indicating that the seed vigor level is high; however, soaking treatment with miR168a solution of the present invention (Example 12) can still improve the vigor level of pakchoi seeds.

Example 13

The nucleotide sequence of miR168a (exogenous miR168a) was artificially synthesized, and then the exogenous miR168a was dissolved in sterile purified water to prepare a 1.0 μg/ml aqueous solution.

Wash the conventional rice (ZR02) seeds produced in our laboratory in 2016 and stored for one year with sterile pure water for 3 to 4 times, then soak in 75% alcohol for 1 min, drain the alcohol, and soak in 3% sodium hypochlorite , shake at 180rpm for 30min, discard the solution, wash the seeds with sterile pure water 4 to 5 times, put them on filter paper to absorb the surface moisture. Place the sterilized seeds in a Φ11.0cm petri dish, soak the seeds in the miR168a solution for 1d [the mass-to-volume ratio of the seeds to the exogenous miR168a solution (W:V=g/ml) is 1:1.5], pour it out after soaking The residual exogenous miR168a solution was then used as the germination matrix in purified water, and placed in a constant temperature incubator at 30±1°C for germination test. After germination treatment for 7 days, the germination rate of seeds, seedling root length and shoot length, Seed vigor index, the results are shown in Table 13.

Comparative example 13

In Example 13, the exogenous miR168a solution soaked in conventional rice ZR02 seeds was replaced with water, and other conditions and operations remained unchanged. The results of seed germination rate, seedling root length and shoot length, and seed vigor index are shown in Table 13.

Table 13 Effect of exogenous miR168a on seed germination of conventional rice (ZR02)

ZR02 is a conventional rice, and its seed germination rate is still 96% after one year of storage (comparative example 13), indicating that its vigor level is high. Therefore, compared with hybrid rice seeds, the seeds of conventional rice ZR02 have better storage resistance. However, soaking treatment with the miR168a solution of the present invention (Example 13) can still further improve the vigor level of ZR02 seeds.

Comparison of DNA genetic diversity of exogenous miR168a and water-treated seedlings after germination

Using ISSR technology, the DNA of seedlings after germination of tomato, cucumber, mung bean, Chinese cabbage and conventional paddy rice seeds is carried out comparative analysis of genetic polymorphism to embodiment 9-13 processing and comparative example 9-13 processing, and its result is shown in Figure 4 shown. It can be seen from Figure 4 that the DNA sequences of the germinated seedlings of different crop seeds soaked in miR168a solution are completely consistent with those of the germinated seedlings treated with water, indicating that exogenous miR168a will not change the genetic material of the seeds Structural composition, no risk of genetic modification.

sequence listing

<110> Hunan Normal University

<120> A treatment for improving seed vigor

<160> 1

<170> SIP Sequence Listing 1.0

<210> 1

<211> 21

<212> RNA

<213>miR168a

<400> 1

ucgcuuggug cagaucggga c 21

Claims (7)

1. A processing method for improving seed vigor, comprising the following steps: 1) Artificially synthesizing miR168a, the nucleotide sequence of which is shown in SEQ ID 1; then dissolving the synthesized miR168a in sterile purified water to obtain an exogenous miR168a solution; 2) The seeds are soaked in the miR168a solution to improve the vigor of the seeds. 2. The treatment method for improving seed vigor according to claim 1, characterized in that, in the step 1), the concentration of the exogenous miR168a solution is 0.5-1.5 μg/ml. 3. The treatment method for improving seed vigor according to claim 1 or 2, characterized in that, in said step 2), the specific steps of soaking and treating seeds in miR168a solution are: sterilizing and cleaning the seeds to be germinated Afterwards, soak in exogenous miR168a solution for 1 day, then take out the seeds, and then use water as the germination substrate to germinate the seeds at a set temperature. 4. The treatment method for improving seed vigor according to claim 1 or 2, characterized in that, in the step 2), the specific steps of soaking the seeds in the miR168a solution are: completely soaking the seeds in the exogenous miR168a solution 2 ~3 hours later, air-dry the seeds to a safe water content, and store in sealed packages; when the seeds are to be germinated, sterilize and clean the seeds, soak them in exogenous miR168a solution for 1 day, and then take out the seeds. Then use water as the germination substrate, and germinate the seeds at a set temperature. 5. according to the processing method of claim 3 or 4 described improving seed vigor, it is characterized in that, the mass volume ratio (W:V=g/ml) of seed and exogenous miR168a solution is 1:1.5 when described germination -2. 6. The treatment method for improving seed vigor according to claim 3 or 4, characterized in that, during the germination, the set temperature is 27-30°C. 7. The treatment method for improving seed vigor according to claim 3 or 4, characterized in that, during the germination, the germination treatment time is 3-7d.