CN102282931B - Method for relieving salt injury of soybean seedlings by using exogenous soybean isoflavone - Google Patents
Method for relieving salt injury of soybean seedlings by using exogenous soybean isoflavone Download PDFInfo
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- CN102282931B CN102282931B CN2011101761487A CN201110176148A CN102282931B CN 102282931 B CN102282931 B CN 102282931B CN 2011101761487 A CN2011101761487 A CN 2011101761487A CN 201110176148 A CN201110176148 A CN 201110176148A CN 102282931 B CN102282931 B CN 102282931B
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Abstract
The invention discloses a method for relieving salt injury of soybean seedlings by using exogenous soybean isoflavone. The method comprises the following steps of: (1) dissolving the soybean isoflavone by using dimethyl sulfoxide (DMSO) to prepare soybean isoflavone aqueous solution containing 1 percent of DMSO, wherein the concentration of the aqueous solution is not more than 1.0mg/L; (2) soaking soybean seeds into the soybean isoflavone aqueous solution in an incubator of 25 DEG C, putting the seeds on a germinating bed after the seeds are soaked for 10 hours, and performing light shaded germination acceleration; (3) sowing the germinated seeds into a porous plastic cup filled with quartz sand, putting the plastic cup into a transfer box filled with 1/2 Hoagland nutrient solution, performing light cultivation, and changing the nutrient solution once every 2 to 3 days; and (4) moving seedlings to a porous foam board when a first pair of main leaves is grown, cultivating the seedlings in a transfer box filled with 100 to 130mmol/L NaCl solution prepared by using 1/2 Hoagland nutrient solution, observing the growth condition of the seedlings, measuring the area of the leaves, taking samples of a root, a stem and a leaf after the seedlings are treated for 6 days, and respectively measuring the soybean isoflavone content of the samples by adopting a high performance liquid chromatography (HPLC) method. By the method, the salt tolerance of the soybean seedlings can be enhanced, the salt injury of the soybean seedling is effectively relieved, and the method has more obvious effect on the soybean material with salt tolerance.
Description
Technical field
The invention belongs to the anti-contrary regulation and control new technical field of crops, relate to a kind of method of alleviating the soybean seedling salt damage, be specifically related to a kind of method of utilizing the external source isoflavones to alleviate the soybean seedling salt damage.
Background technology
Isoflavones (soybean isoflavones) is to extract an isolated class active substance with typical isoflavonoid structure from soybean seed material, is one of the most noticeable functional component in soybean.A large amount of studies show that, isoflavones has pre-anti-cancer, angiocardiopathy and osteoporosis, anti-oxidant and alleviate the important physiological function such as women's climacteric syndrome, thereby caused the extensive concern of academia, become one of the focus (Song Bing etc., 2008) of countries in the world scientist research.
The isoflavones metabolism is the main secondary metabolism approach in the leguminous plants such as soybean, isoflavones can be brought into play multiple important Ecology Action in some leguminous plant opposing adverse circumstance process, as avoid the injury (Bohm of ultra-violet radiation, 1998), at the overground part of plant, isoflavones can be subject to inducing when pathogeny infects plant synthetic, with the invasion and attack of opposing plant pathogenic microorganisms, plant protection element (Harborne et a1., 1976 with broad spectrum antibiotic activity; Adesanya et a1., 1986; Dixon, 2001), the phytophagy insect is had toxic action and avoidance effect (Lane et a1., 1985); At underground part, leguminous plant root system secretion isoflavonoid has chemotaxis to rhizobium, or the inducing substance of rhizobium nodulation gene (Kosslak et a1., 1987; Hirsch, 1992; Tahara and Ingham, 2000).When plant nitrogen lacked, the gene of synthetic isoflavones can be induced to express, and impelled the formation (Subramanian et al., 2004) of root nodule as the signaling molecule of rhizobium thereby produce isoflavone like substance.
Salt stress is that plant meets with a kind of of adverse circumstance, and the isoflavones metabolism may to adapt to the salt marsh environment relevant with plant.Find by the main secondary metabolites that compares in salt-resistant type and salt responsive type soybean, isoflavones and soyasaponin have very large difference in this soybean of two types, can utilize isoflavones and soyasaponin index to identify the salt resistance of soybean (Wu et al., 2008).The not salt tolerant the cultivated soybean kind that in the Salt-tolerance Wild soya seeds, isoflavone content was gathered in the crops far above the local same period, and under controlled cultivation condition, certain density Salinity has the effect that promotes isoflavones accumulation in the wild soybean seed, isoflavones formation for the cultivated soybean has inhibitory action (week is third-class, 2007).In non-leguminous plant, also can improve under some adverse environmental factor as the physical signs of acid rain and Under Drought Stress in Plant by executing isoflavones outward, alleviate the injury effect (Ye Meirong etc., 2008) of adverse circumstance.Yet, relation between inside and outside source isoflavones and soybean salt-tolerance is also indefinite, relevant application method or technology, as the mode by external source isoflavones seed soaking, study it on the impact of soybean plant strain salt resistance or to the physiological effect of salt damage, through retrieving the report that has no at present this respect.
Summary of the invention
Goal of the invention: for the deficiencies in the prior art, the purpose of this invention is to provide a kind of method of utilizing the external source isoflavones to alleviate the soybean seedling salt damage, so that soybean seedling has higher salt resistance, effectively alleviate the soybean seedling salt damage.
Technical scheme: in order to realize the foregoing invention purpose, the technical solution used in the present invention is as follows:
A kind of method of utilizing the external source isoflavones to alleviate the soybean seedling salt damage comprises the following steps:
(1) dissolve isoflavones with DMSO, be configured to contain 1%(V/V) the isoflavones aqueous solution of DMSO, concentration is not more than 1.0 mg/L
(2) soak soya seeds with the isoflavones aqueous solution, 25 ℃ of insulating boxs, seed soaking 10 h are placed on lucifuge vernalization on germinating bed;
(3) get the germination seedling and be sowed in the tool hole plastic cup that fills quartz sand, be placed in the Turnover Box that fills 1/2 Hoagland nutrient solution, illumination cultivation, the every 2 ~ 3d of nutrient solution changes once;
(4) when the 1st pair of true leaf grows, transplant seedlings to the cystosepiment in tool hole, cultivate in the Turnover Box that fills with 100 ~ 130mmol/LNaCl solution of 1/2 Hoagland compounding, observe the growing state of seedling, take pictures and measure its leaf area; After processing 6d, be divided into root, stem, leaf three Subsamplings, adopt the HPLC method to measure respectively its isoflavone content;
Wherein, the isoflavones aqueous solution is daidzin solution or genistin solution.Soya seeds is the weak the cultivated soybean kind N23674 kind of salt resistance, and the wild soybean BB52 population of salt tolerant beach and warp thereof are by filial generation 4076 strains that obtain for selection of salt tolerance.
In step (1), the concentration that contains the isoflavones aqueous solution of 1% DMSO is 0.01 mg/L.
In soybean kernel, two kinds of more isoflavones components of content are daidzin and genistin, and daidzein and genistein content are less.The Salt Tolerance at Seedling Stage coefficient is one of important indicator of soybean salt-tolerance evaluation, and the salt-tolerant callus numerical value is larger, and the expression salt resistance is stronger.The salt tolerant coefficient of isoflavone content in seed and respective material seedling is made correlation analysis, and its correlation coefficient is 0.696, shows to have utmost point marked positive correlation relation between the two.Isoflavones is also to have one of main secondary metabolites of significant difference in salt-resistant type and salt responsive type soybean.Therefore, isoflavones can be used as one of reference index of soybean salt-tolerance evaluation, by measuring isoflavone content in seed, need not to germinate or the cultivation in seedling stage, can carry out batch selection of salt tolerance and evaluation fast to a large amount of germplasms.
Beneficial effect: compare with existing soybean seedling, the outstanding advantages that utilizes method that external source isoflavones seed soaking is alleviated the soybean seedling salt damage to have of the present invention, comprising: (1) is easy and simple to handle, with low cost, relieving effect is obvious; (2) find that endogenous isoflavone content and soybean salt-tolerance have utmost point marked positive correlation, by measuring isoflavones component and content in seed, need not to germinate or the cultivation in seedling stage, can carry out batch selection of salt tolerance and evaluation fast to a large amount of germplasms, can be as one of reference index of soybean salt-tolerance evaluation; (3) external source is added daidzin or genistin when seed soaking, when even concentration is low to moderate 0.01 mg/L, also can effectively alleviate salt stress to the inhibitory action of Soybean Seedlings Growth, effect is remarkable, the cultivated soybean kind effect relatively weak to salt resistance is more obvious, in the production application process, can effectively save cost, have certain practicality.
Description of drawings
Fig. 1 is N23674,4076, BB52 with coerce the seedling of lower growth after the daidzin of variable concentrations or genistin solution seed soaking at 100 mmol/L NaCl; Wherein A, B, C, D represent respectively 0,0.01,0.1,1 mg/L daidzin or the processing of genistin solution seed soaking, and the E representative is without daidzin or genistin seed soaking and salt stress processing;
Fig. 2 coerces the seedling leaf area variation diagram of lower N23674 growth in 100 mmol/L NaCl after variable concentrations daidzin solution seed soaking; D represents daidzin solution, and concentration is respectively 0,0.01,0.1 and 1mg/L, and Cotrol is contrast;
Fig. 3 coerces down the seedling leaf area variation diagram of 4076 growths in 100 mmol/L NaCl after variable concentrations daidzin solution seed soaking; D represents daidzin solution, and concentration is respectively 0,0.01,0.1 and 1mg/L, and Cotrol is contrast;
Fig. 4 coerces the seedling leaf area variation diagram of lower BB52 growth in 100 mmol/L NaCl after variable concentrations daidzin solution seed soaking; D represents daidzin solution, and concentration is respectively 0,0.01,0.1 and 1mg/L, and Control is contrast;
Fig. 5 coerces the seedling leaf area variation diagram of lower N23674 growth in 100 mmol/LNaCl after variable concentrations genistin solution seed soaking; G represents genistin solution, and concentration is 0,0.01,0.1 and 1 mg/L, and Cotrol is contrast;
Fig. 6 coerces down the seedling leaf area variation diagram of 4076 growths in 100 mmol/LNaCl after variable concentrations genistin solution seed soaking; G represents genistin solution, and concentration is 0,0.01,0.1 and 1 mg/L, and Control is contrast;
Fig. 7 coerces the seedling leaf area variation diagram of lower BB52 growth in 100mmol/LNaCl after variable concentrations genistin solution seed soaking; G represents genistin solution, and concentration is 0,0.01,0.1 and 1 mg/L, and Control is contrast;
Fig. 8 is after daidzin (D) and genistin (G) seed soaking (0.01 mg/L) are processed, isoflavone content figure in the cultivated soybean N23674 seedling under salt stress;
Fig. 9 is after daidzin (D) and genistin (G) seed soaking (0.01 mg/L) are processed, isoflavone content figure in wild soybean BB52 seedling under salt stress;
Figure 10 is after daidzin (D) and genistin (G) seed soaking (0.01 mg/L) are processed, isoflavone content figure in filial generation 4076 seedling under salt stress;
Figure 11 is isoflavone content chart in the material seed, in table, and the different letter representation P of same row＜0.05.
Embodiment
The present invention is described further below in conjunction with specific embodiment.
Material and analytical method that following examples are used are as follows:
Material: with the different representative the cultivated soybean kind N23674(Lishui of Jiangsu of salt resistance, salt resistance is weak) and Kenli, soybean population BB52(Shandong, beach open country, salt resistance is strong) and filial generation F5 be test material for strain (4013,4035,4076 and 4111 etc.).In each material seed, isoflavone content as shown in figure 11.
Analytical method: (HPLC) measures isoflavone content in its seed with high performance liquid chromatography; In seed, isoflavones adopts the mild hydrolysis method to extract, and detects (Wang Song etc., 2005) with high-efficient liquid phase chromatogram technology (HPLC).Standard items daidzin (Daidzin), genistin (Genistin), daidzein (Daidzein), genistein (Genistein) purity 〉=99%, sample is qualitative according to the appearance time of standard specimen, according to peak area quantification.The isoflavones total amount is above-mentioned 4 kinds of isoflavone content sums.The statistical analysis measurement result, and the binary spacing variable relevant function method of using in SPSS software is made correlation analysis with isoflavone content and respective material seedling salt tolerant coefficient.
Embodiment 1
Take respectively appropriate daidzin and genistin, first with a small amount of DMSO dissolving, finally be mixed with contain 1%(V/V) daidzin solution and the genistin solution of DMSO, concentration is respectively 0.01 mg/L, 0.1 mg/L and 1.0 mg/L.Get soya seeds, with daidzin solution, genistin solution and the 1%(V/V of above-mentioned variable concentrations) DMSO solution soaks seed respectively, 25 ℃ of insulating boxs, vernalization after 10h.Taking-up is with 1%(V/V) half seed of DMSO solution seed soaking, with deionized water vernalization (Control), second half seed is with 100 mmol/L NaCl solution vernalization (NaCl processes separately), and other processing is all with 100 mmol/L NaCl solution vernalization.Each is processed and to choose the consistent person of germinateing and be sowed in the tool hole plastic cup that fills quartz sand.Except contrast is placed in the Turnover Box that fills 1/2 Hoagland nutrient solution, all the other process the Turnover Box that all is placed in the 1/2 Hoagland nutrient solution that contains 100 mmol/L NaCl.Cultivate at suitable illumination and temperature, the every 2 ~ 3d for the treatment of fluid changes once.Observe the growing state of seedling, take pictures and the Calculate Leaf Area morphological index.As shown in Figure 1, can find out in appearance from Soybean Seedlings Growth, under the coercing of 100 mmol/L NaCl, N23674,4076 and the growth of seedling of BB52 all be suppressed, and through after daidzin or genistin seed soaking, the cultivated soybean N23674 kind and the filial generation 4076 suffered inhibitory action of strain growth of seedling are all obviously alleviated, and wild this effect of soybean BB52 population is not obvious.Leaf area index analysis result such as Fig. 2, Fig. 3, Fig. 4, Fig. 5, Fig. 6 and shown in Figure 7, show the remission effect between three concentration for the treatment of of daidzin or genistin does not reach the significant difference level more, but save the angle of cost from the production application process, preferred least concentration carries out.
Embodiment 2
Result according to embodiment 1, select daidzin solution and the genistin solution of 0.01 mg/L to carry out seed soaking, the seed of every kind of material is used respectively 1%(V/V) the daidzin solution of DMSO solution, 0.01 mg/L, the genistin solution of the 0.01 mg/L 10h that soaks seed in 25 ℃ of insulating boxs, then be placed in lucifuge vernalization on germinating bed, choosing the consistent person of germinateing is sowed in the tool hole plastic cup that fills quartz sand, be placed in the Turnover Box that fills 1/2 Hoagland nutrient solution, cultivate at suitable illumination and temperature, the every 2 ~ 3d for the treatment of fluid changes once.When the 1st pair of true leaf grows, transplant seedlings to the cystosepiment in tool hole, carry out water planting in the Turnover Box that fills 1/2 Hoagland nutrient solution.When seedling grows to the 1st compound leaf expansion, the material that 1% DMSO solution seed soaking is processed is divided into two groups, one group is contrast (Control, 1/2 Hoagland nutrient solution), another group is processed separately (NaCl for NaCl, 130 mmol/L NaCl solution with the 1/2Hoagland compounding), other all carry out water planting with 130 mmol/L NaCl solution of 1/2 Hoagland compounding, the processing called after NaCl+D of daidzin seed soaking, the processing called after NaCl+G of genistin seed soaking.The every 2d of nutrient solution or treatment fluid changes 1 time, after processing 6d, is divided into root, stem, leaf three Subsamplings, adopts the HPLC method to measure respectively its isoflavone content.
Under salt stress, isoflavone content in the soybean seedling tissue contrasts and is significantly increased, two kinds of isoflavones external source seed soakings can make isoflavone content increase De Genggao, on the cultivated soybean N23674 kind and filial generation 4076 strain seedling the performance more obvious, as Fig. 8, Fig. 9 and shown in Figure 10.Under this explanation salt stress and after external source isoflavones seed soaking, all can be by improving the endogenous isoflavones level of soybean seedling, to adapt to salt stress or to alleviate salt damage.
Claims (3)
1. a method of utilizing the external source isoflavones to alleviate the soybean seedling salt damage, is characterized in that, comprises the following steps:
(1) dissolve isoflavones with DMSO, be mixed with and contain the isoflavones aqueous solution that volume ratio is 1% DMSO, the concentration of isoflavones is not more than 1.0mg/L;
(2) soak soya seeds with the isoflavones aqueous solution, 25 ℃ of insulating boxs, seed soaking 10 h are placed on lucifuge vernalization on germinating bed;
(3) get the germination seedling and be sowed in the tool hole plastic cup that fills quartz sand, be placed in the Turnover Box that fills 1/2 Hoagland nutrient solution, illumination cultivation, the every 2 ~ 3d of nutrient solution changes once;
(4) when the 1st pair of true leaf grows, transplant seedlings to the cystosepiment in tool hole, cultivate in the Turnover Box that fills with 100 ~ 130 mmol/L NaCl solution of 1/2 Hoagland compounding, observe the growing state of seedling, take pictures and measure its leaf area; After processing 6d, be divided into root, stem, leaf three Subsamplings, adopt the HPLC method to measure respectively its isoflavone content.
2. the method for utilizing the external source isoflavones to alleviate the soybean seedling salt damage according to claim 1, it is characterized in that: in step (1), the concentration of the isoflavones aqueous solution is 0.01 mg/L.
3. the method for utilizing the external source isoflavones to alleviate the soybean seedling salt damage according to claim 1, it is characterized in that: the described isoflavones aqueous solution is daidzin solution or genistin solution.
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