CN111802017A - Method for improving stress resistance of corn seeds by using exogenous vitamins - Google Patents

Method for improving stress resistance of corn seeds by using exogenous vitamins Download PDF

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CN111802017A
CN111802017A CN202010804263.3A CN202010804263A CN111802017A CN 111802017 A CN111802017 A CN 111802017A CN 202010804263 A CN202010804263 A CN 202010804263A CN 111802017 A CN111802017 A CN 111802017A
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exogenous
vitamins
low temperature
corn
corn seeds
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张海燕
迟宇新
杨丽
杨克军
赵长江
林俊俊
董洁静
李佐同
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Heilongjiang Bayi Agricultural University
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01C—PLANTING; SOWING; FERTILISING
    • A01C1/00—Apparatus, or methods of use thereof, for testing or treating seed, roots, or the like, prior to sowing or planting
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01C—PLANTING; SOWING; FERTILISING
    • A01C1/00—Apparatus, or methods of use thereof, for testing or treating seed, roots, or the like, prior to sowing or planting
    • A01C1/08—Immunising seed

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Abstract

The invention provides a method for improving the stress resistance of corn seeds by using exogenous vitamins, and relates to the technical field of crop cultivation and stress regulation. The invention uses exogenous vitamin VB2And/or VB12The corn seeds are soaked, so that the seed germination is promoted, and the germination capacity and various physiological indexes of the corn seeds under the low-temperature stress with different strengths are obviously enhanced. Compared with the traditional method, the method has the advantages of cost saving, efficiency improvement, time saving, labor saving and ecological safety, and provides a theoretical basis for the alleviation of the low temperature resistance of the seeds by the exogenous vitamins.

Description

Method for improving stress resistance of corn seeds by using exogenous vitamins
Technical Field
The invention relates to the technical field of crop cultivation and stress regulation, in particular to a method for improving the stress resistance of corn seeds by using exogenous vitamins.
Background
In recent years, the climate environment is continuously changed to be an important problem faced by crops in the growing process, and various adverse conditions such as low temperature, waterlogging and the like can be encountered in the germination period of seeds, so that the seeds are pulverized, and finally economic loss is caused. Among them, low temperature is one of the important limiting factors for the production potential of crops in northern areas of China.
When plants are exposed to low temperatures, various physiological processes such as water metabolism, photosynthesis, substance metabolism, and biofilm are affected to various degrees. The low temperature is common in early spring in northeast, so that the germination of corn seeds and the growth of seedlings are inhibited, and 'powder seeds' are caused in severe cases. Therefore, it is necessary to discuss and solve the problem of corn flour seeds sowed in the northeast of China in spring, and has important theoretical and practical significance.
Vitamins are also "essential" for plant growth and development and generally play an important role in plant metabolism. Although the basic biochemical role of vitamins as co-substrates or cofactors is common to most eukaryotes, the metabolic and physiological effects of vitamins can be quite different between plants and animals. In order to explore the efficacy of vitamins in the agricultural field, not only are their effects on plant defense clarified, but also their effects on plant development and environmental responses. Among them, the research on the physiological and biochemical mechanism of the vitamins for relieving the low temperature stress of the corn seeds is rarely reported. In the current production, the low-temperature loss is mainly reduced by the technologies of stress-tolerant variety cultivation, plastic film mulching heat preservation, external application of growth regulator stress relief and the like. However, the variety breeding cycle is long, the cost of mulching film coverage is high, most hormone growth regulators are used after being stressed by adversity, and the cost investment and the labor cost are increased. Therefore, the method selects the regulating substances of the endogenous non-hormonal organic matters of the plants and realizes the low temperature resistance of the sprouts by the growth regulating technology initiated by early seeds, thereby having important theoretical and practical significance for ecological and efficient cultivation in northern cold regions.
Disclosure of Invention
The invention aims to provide a method for improving low-temperature stress resistance of corn seeds by using exogenous vitamins. The invention utilizes exogenous vitamin B2And vitamin B12After the corn seeds are treated, the low temperature resistance of the corn seeds can be obviously improved.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a method for improving the stress resistance of corn seeds by using exogenous vitamins, which comprises the following steps:
soaking corn seeds with exogenous vitamin, wherein the exogenous vitamin is VB2And/or VB12。
In the present invention, the concentration of the exogenous vitamin is 50 to 200mg/L, and more preferably 100 mg/L.
In the invention, the soaking time is 12-36h, and the more preferable soaking time is 24 h.
In the invention, the stress resistance is the low temperature stress resistance.
In the invention, the temperature of the low-temperature stress is 0-10 ℃.
In the present invention, the low temperature stress is applied for 1 to 7 days.
In the invention, the low-temperature stressed corn is recovered for 5-7 days at 22-30 ℃.
In the invention, the corn seeds are soaked in the exogenous vitamins after being disinfected.
In the present invention, the disinfection is performed by using sodium hypochlorite with a concentration of 8% to 15%, and more preferably, by using sodium hypochlorite with a concentration of 10%.
In the invention, the disinfection time of the sodium hypochlorite is 15 min-30 min, and the more preferable disinfection time is 20 min.
The invention has the following beneficial effects:
(1) the invention utilizes exogenous vitamin VB2And/or VB12Soaking semen MaydisOnly promote the seed germination, and obviously enhance the germination capacity of the corn seeds under the low-temperature stress with different strengths. Compared with the traditional method, the method has the advantages of cost saving, efficiency improvement, time saving, labor saving and ecological safety.
(2) The corn seeds soaked by exogenous vitamins are cultured for 1 to 7 days under the condition of low temperature stress at the temperature of between 0 and 10 ℃. VB at Low temperature stress 1d and 3d time2And VB12The germination rate of the corn seeds can be obviously improved by treatment; antioxidant enzyme activity is in VB2And VB12Increasing with increasing number of days of cold stress, H under treatment2O2The more obvious the inhibition effect of the content of oxygen free radical along with the increase of low-temperature stress days is, the vitamin VB is shown2And VB12The treatment can effectively remove active oxygen and reduce the damage of low temperature to the embryo.
(3) The invention researches the B-family water-soluble vitamin VB2And/or VB12The effect and the mechanism of relieving the low-temperature stress provide a theoretical basis for further disclosing the relieving of the exogenous vitamins to the low-temperature resistance of the seeds.
Drawings
FIG. 1 is a graph showing the effect of exogenous vitamin on the germination rate of corn seeds under low temperature stress after being soaked for 1 day;
FIG. 2 is a graph showing the effect of exogenous vitamins on the germination rate of maize seeds under low temperature stress after being soaked for 3 days;
FIG. 3 is a graph showing the effect of exogenous vitamin on the germination rate of maize seeds under low temperature stress after being soaked for 5 days;
FIG. 4 is a graph showing the effect of exogenous vitamin on the germination rate of maize seeds under low temperature stress after being soaked for 7 days;
FIG. 5 is a graph showing the effect of exogenous vitamin seed soaking on maize seed germination index under low temperature stress;
FIG. 6 is a graph showing the effect of exogenous vitamin seed soaking on the root-crown ratio of maize seedlings under low temperature stress;
FIG. 7 is a graph of the effect of exogenous vitamin seed soaking on soluble sugar content in maize embryos under low temperature stress;
FIG. 8 is a graph of the effect of exogenous vitamin seed soaking on proline in maize embryos under low temperature stress;
FIG. 9 shows the hydrogen peroxide (H) in maize germ under low temperature stress by exogenous vitamin seed soaking2O2) The effect of the content;
FIG. 10 shows superoxide anion (O) in maize germ under low temperature stress by exogenous vitamin seed soaking2 -) The effect of the content;
FIG. 11 is a graph of the effect of exogenous vitamin seed soaking on lipid peroxide (MDA) content in maize embryos under low temperature stress;
FIG. 12 is a graph of the effect of exogenous vitamin seed soaking on superoxide dismutase (SOD) activity in maize embryos under low temperature stress;
FIG. 13 is a graph showing the effect of exogenous vitamin seed soaking on Peroxidase (POD) activity in maize embryos under low temperature stress;
FIG. 14 is a graph of the effect of exogenous vitamin soaking on Catalase (CAT) activity in maize embryos under low temperature stress;
FIG. 15 is a graph of the effect of exogenous vitamin seed soaking on Ascorbate Peroxidase (APX) activity in maize embryos under low temperature stress.
Detailed Description
The invention provides a method for improving the stress resistance of corn seeds by using exogenous vitamins, which comprises the following specific steps: soaking corn seeds with exogenous vitamin, wherein the exogenous vitamin is VB2And/or VB12. The invention has no special limitation on the variety of the corn, and can select the corn seeds with consistent size, fullness and no damage. In the specific embodiment of the invention, the corn (Zea Mays L.) variety to be tested is Zhengdan 958, and is bred by grain crop research institute of academy of agricultural sciences in Henan province. In the present invention, the source of the exogenous vitamin is not particularly limited, and a conventional commercially available product may be used.
In the present invention, the concentration of the exogenous vitamin used is 50 to 200mg/L, and more preferably 80 to 150 mg/L.
In the invention, the seed soaking time with the exogenous vitamins is 12-36h, and the more preferable seed soaking time is 18-30 h.
In the invention, the corn seeds are soaked in the exogenous vitamins after being disinfected. The corn seed sterilization may be performed in a manner conventional in the art. In the embodiment of the present invention, the disinfection is performed by using sodium hypochlorite with a concentration of 8% to 15%, and more preferably, by using sodium hypochlorite with a concentration of 10% to 12%. In the invention, the disinfection time of the sodium hypochlorite is 15-30 min, and the more preferable disinfection time is 18-25 min. In the present invention, the source of the sodium hypochlorite is not particularly limited, and a conventional commercially available product may be used.
In the invention, the corn seeds are disinfected and then washed by distilled water for 3-5 times, and then are placed in a ventilated place for natural airing, and the more preferable washing time is 3 times.
The stress resistance is low-temperature stress resistance, and the specific treatment steps are as follows: according to the invention, the corn seeds are soaked by the technical scheme to obtain the corn seeds treated by exogenous vitamins, and then the corn seeds are uniformly sowed in the germination box and placed in the low-temperature incubator for low-temperature stress treatment to simulate outdoor low-temperature adversity. In the present invention, the germination box and the low temperature incubator are not particularly limited, and conventional apparatuses in the art may be used for cultivation.
In the present invention, the low temperature stress treatment is a treatment at 0 to 10 ℃ under dark conditions, and more preferably at 5 ℃. In the invention, the treatment time of low temperature stress is 1-7 d.
In the invention, the low-temperature stressed corn is recovered for 5-7d at the temperature of 22-30 ℃ in a plant growth chamber, and more preferably, the recovery condition is that the corn is recovered for 7d at the temperature of 25 ℃ in the plant growth chamber. In the present invention, the plant growth chamber is not particularly limited, and the plant may be cultured in a culture chamber that is conventional in the art.
The corn seeds are soaked by exogenous vitamins and then subjected to low-temperature stress treatment for 1d, 3d, 5d and 7 d. VB2And VB12The corn seedling under low temperature stress for different time periods is relieved, and the emergence rate, the germination vigor, the plant height, the root length, the dry fresh weight on the ground and the like of the corn seed are improved; has effects in promoting cell permeation regulating substance content, antioxidant substance content and antioxidant enzyme activity, and reducing embryoH in bud2O2And oxygen free radical content, which is beneficial to regulating internal osmotic potential and promoting the growth of seedlings of plants.
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The corn seed variety used in the following examples is Zhengdan 958, and was selected and bred by the institute of food crops, academy of agricultural sciences, Henan province. Other varieties of corn may also be used, as the present invention is not limited in this regard.
Example 1
Selecting corn seeds with consistent size, plump shape and no damage, sterilizing with 8% sodium hypochlorite, washing with distilled water for 3 times after sterilization, and naturally drying in the ventilated place.
Using 50mg/L vitamin B2(VB2) Soaking seeds for 36h in germination boxes, 30 seeds at a time, repeating for 3 times. Setting artificial climate box in dark at 5 deg.C, low temperature stress treating for 1, 3, 5, 7d, recovering at 22 deg.C in plant growth chamber for 7d, and placing in artificial climate box for germination.
Example 2
Selecting corn seeds with consistent size, plump shape and no damage, sterilizing with 8% sodium hypochlorite, washing with distilled water for 3 times after sterilization, and naturally drying in the ventilated place.
Using 50mg/L vitamin B12(VB12) Soaking seeds for 36h in germination boxes, 30 seeds at a time, repeating for 3 times. Setting artificial climate box in dark at 5 deg.C, low temperature stress treating for 1, 3, 5, 7d, recovering at 22 deg.C in plant growth chamber for 7d, and placing in artificial climate box for germination.
Example 3
Selecting corn seeds with consistent size, plump shape and no damage, sterilizing with sodium hypochlorite with concentration of 15%, washing with distilled water for 4 times after sterilization, and naturally drying in the ventilated place.
Using 200mg/L vitamin B2(VB2) Soaking the seeds for 12h in a germination box, 30 seeds at a time, and repeating for 3 times. Setting artificial climate box in dark at 10 deg.C, low temperature stress treating for 1, 3, 5, 7d, recovering 3d at 28 deg.C in plant growth chamber, and placing in artificial climate box for germination.
Example 4
Selecting corn seeds with consistent size, plump shape and no damage, sterilizing with sodium hypochlorite with concentration of 15%, washing with distilled water for 4 times after sterilization, and naturally drying in the ventilated place.
Using 200mg/L vitamin B12(VB12) Soaking the seeds for 12h in a germination box, 30 seeds at a time, and repeating for 3 times. Setting artificial climate box in dark at 10 deg.C, low temperature stress treating for 1, 3, 5, 7d, recovering 3d at 28 deg.C in plant growth chamber, and placing in artificial climate box for germination.
Example 5
Selecting corn seeds with consistent size, plump shape and no damage, sterilizing with 10% sodium hypochlorite, washing with distilled water for 3 times, and naturally drying in the air.
Using 100mg/L vitamin B2(VB2) Soaking seeds for 24h in germination boxes, 30 seeds at a time, repeating for 3 times. Setting artificial climate box in dark at 5 deg.C, low temperature stress treating for 1, 3, 5, 7d, recovering at 25 deg.C in plant growth chamber for 7d, and placing in artificial climate box for germination.
Example 6
Selecting corn seeds with consistent size, plump shape and no damage, sterilizing with 10% sodium hypochlorite, washing with distilled water for 3 times, and naturally drying in the air.
Using 100mg/L vitamin B12(VB12) Soaking seeds for 24h in germination boxes, 30 seeds at a time, repeating for 3 times. Setting artificial climate box in dark at 5 deg.C, low temperature stress treating for 1, 3, 5, 7d, recovering at 25 deg.C in plant growth chamber for 7d, and placing in artificial climate box for germination.
Comparative example 1
Mixing vitamin B2Replacement with vitamin H (V)H) Otherwise, the same procedure as in example 5 was repeated.
Comparative example 2
Mixing vitamin B2Replacement with vitamin PP (V)PP) Otherwise, the same procedure as in example 5 was repeated.
Comparative example 3
Mixing vitamin B2The procedure was repeated except for using distilled water, and the procedure was as in example 5. (CK treatment)
Example 7
The germination conditions of the corn seeds of the examples 5-6 and the comparative examples 1-3 are observed every day, the germination rate of the seeds is counted, 10 grains are taken from each example after 7 days of recovery to measure the length of the corn embryo, the length of the embryonic root, the fresh weight of the overground part and the fresh weight of the underground part, and the samples are taken to carry out the measurement of physiological and biochemical indexes.
1. And (3) measurement of germination indexes:
the germination rate is (number of germinated seeds/number of tested seeds) × 100%;
germination potential (total germination number of the first 3 d/total number of seeds to be tested) × 100%;
germination Index (GI) ═ Σ Gt/Dt (Gt is the number of normal germinating grains on the respective day and Dt is the number of the respective germinating days).
As shown in FIG. 1, in the case of the low-temperature treatment 1d, vitamin VB was exogenous2And VB12The low-temperature germination promoting effect is more obvious and is higher than VHAnd VPPCompared with the low-temperature control CK treatment, the treatment rate is respectively improved by 34.61 percent and 39.29 percent. As shown in FIG. 2, in the case of the low-temperature treatment for 3d, vitamin VB was exogenous2And VB12The low-temperature germination promoting effect is obvious, and the low-temperature germination promoting effect is respectively improved by 37.04 percent and 41.38 percent compared with the low-temperature control CK treatment, although VHAnd VPPThe treatment also promoted the germination rate of the seeds, but the effect was not significant. Similarly, according to FIGS. 3 and 4, in the cases of 5d and 7d, the low temperature treatment resulted in a higher cold germination-promoting effect of the test 4 vitamins than the control, and VB2And VB12The promoting effect is still better than that of VHAnd VPP. Indicating the vitamin VB to be tested2And VB12Can obviously promote the germination of corn seeds under low temperature stress, and has better promotion effect on the germination of the seeds under short-term low temperature stress.
As shown in FIG. 5, VB at low temperatures of 1, 3, 5 and 7d compared to CK treatment2The seed soaking treatment can promote the germination indexes of the corn seeds at the early stage to be respectively improved by 32.39%, 37.13%, 33.29%, 40.89% and VB12The seed soaking treatment is respectively improved by 28.19%, 28.78%, 39.02% and 32.94%. It was shown that VB was observed in the case of the low-temperature treatment for different days2And VB12Can promote the germination index of the early stage of seeds.
2. Measurement of growth indexes: 10 corn seeds were taken from each example, and the germ length and the radicle length were measured; separating corn germ and radicle, and measuring the fresh weight of overground part (germ) and underground part (radicle); then placing in a drying oven at 105 ℃ for deactivating enzymes for 15min, drying to constant weight at 80 ℃, and weighing the dry weight.
As shown in Table 1, vitamin VB was added at a concentration of 100mg/L2And VB12Under the seed soaking treatment, the length of the embryo axis, the length of the embryo, the fresh weight of the embryo axis and the fresh weight of the embryo of the seed can be obviously improved, the water content in the plant body is increased, and the total biomass accumulation is increased.
TABLE 1 Effect of vitamin pretreatment on maize seed embryo Length, radicle Length, shoot fresh weight, and root Dry weight under Low temperature stress
Figure BDA0002629036450000081
Note that according to the Duncan test. The different letter representations after the same column of data differed significantly at the 0.05 level. The values in the table are the mean of 3 replicates. + -. SE.
3. Determination of root-to-crown ratio: the ratio of the dry weight of the underground portion to the above-ground portion. (i.e., R/S is root dry weight/above-ground dry weight)
As shown in FIG. 6, VB at low temperatures of 1, 3, 5 and 7d2The root-crown ratio of the corn seedlings can be remarkably improved, and is respectively increased by 38.49%, 20.59%, 24.6% and 23.05% compared with CK treatment; VB at low temperature of 1, 3, 5, 7d12The treatment can obviously improve the ratio of the root to the crown of the corn seedlingCompared with CK treatment, the addition amounts are respectively 31.19%, 37.73%, 12.32% and 30.49%. And V at low temperature of 1, 3, 5, 7dH，VPPTreatment below VB2，VB12Treatment in which V is stressed at 3d at low temperatureH，VPPThe treatment is lower than that of a control group, and the treatment is respectively reduced by 25.85 percent and 15.1 percent; v under low temperature stress of 5dPPCompared with CK, the corn seedling root cap is obviously inhibited, and the reduction is 23.07 percent. Illustrating VB under low temperature stress2，VB12Can effectively relieve and improve the influence of the root-crown ratio of the corn seedlings.
4. Determination of soluble sugar and protein content: measured according to anthrone colorimetry. Taking 0.05g of dried sample, adding 10ml of distilled water, reacting in a boiling water bath for 20min, pouring the supernatant into a 100ml volumetric flask, and fixing the volume to the scale. Adding 5ml anthrone (1g anthrone dissolved in 1000ml 80% concentrated sulfuric acid) reagent into 1ml solution to be detected, boiling for 10min, and measuring the light absorption value at 620 nm.
Soluble protein content was determined with reference to the Bradford method.
As shown in FIG. 7, VB was observed at low temperatures of 1, 3, 5 and 7d2The soluble sugar content in the corn germ after seed soaking treatment is obviously increased and is respectively increased by 4.02%, 5.97%, 13.25% and 1.18% compared with CK treatment. VB at low temperature of 1, 3, 5, 7d12The soluble sugar content of the corn germ after seed soaking treatment is obviously increased and is respectively increased by 2.19%, 8.75%, 12.98% and 4.17% compared with CK treatment. But V at low temperatures of 1, 3, 5, 7dH，VPPThe soluble sugar in the maize germ is obviously inhibited under the treatment, and is reduced by 7.42-24.16% compared with the CK treatment. And VB2And VB12And VHAnd VPPThere was a significant difference between treatments. Description of VB2，VB12Can effectively relieve and improve the accumulation of soluble regulating substances of the corn seedlings under low-temperature stress, reduce the cell osmotic potential and improve the water absorption capacity of the cells.
5. Determination of proline content: free proline was determined with reference to the Bates method. Weighing 0.1g of dried sample, adding 5ml of 3% sulfosalicylic acid solution, grinding, centrifuging at 3000r/min for 5min, taking 2ml of supernatant, adding 2ml of glacial acetic acid and 2ml of acid ninhydrin reagent, boiling in a water bath for 30min, cooling, adding 4ml of toluene, and carrying out color comparison on the supernatant at 520nm respectively.
According to the graph shown in FIG. 8, the proline content in the low-temperature stress 1 and 3d shows a rising trend according to different changes of the affected substance content, wherein VB2The treatment effect is most obvious, and is respectively increased by 32.72 percent and 47.27 percent compared with CK treatment; the proline content is increased and then decreased when the stress is 5 and 7 days at low temperature, wherein VB2And VB12The treatment can promote proline content in corn germ, and is at VHAnd VPPThe content of proline in the seed soaking treatment is obviously reduced, and is respectively reduced by 7.23%, 27.3%, 21.67% and 20.98% compared with CK.
6、H2O2And (3) content determination: determination of H with reference to the method of Velikova2O2. Collecting leaf 0.5g, adding 5ml 0.1% trichloroacetic acid, grinding in ice bath, centrifuging at 12000r/min at 4 deg.C for 15min, collecting supernatant of 1-2ml, adding 0.5ml 10 mmol. L-1Potassium phosphate buffer (pH 7.0) and 1ml of 1 mol. L-1KI was colorimetric at 390nm and 1ml of 0.1% trichloroacetic acid was used instead of the supernatant as a control.
H2O2Is an important active oxygen in plants. As shown in FIG. 9, VB was observed under low temperature stress at each time period2，VB12All treatments reduce H2O2The content is reduced by 54.36 percent and 59.69 percent respectively compared with CK treatment, wherein the effect is most remarkable when the stress is carried out for 5d at low temperature. Description of VB2，VB12Can obviously reduce the accumulation of active oxygen of corn seedlings under low-temperature stress and further reduce the damage caused by membrane lipid peroxidation in plant cells.
Superoxide anion free radicals are accumulated in a plant body in a large amount in the form of active oxygen, and can be changed once being stressed differently, so that membrane mutation occurs in the plant body, and the oxidative damage is generated to the plant. According to FIG. 10, VB in different time periods under low temperature stress2The oxygen free radicals in the maize germ are reduced under the treatment, and are respectively reduced by 34.4%, 33.47%, 14.15% and 13.83% compared with CK. VB12Respectively 16.23% lower than CK,44.57%, 25.44% and 60.72%. Illustrating VB under Low temperature stress treatment2，VB12Can relieve and inhibit oxygen free radical content in corn germ, improve plant oxidation resistance, and reduce active oxygen damage to plant.
7. And (3) measuring the MDA content: MDA was determined by reference to the method of Hodges. Taking 0.5g of leaves, adding 5ml of 5% trichloroacetic acid, grinding into homogenate, centrifuging at 12000r/min for 15min, taking 2ml of supernate, adding 2ml of 20% TCA solution containing 0.5% thiobarbituric acid, carrying out boiling water bath for 15min, and carrying out color comparison on the supernate at 450nm, 532nm and 600nm respectively.
MDA is an important index of the degree of reactive membranous peroxidation damage. As shown in FIG. 11, VB was observed under low temperature stress of 1d2The treatment is increased and is 2.18 times of CK; VB12The treatment showed a downward trend, 16.24% lower than the CK treatment. The rest treatments all show an ascending trend, and VB is generated when the temperature is stressed for 3d2，VB12The treatment effect is more obvious, and is respectively increased by 43.06 percent and 43.86 percent compared with CK treatment.
8. Determination of SOD Activity: see methods and modifications of Jiang, Giannopolitis and Ries. Reaction System 3ml containing 50 mmol. multidot.L-1Potassium phosphate buffer (pH 7.8), 75. mu.M nitroblue tetrazolium, 2. mu. mol. multidot.L-1Riboflavin, 13 mmol. L-1Methionine, 0.1 mmol. multidot.L-1EDTA-Na2, 100 mul enzyme crude extract, vibrating, placing in an intelligent illumination incubator at 25 deg.C and 450 mul mol m-2·s-1Irradiating for 30min under the condition, adjusting zero, and comparing color at 560 nm. SOD enzyme activity unit (U) is the amount of enzyme required to inhibit NBT reduction by 50% per mg protein in l min.
As shown in FIG. 12, SOD activity in maize embryos was at VB for different time periods under low temperature stress2Compared with CK, the treatment rate is respectively improved by 27.02%, 26.82%, 32.79% and 44.32%. VB12The treatment efficiency is respectively improved by 34.36%, 12.62%, 2.11% and 22.78% compared with CK; at VH，VPPNo significant difference was observed under the treatment, but V was observed under low temperature stress of 1dHThe treatment has 4.07% lower than CK, and V is reduced at low temperature of 3, 5 and 7 daysPPThe treatment is reduced by 5.39 percent and 4.28 percent compared with CKAnd 1.98%. Description of VH，VPPHas no obvious effect on SOD activity in corn germ, but has vitamin B2，B12After treatment, SOD activity in corn germ can be relieved and improved, wherein VB2Shows a linear increase indicating VB2Has certain influence on SOD activity.
9. Measurement of POD Activity: reference is made to Britton and Mehley methods and modifications. Weighing plant material 0.5g, adding 20mmol/LKH2PO45ml, slurried in a mortar, centrifuged and the supernatant collected in a cold place, and the resulting residue was again slurried with 5ml KH2PO4Extracting the solution once, combining the two supernatants, and measuring OD value at 470nm for color comparison.
As shown in FIG. 13, VB was observed under the duress treatment for each time period2The effect is most remarkable, and the treatment results are respectively 39.61%, 32.78%, 39.08% and 39.71% higher than that of CK treatment. VB12The activity of POD in the maize germ is promoted, and is respectively increased by 23.95%, 25.43%, 29.48% and 35.36% compared with CK treatment. VH，VPPHas no significant difference on POD activity promoting effect in corn embryo, wherein V is subjected to low temperature stress for 3dPPPOD activity showed inhibition under treatment, which was 25.08% lower than CK. Description via VB2，VB12After treatment, POD activity in corn germ is remarkably increased, and VB2The effect is more obvious.
10. Determination of CAT Activity: refer to the method of Aebi and improve. 3ml of the reaction system contained 50mM potassium phosphate buffer, 10mM H2O2, 200. mu.l of the enzyme crude extract. Adding H last2O2To start the reaction, the decrease in OD240 was read every 1 min.
FIG. 14 shows VB under low temperature stress2，VB12The CAT content in the maize germ can be improved by treatment, wherein the low-temperature 3d improving effect is the most obvious effect among groups, and the low-temperature 3d improving effect is respectively improved by 4.03 percent and 3.92 percent compared with CK. And between low temperature stresses VH，VPPThe treatment obviously inhibits the CAT activity in the maize germ, and the CAT activity is respectively reduced by 7.11%, 7.62%, 5.84%, 4.15%, 18.75%, 16.99%, 50.97% and 65.24% compared with the CK treatment. Description of the inventionDespite VB under low temperature stress2，VB12Treatment did not significantly affect CAT activity in corn germ, but was still promoting. And VH，VPPThe treatment can inhibit CAT content, thereby affecting the antioxidant system of corn germ and normal growth and development of plants.
11. Determination of Ascorbate Peroxidase (APX) activity: reference is made to the method of Nakano and Asada and modified. 3ml of a reaction mixture containing 1.5ml of 50 mmol. multidot.L-1Potassium phosphate buffer (pH 7.0), 0.1ml 15 mmol. multidot.L-1ASA，0.3ml1mmol·L-1H2O21ml of distilled water, finally adding 100 mul of enzyme crude extract to start the reaction, reading the reduction value of OD290 every 10s, taking 10s-60s in time period, replacing the enzyme crude extract with enzyme extraction buffer solution as a control, and zeroing with distilled water during color comparison. APX enzyme activity units (U) is the amount of enzyme required to reduce OD290 by 0.01 units per mg protein/min.
According to FIG. 15, VB is shown at different time periods under low temperature stress2The treatment obviously promotes the APX content in the maize germ, and the APX content is respectively increased by 50.64%, 40.62%, 60.5% and 27.19% compared with CK. VB12Treatment increased 55.42%, 41.27%, 59.21% and 44.14% compared to CK, respectively. In addition, stress 1, 3dV at low temperatureHThe treatment inhibited APX activity in corn germ, which was reduced by 34.73% and 6.71% compared to CK treatment, and there was no significant difference between the other treatments. Illustrating VB under low temperature stress2，VB12Can obviously improve the APX activity in the maize germ, and VH，VPPThe treatment did not make any significant difference in increasing APX activity.
A combination of the above example data shows VB2，VB12The corn seedling under low temperature stress for different time periods is relieved, and the emergence rate, the germination vigor, the plant height, the root length, the dry fresh weight on the ground and the like of the corn seed are improved; has effects in promoting cell permeation regulating substance content, antioxidant substance content and antioxidant enzyme activity, and reducing H in embryo bud2O2And oxygen free radical content, which is beneficial to regulating internal osmotic potential and promoting the growth and development of plants. But in no longerSimultaneous period of low temperature stress VH，VPPUnder seed soaking treatment, the plant height, root length, dry and fresh weight on the ground, proline, soluble sugar and enzyme activity of the maize germ all show a descending trend, and are obviously lower than those of a control group. Description of VB2And VB12Can effectively enhance the low temperature stress resistance of the corn seeds.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims (10)

1. A method for improving the stress resistance of corn seeds by using exogenous vitamins is characterized in that the corn seeds are soaked by the exogenous vitamins, and the exogenous vitamins are VB2And/or VB12。
2. The method for improving stress tolerance of corn seeds with exogenous vitamins of claim 1, wherein said exogenous vitamins are present in a concentration of 50-200 mg/L.
3. The method for improving the stress tolerance of corn seeds with exogenous vitamins according to claim 1, wherein the soaking time is 12-36 hours.
4. The method of utilizing exogenous vitamins to increase stress tolerance of a corn seed as claimed in claim 1 wherein said stress tolerance is low temperature stress tolerance.
5. The method for improving stress tolerance of corn seeds with exogenous vitamins according to claim 4, wherein the low temperature stress is at a temperature of 0-10 ℃.
6. The method for improving stress tolerance of corn seeds with exogenous vitamins according to claim 5, wherein the time period of low temperature stress is 1-7 days.
7. The method for improving stress tolerance of corn seeds with exogenous vitamins according to claim 5 or 6, wherein the low temperature stressed corn is restored at 22-30 ℃ for 5-7 days.
8. The method for improving stress tolerance of corn seeds with exogenous vitamins of claim 1 or 2, wherein said corn seeds are sterilized and then soaked with said exogenous vitamins.
9. The method for improving stress tolerance of corn seeds with exogenous vitamins according to claim 8, wherein the sterilization is performed by using sodium hypochlorite with a concentration of 8-15%.
10. The method for improving stress tolerance of corn seeds with exogenous vitamins according to claim 9, wherein the time for disinfecting the sodium hypochlorite is 15min to 30 min.
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