CN104904368A - Method for corn seed germination under exogenous sugar seed soaking salt stress alleviation - Google Patents
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Abstract
Relating to the technical field of agricultural application, the invention provides a method for corn seed germination under exogenous sugar seed soaking salt stress alleviation. The operation steps include: a. disinfection: soaking corn seeds with a 5%-15% of disinfectant solution for 10-20min, conducting rinsing with distilled water and then performing airing to obtain disinfected corn seeds; and b. sugar-soaking: soaking the corn seeds with a 0.1-1mmol.L<-1> sugar solution for 30-50h to obtain salt tolerant corn seeds. The method for promoting corn seed germination under salt stress provided by the invention carries out glucose or sucrose seed soaking treatment on corn seeds, significantly improves the corn seed early germination rate and germination index, and shows that application of proper concentration exogenous sugar can promote corn seed germination. Seed soaking treatment with glucose or sucrose on corn seeds can effectively alleviate the inhibition of salt stress on corn seed germination, improves the seed germination rate and germination index to different degrees, and promotes the increase of corn sprout length, root length and dry weight.
Description
Technical field
The present invention relates to agriculture applied technology field, particularly relate to the method that maize under NaCl stress seed germination is alleviated in the seed soaking of a kind of exogenous sugar.
Background technology
The soil salinization is a global resource and ecological problem, there is varieties in saline-alkali areas 3.6 × 107 hm2 in China, account for 1/10th of total area under cultivation, owing to irrigating improper and using the reasons such as chemical fertilizer in a large number, Secondary Saline soil erosion also expands in continuation, thus cause arable area to decline year by year, there is research to point out that arable area will reduce 30% in 25 years of future, produce and improvement of the ecological environment so the soil salinization seriously governs agriculture and forestry.Due to sulphate, chloride, carbonate and heavy carbonate containing sodium, potassium, calcium, magnesium etc. in saliferous soil, and content is higher, therefore the impact for corn seed is very serious, saliferous soil can affect early stage germination rate and the germination index of corn seed, hinder Course of Corn Seed Germination, reduce antioxidase activity and Antioxidants in corn seed, affect born of the same parents' internal oxidition reduction potential and K
+/ Na
+ionic equilibrium, produces injury to maize cell, and then causes corn seed germination rate extremely low, even do not germinate.
Summary of the invention
The present invention, for above-mentioned the deficiencies in the prior art, provides the method that maize under NaCl stress seed germination is alleviated in the seed soaking of a kind of exogenous sugar.
The method of maize under NaCl stress seed germination is alleviated in exogenous sugar seed soaking of the present invention, and its operating procedure is as follows:
A, sterilization: with the medicining liquid dipping corn seed 10 ~ 20min of 5% ~ 15% weight concentration, dry after rinsing well with water, obtain sterilization maize seed;
B, leaching sugar: with 0.1 ~ 1mmolL
-1sugar juice soak the obtained sterilization maize seed 30 ~ 50h of a step, obtain haloduric corn kind.
As a further improvement on the present invention, its operating procedure is as follows:
A, sterilization: with the medicining liquid dipping corn seed 10 ~ 15min of 8% ~ 12% weight concentration, dry after rinsing well with water, obtain sterilization maize seed;
Leaching sugar: with 0.3 ~ 0.8mmolL
-1sugar juice soak the obtained sterilization maize seed 40 ~ 50h of a step, obtain haloduric corn kind.
As a further improvement on the present invention, described thimerosal is liquor natrii hypochloritis.
As a further improvement on the present invention, described sugar juice is glucose solution or sucrose solution.
The method of maize under NaCl stress seed germination is alleviated in exogenous sugar seed soaking of the present invention, by carrying out the seed soaking of glucose solution or sucrose solution to corn seed, significantly improve the early stage germination rate of corn seed and germination index, facilitate the sprouting of corn seed, and antioxidase activity and Antioxidants can be improved, maintain born of the same parents' internal oxidition reduction potential and K
+/ Na
+ionic equilibrium, alleviates salt stress to the injury of maize cell, Course of Corn Seed Germination under promotion salt adverse circumstance.
Accompanying drawing explanation
Fig. 1 is the impact of Glc and Suc pretreatment on maize under NaCl stress seed growth;
Fig. 2 is the impact of Glc and Suc pretreatment on maize under NaCl stress percentage of seedgermination;
Fig. 3 is the impact of Glc and Suc pretreatment on maize under NaCl stress seed germination index;
Fig. 4 is the impact of Glc and Suc pretreatment on salt stress maize germ TBARS content;
Fig. 5 is that Glc and Suc pretreatment is to salt stress maize germ H
2o
2the impact of content;
Fig. 6 is the impact of Glc and Suc pretreatment on maize under NaCl stress plumule SOD activity;
Fig. 7 is the impact of Glc and Suc pretreatment on maize under NaCl stress plumule APX activity;
Fig. 8 is the impact of Glc and Suc pretreatment on maize under NaCl stress plumule GPX activity;
Fig. 9 is the impact of Glc and Suc pretreatment on maize under NaCl stress plumule GR activity;
Figure 10 is the impact of Glc and Suc pretreatment on maize under NaCl stress plumule G6PDH activity;
Figure 11 is that Glc and Suc pretreatment is to maize under NaCl stress plumule Na
+, K
+the impact of content;
Figure 12 is that Glc and Suc pretreatment is to maize under NaCl stress plumule K
+/ Na
+the impact of ratio.
Embodiment
Embodiment 1
The method of maize under NaCl stress seed germination is alleviated in exogenous sugar seed soaking of the present invention, and its operating procedure is as follows:
A, sterilization: with liquor natrii hypochloritis's soaking corn seed 10min of 5% weight concentration, dry after rinsing well with water, obtain sterilization maize seed;
B, leaching sugar: use 0.1mmolL
-1glucose solution soak the obtained sterilization maize seed 30h of a step, obtain haloduric corn kind.
Embodiment 2
A, sterilization: with liquor natrii hypochloritis's soaking corn seed 20min of 15% weight concentration, dry after rinsing well with water, obtain sterilization maize seed;
B, leaching sugar: use 1mmolL
-1sucrose solution soak the obtained sterilization maize seed 50h of a step, obtain haloduric corn kind.
Embodiment 3
A, sterilization: with liquor natrii hypochloritis's soaking corn seed 10min of 8% weight concentration, dry after rinsing well with water, obtain sterilization maize seed;
B, leaching sugar: use 0.3mmolL
-1glucose solution soak the obtained sterilization maize seed 40h of a step, obtain haloduric corn kind.
Embodiment 4
A, sterilization: with liquor natrii hypochloritis's soaking corn seed 15min of 12% weight concentration, dry after rinsing well with water, obtain sterilization maize seed;
B, leaching sugar: use 0.8mmolL
-1sucrose solution soak the obtained sterilization maize seed 50h of a step, obtain haloduric corn kind.
Embodiment 5
A, sterilization: with liquor natrii hypochloritis's soaking corn seed 10min of 10% weight concentration, dry after rinsing well with water, obtain sterilization maize seed;
B, leaching sugar: use 0.5mmolL
-1glucose solution soak the obtained sterilization maize seed 48h of a step, obtain haloduric corn kind.
Embodiment 6
A, sterilization: with liquor natrii hypochloritis's soaking corn seed 10min of 10% weight concentration, dry after rinsing well with water, obtain sterilization maize seed;
B, leaching sugar: use 0.5mmolL
-1sucrose solution soak the obtained sterilization maize seed 48h of a step, obtain haloduric corn kind.
Below for embodiment 5 and embodiment 6, by contrast experiment, indices detection is carried out to the corn seed processed:
The selection of control group: in the same size and unabroken corn (Zea Mays L.) crossbreed Kenyu No.6 (being provided by corn breeding research department of Heilongjiang Bayi Agricultural Reclamation University) is provided, and carry out process corn seed with the method for embodiment 5 and embodiment 6, be sowed on the 6% water agar containing 150 mM NaCl, salt stress process 5 d is carried out to corn seed, using no salt added water agar as non-Stress treatment, normal growth contrast (the i.e. non-Stress treatment of water logging kind is respectively for examination 7 process, CK), Glc soaks seed non-Stress treatment (G, the corn seed that embodiment 5 is obtained), Suc soaks seed non-Stress treatment (T, the corn seed that embodiment 6 is obtained), water logging kind salt stress process (S), Glc(glucose) salt stress process (G+S) of soaking seed, Suc(sucrose) salt stress process (T+S) of soaking seed, mannitol seed soaking salt stress process (M+S), each process repeats for 3 times, each 100 seeds, culture dish is placed in climatic cabinate, 25 DEG C of dark germinations, relative moisture is 60%, namely the half that seed bud reaches seed length is considered as germinateing, record the germinative number of each process seed every day, calculating germination rate and germination index are as the dynamic (dynamical) parameter of sprouting, get maize germ in Ficus caricaL the 5th d and measure every physiological and biochemical index, its detection method is as follows:
1, the mensuration of growth indexes: Ficus caricaL the 5th d, 10 strain corns are got in each process, measure its bud length, root is long.Maize germ is separated with radicle, is placed in 105 DEG C of baking ovens and completes 15 min, dry to constant weight, then take dry weight, and calculate every strain seedling overground part and underground part dry weight respectively for 80 DEG C; As shown in Figure 1, it is the impact of Glc and Suc pretreatment on maize under NaCl stress seed growth; And Glc and Suc pretreatment is long on salt stress maize germ, the impact of radicle length, shoot dry matter and root dry weight sees the following form 1:
Note: each process as shown in Figure 1.Different letter representations after same column data significant difference in 0.05 level.In table, numerical value is repeat mean value ± 3 times
sE.Following table herewith.
As shown in Figure 1, different disposal corn seedling plumule lives forever at notable difference.As shown in Table 1, under non-salt stress, the every growth indexes between CK, G, T process without significant difference, 150 mmolL
-1naCl coerces has obvious inhibitory action to corn growth, and compared with CK, after S process 5 d, maize germ and radicle length reduce 63.64% and 66.51%, and shoot dry matter and root dry weight reduce 72.08% and 60.59% respectively, significant difference, G+S, the corn of T+S process is all significantly increased than each growth indexes with S-phase, wherein the maize germ of G+S process is long, radicle is long, plumule dry weight, radicle dry weight is 1.54 times of S process, 1.30 doubly, 2.09 doubly, 1.79 doubly, T+S process is respectively 1.67 times of S, 1.31 doubly, 2.69 doubly, 1.94 doubly, and T+S process plumule dry weight is significantly higher than G+S process, although each growth indexes of M+S process is higher than S process, but difference is not remarkable, illustrate that salt stress has obvious inhibitory action for corn seed early growth, and Glc and Suc pretreatment can alleviate the injury of salt stress to corn growth, promote the dry-matter accumulation in Course of Corn Seed Germination stage, in addition, isotonic treatment with mannitol fails to cause identical effect, illustrates that this remission effect is not because osmosis causes, and is to be soaked seed by sugar to cause.
As shown in Figure 2 and Figure 3, compared with CK, 0.5 mmolL
-1glc, Suc seed soaking all can improve Course of Corn Seed Germination under normal condition to some extent, and early stage (germinate the 2nd germination rate d) and germination index.Maize under NaCl stress percentage of seedgermination and germination index significantly decline, and compared with S process, G+S, T+S process all can significantly improve the early stage germination rate of maize under NaCl stress seed and germination index, wherein Suc seed soaking best results.In addition, under salt stress, its germination rate of mannitol pretreatment and germination index are in each process minimum, and illustrating that Glc, Suc of debita spissitudo soak seed in advance (is respectively 0.5 mmolL
-1) can significantly alleviate 150 mmolL
-1naCl coerces the inhibitory action to Course of Corn Seed Germination.
2, thiobarbituric acid reaction thing (TBARS) and hydrogen peroxide (H
2o
2) mensuration of content: the method with reference to Hodges etc. measures TBARS, get 0.1 g plumule and dry sample, the trichloroacetic acid (TCA) adding 5 ml 5% grinds to form homogenate, in centrifugal 15 min of 12000 g, get 2 ml supernatants and add the 20% TCA solution of 2 ml containing 0.5% thiobarbituricacidα-(TBA), boiling water bath 15 min, supernatant is respectively at 450 nm, 532 nm, 600 nm place colorimetrics; Measure H
2o
2, get 0.1 g plumule and dry sample, add 5 ml 0.1% TCA ice bath grindings, 12000 g, in 4 DEG C of centrifugal 15 min, get 1 ml ~ 2 ml supernatant and add 0.5 ml 10 mmolL
-1kaliumphosphate buffer (pH 7.0) and 1 ml 1 molL
-1kI, in 390 nm place colorimetrics, replaces supernatant in contrast with 1 ml 0.1% TCA.
TBARS is the important indicator of reacting cells membrane damage degree, and shown in Fig. 4, Fig. 5, the TBARS content in maize under NaCl stress plumule significantly raises, and is 1.90 times of CK. compared with S process, and 0.5mmolL
-1glc and Suc pretreatment obviously can reduce the TBARS content of maize under NaCl stress plumule, comparatively S process reduces by 28.78%, 30.35% respectively.H
2o
2it is the active oxygen that in plant corpus, a class is important.The H of each process plumule under salt stress
2o
2content obviously raises, the H of wherein S process
2o
2content is the H that 2.81 times of .G+S and T+S process of CK significantly reduce maize under NaCl stress plumule
2o
2content, reduces by 24.9%, 20.59% respectively compared with S process, and without significant difference between G+S process and T+S process.In addition, isotonic mannitol seed soaking fails to cause TBARS content and H in maize under NaCl stress body
2o
2the decline of content, illustrates that exogenous sugar is soaked seed the ROS content that can reduce in maize germ, maintains the stability of maize under NaCl stress embryo cell film.
3, the mensuration of activities of antioxidant enzymes: take fresh plumule 0.5 about g, put into the mortar of precooling, with the Extraction buffer (K of 10 ml precoolings
2hPO
4-KH
2pO
4, pH 7.0,1 mM EDTA, 1% PVP, 1 mM ASC) and grind homogenate, 15000 g are in 4 DEG C of centrifugal 20 min, and supernatant is enzyme extract; Superoxide dismutase (SOD) is with reference to the method for Giannopolitis and Ries, ascorbate peroxidase enzyme (APX) is with reference to the method for Nakano and Asada, glutathione peroxidase (GPX) is with reference to the method for Egley etc., and glutathione reductase (GR) reference Schaedle and Bassham method is also improved. and soluble protein content measures with reference to Bradford method. and the enzyme activity unit number that activities of antioxidant enzymes has with every mg protein represents.
SOD is the antioxidase that in plant corpus, a class is important, can catalysis O
-2, reaction generates O
2and H
2o
2, APX, GPX, GR are the key enzymes in plant in ASA-GSH oxidoreduction approach, effectively can remove the H in plant corpus
2o
2, shown in Fig. 6, Fig. 7, Fig. 8, Fig. 9,150 mmolL
-1naCl coerces and significantly reduces maize germ activities of antioxidant enzymes, and SOD, APX, GPX, GR activity of wherein S process comparatively CK declines 55.4%, 46.28%, 29.33%, 37.46% respectively.Under condition of salt stress, external source Glc, Suc seed soaking can effectively improve maize germ antioxidase activity, wherein SOD, APX, GPX, GR activity of G+S process is respectively 1.66 times, 1.62 times, 1.32 times, 1.38 times of S process, T+S process is respectively 1.67 times, 1.60 times, 1.29 times, 1.38 times of S process, and without significant difference between G+S process and T+S process, as can be seen here, Glc and Suc pretreatment can improve maize germ oxidation resistance, effectively reduce the accumulation of active oxygen, thus reduce active oxygen radical to the injury of plant cell.
4, the mensuration of ascorbic acid, glutathione content: get 0.1 g plumule and dry sample, add 2.5 ml 5% sulfosalisylic acid ball milling homogenate, 20000 g are centrifugal 20 min at 4 DEG C, and supernatant is used for the mensuration of reduced form ascorbic acid (ASA), glutathione (GSH) and oxidized type dehydroascorbic acid (DHA), glutathione (GSSG) content:
Table 2 is Glc and Suc pretreatment impacts on salt stress maize germ ascorbic acid content, glutathione content, reduced form ascorbic acid/oxidized type dehydroascorbic acid and reduced glutathione/oxidized form of glutathione
ASA, GSH are non-enzyme antioxidants important in plant corpus, can remove H specifically
2o
2.As shown in Table 2, after salt stress 5 d, ASA+DHA, GSH+GSSG content in maize germ significantly declines, 21.69%, 26.7% is declined respectively compared with CK, and Glc, Suc process all can significantly improve maize under NaCl stress plumule ASA+DHA, GSH+GSSG content, the particularly content of ASA, GSH, and then improve maize germ ASA/DHA, GSH/GSSG; Wherein G+S process ASA content, GSH content, ASA/DHA and GSH/GSSG comparatively S process improve 20.08%, 13.6%, 38.05%, 15.22% respectively, T+S process improves 18.94%, 14.03%, 23.32%, 17.61% respectively, and without significant difference between G+S process and T+S process.In addition, the accumulation that this experiment also finds external source Glc under home, Suc seed soaking can increase plumule ASA, GSH, improves ASA/DHA ratio, GSH/GSSG ratio, illustrates that exogenous sugar can induce the synthesis of ASA, GSH.
5, the mensuration that G 6 PD (G6PDH) is active: get the fresh plumule of 0.5g, add zyme extract (0.02 molL of 5ml precooling
-1tris-HCl, pH 7.5,0.42 molL
-1mannitol, 5 mmolL
-1kCl, 5 mmolL
-1mgSO
4) ice bath grinds to form homogenate, homogenate filtered with 4 layers of gauze, centrifugal 10 min of filtrate 3000 r at 0 DEG C, draw 100 ul supernatants, add 2ml reactant liquor and (include 0.5 mmolL
-1nADP, 5 mmolL
-1g-6-P, 5 mmolL
-1mgCl
2, 0.05 mmolL
-1tris-HCl, pH 8.2) in 340 nm place colorimetrics, after 5 min, measure the change of OD, G6PDH specific activity of enzyme is weighed with the optical density change of every mg zymoprotein in the unit interval; As shown in Figure 10, G6PDH under condition of salt stress in maize germ is active significantly to decline, decline 46.78% compared with CK, and G+S, T+S process all can significantly improve maize under NaCl stress plumule G6PDH vigor, comparatively S process improves 50.46%, 50.41% respectively, and without significant difference between G+S process and T+S process; In addition, under home, Glc, Suc process fails to cause the change of G6PDH activity.
6, Na
+, K
+the mensuration of ion concentration: get 0.1 g plumule and dry sample, add 5 ml sulfuric acid, 1 ml H
2o
2, sand-bath 2 h, then 300 DEG C, electric furnace carries out disappearing boiling, and adds 1 ml H every 30 min
2o
2, until mixed liquor water white transparency, be then settled to 100 ml with distilled water, measure Na with atomic absorption spectrophotometer (TAS-990 Super, general analyse general)
+, K
+ion concentration, is shown in that Figure 11, Figure 12 are known, 150 mmolL
-1naCl coerce 5 d after, Na in maize germ
+content significantly increases, K
+content and K
+/ Na
+than significantly declining, wherein S process Na
+content is 10.31 times of CK, K
+content and K
+/ Na
+comparatively CK declines 51.95%, 95.31% respectively.External source Glc and Suc can significantly improve K under salt stress
+content, effectively reduces Na
+content, and then improve K in maize germ
+/ Na
+, wherein G+S and T+S processes Na
+content is S process decline 26.72%, 28.9%, K comparatively
+content is respectively 1.73,1.73 times of S process, K
+/ Na
+than being respectively 2.34 times, 2.42 times of S process, without significant difference between two process.In addition, compared with S process, M+S process fails to cause Na
+, K
+content and K
+/ Na
+there is any marked change.
In sum, Glc and Suc seed soaking effectively can be alleviated 150 mmol/L NaCl and coerces suppression to Course of Corn Seed Germination, improves percentage of seedgermination, germination index to some extent, and promotes that maize bud is long, root is long and the increase of dry weight.
By to Antioxidant Enzymes analysis, show that dextrose plus saccharose pretreatment significantly improves the vigor of superoxide dismutase (SOD), glutathione peroxidase (GPX), ascorbate peroxidase enzyme (APX), glutathione reductase (GR), thus reduce maize under NaCl stress plumule thiobarbituric acid reaction thing (TBARS) and hydrogen peroxide (H
2o
2) content, maintain the stable of cell membrane under salt stress; Simultaneously, dextrose plus saccharose seed soaking can significantly improve ascorbic acid (ASA) in maize under NaCl stress plumule, glutathione (GSH) content and reduced form ascorbic acid/oxidized type dehydroascorbic acid (ASA/DHA), reduced glutathione/GSSG (GSH/GSSG) ratio, improves intracellular oxidation-reduction potential.
In addition, the K in maize under NaCl stress plumule
+content significantly reduces, Na
+content significantly increases, K
+/ Na
+be starkly lower than control treatment, Exogenous Glucose and sucrose can significantly improve K in maize under NaCl stress plumule
+content, and reduce Na
+content, K
+/ Na
+than significantly increasing.
Visible, under salt stress, glucose, sucrose seed soaking are by improving antioxidase activity and Antioxidants, maintain born of the same parents' internal oxidition reduction potential and K
+/ Na
+ionic equilibrium, alleviates salt stress to the injury of maize cell, Course of Corn Seed Germination under promotion salt adverse circumstance.
Claims (4)
1. the method for maize under NaCl stress seed germination is alleviated in exogenous sugar seed soaking, and its operating procedure is as follows:
Sterilization: with the medicining liquid dipping corn seed 10 ~ 20min of 5% ~ 15% weight concentration, dry after rinsing well with water, obtain sterilization maize seed;
Leaching sugar: with 0.1 ~ 1mmolL
-1sugar juice soak the obtained sterilization maize seed 30 ~ 50h of a step, obtain haloduric corn kind.
2. the method for maize under NaCl stress seed germination is alleviated in exogenous sugar seed soaking as claimed in claim 1, and its operating procedure is as follows:
Sterilization: with the medicining liquid dipping corn seed 10 ~ 15min of 8% ~ 12% weight concentration, dry after rinsing well with water, obtain sterilization maize seed;
Leaching sugar: with 0.3 ~ 0.8mmolL
-1sugar juice soak the obtained sterilization maize seed 40 ~ 50h of a step, obtain haloduric corn kind.
3. the method for maize under NaCl stress seed germination is alleviated in exogenous sugar seed soaking as claimed in claim 1 or 2, it is characterized in that described thimerosal is for liquor natrii hypochloritis.
4. the method for maize under NaCl stress seed germination is alleviated in exogenous sugar seed soaking as claimed in claim 1 or 2, it is characterized in that described sugar juice is glucose solution or sucrose solution.
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| CN107172919B (en) * | 2017-06-06 | 2020-07-31 | 山东省农业科学院玉米研究所 | Method for improving saline-alkali resistance of corn germplasm resource seeds |
| CN110383975A (en) * | 2018-04-18 | 2019-10-29 | 江苏省农业科学院 | A method of promoting the enrichment of corn kernel lutein |
| CN110383975B (en) * | 2018-04-18 | 2021-08-17 | 江苏省农业科学院 | Method for promoting lutein enrichment of corn kernels |
| CN108887125A (en) * | 2018-08-17 | 2018-11-27 | 界首市鑫康家庭农场 | A kind of cultural method of the high aldehydes matter content Radish Sprouts of high yield |
| CN108887125B (en) * | 2018-08-17 | 2020-08-18 | 界首市鑫康家庭农场 | Cultivation method for high-yield high-phenolic-substance-content radish sprout vegetables |
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