CN113057079A

CN113057079A - Method for improving waterlogging tolerance of sweet sorghum in seedling stage

Info

Publication number: CN113057079A
Application number: CN202110270793.9A
Authority: CN
Inventors: 郭彦军; 邓苛莉; 倪郁
Original assignee: Southwest University
Current assignee: Southwest University
Priority date: 2021-03-12
Filing date: 2021-03-12
Publication date: 2021-07-02

Abstract

The invention relates to the technical field of crop planting, and discloses a method for improving waterlogging tolerance of sweet sorghum in a seedling stage. According to the invention, by improving the activity of antioxidant enzymes of plants in seedling stage and reducing the accumulation of malonaldehyde, the influence of flooding on sweet sorghum is reduced, the growth of sweet sorghum plants under flooding conditions is improved, and the flooding tolerance of sweet sorghum in seedling stage is finally improved.

Description

Method for improving waterlogging tolerance of sweet sorghum in seedling stage

Technical Field

The invention relates to the technical field of crop planting, in particular to a method for improving waterlogging tolerance of sweet sorghum in a seedling stage.

Background

Sorghum (Sorghum bicolor L) is an annual herbaceous plant of the family gramineae, is the fifth crop of grains worldwide, and has second only to maize, wheat, rice and barley in yield. Sweet sorghum is a variety of sorghum, is widely used as feed and industrial raw material crops due to high biological yield and rich sugar in stalks, and occupies an extremely important position in global arid and semi-arid agricultural production. However, natural disasters caused by climate change, such as high temperature, drought, flooding, etc., seriously affect the production of sweet sorghum. In abiotic stress, waterlogging has been considered as one of the most important factors affecting the growth, distribution, productivity and survival of vegetation worldwide, affecting not only the natural ecosystem, but also agricultural production. Along with global climate change, the intensity and frequency of abnormal climate phenomena such as extreme rainfall and the like are increased more and more, so that more and more areas face the potential danger of flood disasters. Under the stress of flooding, the plant morphology changes obviously, and the affected symptoms are mainly manifested as leaf yellowing, water stain, wilting, falling, rotting, plant lodging and the like. Under the flooding condition, the formation of new leaves of some plants is blocked, the number of the leaves and the area of the leaves are reduced, and simultaneously, the withering and the shedding of partial leaves are accompanied, so that the biomass is sharply reduced, and great economic loss is caused. During the long-term evolution, plants have formed a complete and complex antioxidant system to maintain the homeostasis of reactive oxygen metabolism in the body. Antioxidant systems can be divided into two major classes, non-enzymatic and enzymatic systems, which scavenge reactive oxygen species in the body by non-enzymatic and enzymatic reactions, respectively. Under the stress of adverse environment, the antioxidant protection system of the plant is damaged, so that a large amount of free radicals are accumulated, the content of malondialdehyde is increased, and the enzyme activity is changed. The high level of malondialdehyde directly reflects the degree of plant damage.

In production, the waterlogging resistance of crops is generally improved by taking some cultivation measures (such as selecting waterlogging-resistant varieties, draining waterlogging, spraying medicines and the like). Research shows that the waterlogging resistance measures can improve the activity of antioxidant enzymes of plants and reduce the content of malondialdehyde. Seed priming refers to a seed treatment technology that the seeds slowly absorb water under a specific condition, so that the seeds slowly return to the dry state after the seeds enter a stage of germination and radicle does not break through the seed coat. A large number of researches show that the initiation treatment can break dormancy, improve germination speed and germination rate and promote the growth of seedlings. The initiation treatment can also improve the stress resistance of the seeds under the abiotic stress conditions of low temperature, waterlogging, drought, salt and alkali and the like. Epibrassinolide (EBL) is a natural product widely present in plant physiological processes and is considered as the sixth major plant hormone. Researches show that after the EBL is externally applied to the plant, the cell membrane permeability can be improved, and the EBL is supposed to regulate and control the capability of the plant for resisting abiotic stress by regulating the plant cell permeability. Therefore, the inventor develops a method for improving the waterlogging tolerance of the sweet sorghum in the seedling stage.

Disclosure of Invention

Based on the problems, the invention provides a method for improving the waterlogging tolerance of sweet sorghum in the seedling stage.

In order to solve the technical problems, the invention provides the following technical scheme:

a method for improving waterlogging tolerance of sweet sorghum in seedling stage comprises the following steps:

s1: seed priming

Selecting healthy and plump sweet sorghum seeds, weighing, soaking in 0.01 mu M of epibrassinolide solution at 25 ℃ for 24 hours, washing the seeds with distilled water, and airing the seeds in a dry environment until the weight of the seeds reaches the weight of the seeds before initiation for later use;

s2: accelerating germination

Taking a culture dish 24 hours before sowing, paving two layers of qualitative filter paper at the bottom of the culture dish, wetting the qualitative filter paper with distilled water, paving the sweet sorghum seeds processed in the step S1 on the qualitative filter paper in the culture dish, covering the cover, and placing the culture dish in a 25 ℃ illumination incubator for germination acceleration;

s3: potting soil

Collecting surface soil from rape field, naturally drying the surface soil, removing root system and stone in the surface soil, grinding the surface soil, sieving to obtain soil with particle size of 2mm, mixing the soil and turfy soil at a ratio of 6:4, and potting with the specification of 15cm × 20 cm;

s4: seeding

Sowing the pregermination seeds obtained in the step S2 in the pots obtained in the step S3, wherein the sowing depth is 2cm, 8 seeds are sowed in each pot, and the seeds are watered thoroughly; then, water is supplemented every 5 days to ensure that the water content of the soil is 70-80% of the water holding capacity of the field;

s5: management of field

Culturing the pot processed in step S4 in a dry shed at 20-25 deg.C and air relative humidity of 75-85%; after seedling emergence, thinning the seedlings to 4 plants in each pot, removing weeds in time without fertilizing;

s6: flooding treatment

After seedling emergence, when a first true leaf and a third true leaf grow on sorghum respectively, flooding the pot until the soil surface reaches 5cm, supplementing tap water for 2 times every morning and afternoon, ensuring that the flooding height is 5cm away from the soil surface, and maintaining the flooding time for 10 days.

Further, the surface soil in step S3 is cultivated soil of 0-20cm soil layer of the rape field.

Compared with the prior art, the invention has the beneficial effects that: according to the invention, by improving the activity of antioxidant enzymes of plants in seedling stage and reducing the accumulation of malonaldehyde, the influence of flooding on sweet sorghum is reduced, the growth of sweet sorghum plants under flooding conditions is improved, and the flooding tolerance of sweet sorghum in seedling stage is finally improved.

Drawings

FIG. 1 is a graph showing the effect of initiation treatment on plant height of sweet sorghum according to an embodiment of the present invention;

FIG. 2 is a pictorial representation of the effect of primed and unprimed treatments on sweet sorghum in accordance with an embodiment of the present invention;

FIG. 3 is a graph showing the effect of priming treatment on the malondialdehyde content of sweet sorghum in accordance with an example of the invention;

FIG. 4 is a graph showing the effect of priming treatment on sweet sorghum peroxidase activity according to an example of the present invention;

FIG. 5 is a graph showing the effect of example priming treatments on sweet sorghum catalase activity.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples and accompanying drawings, and the exemplary embodiments and descriptions thereof are only used for explaining the present invention and are not meant to limit the present invention.

Example (b):

the sweet sorghum variety selected in this example is Murray 8000 from Krowo (Beijing) ecological technology Co., Ltd., and the epi-brassinolide is 24-epi-brassinolide (a Coolaber product), and this example studies whether the epi-brassinolide as an initiator can improve the waterlogging tolerance of the sweet sorghum at the seedling stage.

The main experimental process is as follows:

the seed priming method comprises the following steps: selecting healthy and plump sweet sorghum Mungle 8000 seeds, weighing, soaking in 0.01 mu M of surface brassinolide solution at 25 ℃ for 24 hours, washing the seeds with distilled water, and airing the seeds in a dry environment until the weight of the seeds reaches the weight of the seeds before initiation for later use;

non-priming seed treatment mode: selecting healthy and plump sweet sorghum Murray 8000 seeds, soaking in distilled water for 24h at 25 ℃, and then airing in a dry environment until the weight of the seeds reaches the weight of the seeds before soaking for later use;

accelerating germination: taking a culture dish 24 hours before sowing, paving two layers of qualitative filter paper at the bottom of the culture dish, wetting the qualitative filter paper by distilled water, paving initiated sweet sorghum seeds and non-initiated sweet sorghum seeds on the qualitative filter paper in the culture dish, covering the cover, and placing the culture dish in a 25 ℃ illumination incubator for germination acceleration;

potting and soil filling: collecting surface soil from a rape field, wherein the surface soil is cultivated soil of a rape field with a soil layer of 0-20cm, naturally drying the surface soil, removing roots and stones in the surface soil, crushing the surface soil, sieving to obtain soil with a particle size of 2mm, mixing the soil and turfy soil according to a ratio of 6:4, and potting, wherein the specification of the pot is 15cm multiplied by 20cm for 24 pots;

sowing: sowing the germination accelerating seeds in the pots filled with the soil, wherein the sowing depth is 2cm, 8 seeds are sown in each pot, and the seeds are watered thoroughly; then, water is supplemented every 5 days to ensure that the water content of the soil is 70-80% of the water holding capacity of the field; wherein the seeds with initiation are sown in 12 pots, and the seeds without initiation are sown in 12 pots;

field management: culturing the above sowed pots in dry shed at 20-25 deg.C and air relative humidity of 75-85%; after seedling emergence, thinning the seedlings to 4 plants in each pot, removing weeds in time without fertilizing;

and (3) flooding treatment: after seedling emergence, when a first true leaf and a third true leaf grow on sorghum respectively, selecting 3 priming seed treatment groups and 3 non-priming treatment groups, flooding the pots until the soil surface is 5cm, supplementing tap water for 2 times every morning and afternoon, ensuring that the flooding height is 5cm away from the soil surface, and maintaining the flooding time for 10 days. Simultaneously selecting a 3-pot initiated seed treatment group and a 3-pot non-initiated treatment group as a control, not flooding, and maintaining the water content of the soil to be 70-80% of the water holding capacity of the field by water supplement; the flooding time lasts for 10 days, and then the height of the plants is measured by using a ruler; meanwhile, the overground part sample is collected and directly used for measuring the activities of malondialdehyde and antioxidant enzyme.

The method for measuring the content of Malondialdehyde (MDA) adopts a thiobarbituric acid method (TBA) to measure: placing 0.1g of leaf in a precooling mortar, adding a small amount of liquid nitrogen for grinding, adding 10ml of 5% trichloroacetic acid (TCA), and centrifuging at the rotating speed of 12000r/min and the temperature of 4 ℃ for 20 min; collecting 2ml of supernatant, adding 2ml of 0.67% thiobarbituric acid, boiling in water bath for 30min, rapidly cooling, centrifuging at 10000r/min at 4 deg.C for 10min, collecting supernatant, and measuring light absorption value at wavelength of 532nm and 600 nm; and calculating the content of malondialdehyde in the extracted sample liquid according to a formula.

Determination of superoxide dismutase and catalase activities: enzyme solution extraction comprises adding 0.1g fresh plant leaf into 1.6ml 0.05mol/L phosphate buffer solution (pH 7.8) in a precooled mortar, adding a small amount of liquid nitrogen, and grinding into homogenate. Transferring the homogenate into a centrifuge tube, centrifuging for 20min at 12000g and 4 ℃, and extracting supernatant, wherein the supernatant is the required enzyme solution; superoxide dismutase (SOD) activity determination adopts nitrotetrazene blue chloride (NBT) color development method to prepare 3ml reaction system, 13mmol/L methionine, 75nmo1/L EDTA-Nat, 50 μmo1/L Nitrogen Blue Tetralin (NBT), 1.3 μmo1/L riboflavin, 50 μmol/L phosphate buffer solution (pH 7.8, 50 μ L enzyme extract is added, the mixed solution under the system is reacted for 20min under illumination condition, SOIL enzyme liquid in 3ml reaction system is changed into SOIL phosphate buffer solution (pH 7.8 is used as contrast, color comparison is carried out at 560nm wavelength, and absorbance is measured, and the enzyme quantity for inhibiting 50% NBT photoreduction is used as an enzyme activity unit.

Peroxidase (POD) activity assay: a3 ml reaction system was prepared with 0.2mol of L-1 phosphate buffer (pH6.0) and 30% (v/v) of H ₂0₂Adding 50mL of enzyme extract into guaiacol stock solution, uniformly mixing, measuring the absorbance at 470nm, continuously recording for 5min, changing the SOIL enzyme solution in 3mL of reaction system into phosphoric acid buffer solution of SOIL (pH6.0 as a control, reading every 30s, and taking the change value of absorbance per minute of 0.01 as an enzyme activity unit.

Catalase (CAT) assay: when measured, the reaction mixture and 750mmol/LH₂O₂) Preheating in 25 deg.C water bath, collecting 2.900ml reaction mixture, adding 50 μ L enzyme solution, sealing, preheating in 25 deg.C water bath for 5min, and adding 750mmol/LH when the solution is at the end₂O₂mu.L (final concentration 12.5mmol/L) to start the reaction, a final volume of 3ml, OD read every 20s₂₄₀The reduction value of (2) is calculated by using data in a time period of 10-110 s.

Referring to fig. 1, it can be seen that the flooding treatment significantly suppressed the plant height in the first and third leaf stages, with the uninitiated treatment decreased by 28.4% compared to the control in the first leaf stage and the hormone-induced treatment decreased by 16.1% compared to the control in the first leaf stage; in the three-leaf stage, the unprimed treatment decreased by 35.2% compared to the control, whereas the hormone-primed treatment decreased by 34.5% compared to the control. The growth conditions of plants which are not initiated in the three-leaf period and are initiated by hormone are shown in the attached figure 2, the growth conditions of the sweet sorghum are improved by the initiation of the hormone, the growth vigor of the plants is obviously better than that of the plants which are not initiated, and the leaf color of the plants which are initiated by the hormone under the flooding condition is greener than that of the plants which are not initiated. The brassinolide initiates seeds, the growth of plants under the flooding condition can be improved, and the effect of the first leaf stage is better than that of the third leaf stage.

Referring to the attached figure 3, the flooding treatment can be seen to remarkably improve the malondialdehyde content of the sweet sorghum leaves, which indicates that after flooding, the plant antioxidant protection system is damaged, and the membrane lipid permeability is increased; in the first leaf stage, the unprimed treatment increased significantly by 294% compared to the control, while the hormone-primed treatment increased by 93% compared to the control; in the trefoil stage, the unprimed treatment increased 197% over the control, while the hormone-primed treatment decreased 107% over the control. The brassinolide initiated seeds can improve the oxidation resistance of plant membrane lipids under a flooding condition, and the effect of the one-leaf stage is better than that of the three-leaf stage.

As shown in the attached figure 4, after flooding, the peroxidase activity of the plant leaves is obviously improved, which shows that the plant improves the adaptability to the flooding environment by improving the self oxidation resistance under the adverse conditions. In the first leaf stage, there was a significant 92% increase in unprimed treatment over control, while there was a 279% increase in hormone-primed treatment over control; in the trefoil stage, the unprimed treatment increased 48% over the control, while the hormone-primed treatment decreased 95% over the control. It is described that brassinolide initiated seeds can scavenge free radicals and reduce malondialdehyde accumulation by increasing plant peroxidase activity under flooding conditions.

Referring to the attached figure 5, after the flooding treatment, the catalase activity of the sweet sorghum leaves is also obviously increased, in the first leaf stage, the uninitiated treatment is obviously increased by 66 percent compared with the control, and the hormone-initiated treatment is increased by 180 percent compared with the control; in the trefoil stage, there was a 126% increase in unprimed treatment compared to control, and a 140% decrease in hormone-primed treatment compared to control. It is described that brassinolide-initiated seeds can scavenge free radicals and reduce malondialdehyde accumulation by increasing the catalase activity of plants under flooding conditions.

The experiment results show that after the sweet sorghum seeds are initiated by the epibrassinolide, the accumulation of malondialdehyde can be reduced by improving the activity of antioxidant enzymes of the plants in the seedling stage, the influence of flooding on the sweet sorghum is reduced, the growth of the plants is finally promoted, the plant of the initiated seeds is higher than that of the seeds which are not initiated, and the method for initiating the seeds to cultivate can improve the waterlogging tolerance of the sweet sorghum in the seedling stage.

The above is an embodiment of the present invention. The embodiments and specific parameters in the embodiments are only for the purpose of clearly illustrating the verification process of the invention and are not intended to limit the scope of the invention, which is defined by the claims, and all equivalent structural changes made by using the contents of the specification and the drawings of the present invention should be covered by the scope of the present invention.

Claims

1. A method for improving waterlogging tolerance of sweet sorghum in seedling stage is characterized by comprising the following steps: