CN101881747A - Plant salt tolerance rapid detection method based on SIET (Scanning Ion-selective Electrode Technique) - Google Patents
Plant salt tolerance rapid detection method based on SIET (Scanning Ion-selective Electrode Technique) Download PDFInfo
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Abstract
The invention relates to a plant salt tolerance rapid detection method based on an SIET (Scanning Ion-selective Electrode Technique), comprising the following steps of: selecting a sample of a plant root at the germination stage or the seedling stage to serve as a detected object; putting the sample in a culture solution and detecting the sample by utilizing the SIET to obtain the flow directions and the flow speed of K<+> ions of the sample; after a certain time, adding a salt solution to the culture solution; after sodium chloride in the salt solution reaches a certain final concentration, continuing detecting the flow directions and the flow speed of the K<+> ions; judging the retaining capacity of a plant on the K<+> ions under a salt stress condition according to the detected flow directions and the flow speed, and further serving as the basis for evaluating the plant salt tolerance. The invention realizes the rapid non-invasive detection on the plant salt tolerance, can also realize plant reutilization after detection is ended while saving varieties and materials, eliminates errors caused by individual difference, and improves the detection accuracy.
Description
Technical field
The present invention relates to the plant technical field of nondestructive testing, relate in particular to a kind of plant salt endurance method for quick based on SIET.
Background technology
Salt damage is one of unfavorable factor that influences agricultural production and saline and alkaline regional ecological recovery, the about 20% arable land salination in the whole world.China has 2.0 * 10
7Hm
2Salt wasteland and 6.67 * 10
6Hm
2Salinization soil can account for 25% of cultivated area approximately.Existing plant germplasm resource is carried out the brine tolerance evaluation, and the screening salt-tolerant plant is to making full use of the large tracts of land saline-alkali soil of agricultural potentiality, and it is significant to recover the saline and alkaline regional ecosystem.
Generally speaking, the ability of regulation and control of the brine tolerance of plant and its absorption to different ions, transportation, distribution has direct relation, and thinks that the germination period of plant and seedling stage are the most responsive to salinity.Under condition of salt stress, keep Na lower in the body
+/ K
+Significant to keeping normal growth, so plant is to K
+The ability of possessing of ion becomes the key character of salt-tolerant plant.
The non-damage scan ion is selected electrode technology (Scanning Ion-selective ElectrodeTechnique, SIET) be the scan ion technology of utilizing ion and molecular selectivity microelectrode to constitute, can measure the ion of turnover biomaterial and the information such as movement rate, direction and concentration of molecule in non-invasive mode in conjunction with computer-controlled automatic positioning measurment system.This technology has been applied to fields such as fundamental biological knowledge, physiology, medical science, eagroforestry and drug action mechanism gradually.The Shabala group of Australia tower mulberry Germania university utilizes close with it non-damage ion flow detection technique (Non-invasive Microelectrode Ion FluxMeasuring Technique, MIFE) in the anti-salt research of barley, do many work, proved the K of barley seedlings root system under the salt stress
+Ion flow velocity and its brine tolerance index output correlativity can reach 0.85 (see also Crop Science, 2008,48:1382-1388.).Cuin studies show that on wheat, 80mMNaCl coerces the K after 1 hour
+Also exist great correlativity (to see also Journal of ExperimentalBotany, 2008,59 (10): 2697-2706) between ion flow velocity and the every physical signs.
To the more existing reports of the brine tolerance evaluation of plant germplasm, material therefor has paddy rice, tomato, sweet potato etc. at present.Estimate used method and mainly comprise germination index method, form damage evaluation method and increment relative method etc. under salt/alkaline stress, perhaps utilize tissue culture to screen the plant and the mutant of salt tolerant with salinity.Yin Weilun application number be in 200310113317.8 the Chinese patent application according to the plant cell membrane degree of injury, the method to the quantitative evaluation of plant salt tolerance ability has been proposed.Zhu Weimin application number be in 200610118700.6 the Chinese patent application by the calculating of seedling salt damage investigation under to certain salinity and salt stress index, carry out the tomato variety salt tolerant and screen.Liu Qingchang etc. also utilize the tissue culture method screening sweet potato salt-tolerant mutant under the salt stress in application number is 200710062936.7 Chinese patent application.Sun Zhenyuan application number be in 200910300988.2 the Chinese patent application then according to the characteristic of clone plant, the clonal plant salt endurance appraisement method based on physiological integration ability has been proposed.
Yet in the above method, the evaluation of physical signs need be carried out destructive sampling test, thereby judges its salt damage degree, for example plant cell membrane degree of injury.At present salt tolerant kind and material rareness (for example changeing the material of resistant gene of salt), or can't follow the tracks of the problem that detects same sample after the destructiveness sampling, accomplish Non-Destructive Testing for saving germ plasm resource, it is very necessary to improve the detection degree of accuracy.Utilize under the condition of tissue culture salt stress method for screening to have then that cycles consumed is long, the process complicated problems.
Summary of the invention
(1) technical matters that will solve
The technical problem to be solved in the present invention is: overcome destructive sampling of detection method of the prior art and the long defective of sense cycle.
(2) technical scheme
At the deficiencies in the prior art, the invention provides a kind of plant salt endurance method for quick based on SIET, may further comprise the steps:
S1, the sample of root system of plant of choosing germination period or seedling stage place nutrient solution to utilize the non-damage scan ion to select electrode technology SIET that this sample is detected in sample as detected object, obtain the K of sample
+The flow direction of ion and flow velocity;
S2 after the certain hour, adds salt solusion in nutrient solution, treat that NaCl reaches certain final concentration in the nutrient solution after, continue to detect and to obtain described K
+The flow direction of ion and flow velocity, according to the detected flow direction and flow velocity judge plant under condition of salt stress to described K
+The ability of possessing of ion, and then the foundation of the brine tolerance of plant is estimated in conduct.
Wherein, in step S1 or S2, detect and obtain described K
+The flow direction of ion and the method for flow velocity are specially: utilize the voltage difference between 2 on the SIET test sample, and the ion concentration that this voltage difference is converted between these 2 is poor, then this ion concentration differential conversion is become the flow direction and flow velocity between these 2.
Wherein, the composition of described nutrient solution comprises 0.5mM KCl and 0.1mM CaCl
2
Wherein, used microelectrode is K in described SIET
+The ion selectivity glass microelectrode.
Wherein, the index that is detected is the root system K under instantaneous NaCl induces
+Ion flow, certain final concentration that NaCl reached is 50-150mM.
Wherein, described certain hour is 10 minutes.
Wherein, utilize described K
+The slope that selectivity micro-electrode obtains after proofreading and correct is poor with the ion concentration that this voltage difference is converted between these 2.
Wherein, utilize Fick first diffusion law formula: the J
0=-D dc/dx becomes the flow direction and flow velocity between these 2 with this ion concentration differential conversion.
Wherein, described SIET is the SIET in the non-damage micrometering system.
Wherein, described plant comprises monocotyledon and dicotyledon; Described monocotyledon comprises wheat, corn and paddy rice, and described dicotyledon comprises tomato and soybean.
(3) beneficial effect
Technical scheme of the present invention based on the non-damage scan ion select electrode technology carry out germination period or seedling stage root system of plant K
+The flow direction of ion and flow speed data detect, with root system of plant under the salt stress to K
+The foundation estimated as brine tolerance of the ability of possessing, and to select instantaneous salt stress scope be 50-150mMNaCl, realized to plant salt endurance fast (from the seed germination to the detection, only need 3-6 days) Non-Destructive Testing, when saving kind and material, the error that causes because of individual difference has been eliminated in plant recycling after can also realizing detecting, has improved the accuracy that detects.
Description of drawings
The method flow diagram of Fig. 1 embodiment of the invention;
Fig. 2 shows in the test that the method for utilizing the embodiment of the invention does, and induces continent unit 9369 and Changwu 134 root system K down at 80mMNaCl
+The change in flow of ion;
Fig. 3 shows in the test that the method for utilizing the embodiment of the invention does, and 80mMNaCl coerces continent unit 9369 and Changwu 134 root system K
+The influence of ion mean flow rate.
Embodiment
Below in conjunction with drawings and Examples, the specific embodiment of the present invention is described in further detail.Following examples are used to illustrate the present invention, but are not used for limiting the scope of the invention.
In the embodiments of the invention, utilize the non-damage scan ion to select electrode technology to detect the brine tolerance of Wheat Cultivars.Its process flow diagram as shown in Figure 1.
1. experimental technique
Material is a wheat, and kind is common high yield winter wheat (Triticum aestivum L.) continent unit 9369 (already provided by Laizhou City's Jin Hai kind, be called for short 9369); And drought-enduring variety winter wheat (Triticum aestivum L.) Changwu 134 (provided by Ministry of Water Resources of Chinese Academy of Sciences research soil and water conservation, be called for short 134).
Sterilized 15 minutes in winter wheat Changwu 134 and continent unit 9369 usefulness, 3% sodium hypochlorite, presoaking and germinating was selected and is sprouted consistent seed after 24 hours respectively, place the germination box that is covered with individual layer filter paper to grow, filter paper and nutrient solution are changed every day, and nutrient solution is (0.5mM KCl, 0.1mM CaCl
2).In incubator, cultivate after 6 days, be used for root system K
+Ion flow detects.
K
+Ion flow detects and utilizes non-damage micrometering system (BIO-001B, Younger USA Sci.﹠amp; Tech.Corp., the non-damage scan ion in USA) selects electrode technology to carry out.Detect dynamically with scan ion selectivity micro-electrode technology, obtain the K of turnover sample
+The information of ion concentration (mM mM level), flow velocity and the flow direction (three-dimensional motion direction).K
+Ion-selective microelectrode front end filling 180 μ m K
+Liquid exchanger (LIX) fluid column of ion, rear end filling have the electrolyte column (100mM KCl) about 15-20mm, and the Ag/AgCl silk on the electrode fixator is inserted from the electrode back, and it is contacted with electrolytic solution.Contrast electrode is a solid electrode.
Seedling is transferred to balance 1h in the testing cassete before measurement, the test fluid composition is 0.5mMKCl, 0.1mM CaCl
2, measuring the site is apart from the root system maturation zone about tip of a root 10mm.Voltage difference between 2 of distance root surface 10 μ m place's vertical surveies, 2 displacements of electrode, 30 μ m.Behind the stably measured 10min, add the NaCl solution of equal-volume by the 160mM concentration of test fluid preparation, make its final concentration reach 80mM after, continue to detect K in the 60min
+The moving situation of ion.K in the solution
+The ions diffusion equilibration time is about 1-2min, so Nei data abandon during this period of time.Each is handled and detects three young plants.Utilize K
+The slope (Nernst slope) that obtains after ion-selective microelectrode is proofreaied and correct is poor with the ion concentration that the voltage difference between 2 o'clock is converted between 2.In the computation process of clean ion flow, think root system K basically
+Ion flow meets cylinder diffusion geometric model.The ion concentration difference is used Fick first diffusion law formula: the J to the conversion of the flow velocity and the flow direction
0=-D dc/dx finishes.
2. experimental result
As shown in Figure 2, under the normal growth condition, the 9369 root system maturation zone K of wheat continent unit
+Flow velocity is a negative value, shows near the K of root system
+Interior stream, shock range is bigger, and mean flow rate is-802pmol.cm
-2.s
-1(Fig. 3).Changwu 134 root system K
+Flow velocity shock range less (Fig. 2) shows the K that passes in and out root system
+The ion flow velocity is less, and mean flow rate only is-117pmol.cm
-2.s
-1(Fig. 3).After adding 80mM NaCl in the test fluid, the root K of two product grow wheats
+Flow velocity be on the occasion of, show root system surface K
+Ion flow effluxes to transferring to.The 9369 root K of continent unit
+It is bigger that ion effluxes flow velocity, and stable maintenance is at 3000-4000pmol.cm
-2.s
-1, mean flow rate is 3533pmol.cm
-2.s
-1Induce down Changwu 134 root system K in that NaCl is instantaneous
+The flow direction also shows as and effluxes, and flow velocity increases simultaneously, and peak flow rate (PFR) can reach 1890pmol.cm
-2.s
-1, but in 3-4 subsequently minute, its flow velocity quickly falls to 600-900pmol.cm
-2.s
-1(Fig. 2), mean flow rate is 776pmol.cm
-2.s
-1, only be 1/5 of continent unit 9369.This shows at NaCl coerces down the K of Changwu 134 root systems
+It is little than continent first 9369 to efflux flow velocity, and its root system is to K
+The ability of possessing is stronger, and its brine tolerance is stronger.Wherein, " CK " among Fig. 3 is meant no NaCl contrast test fluid, and this unblanketed box indicating utilization contains the resulting test findings of test fluid of the NaCl concentration of 0mM.
As can be seen from the above embodiments, technical scheme of the present invention based on the non-damage scan ion select electrode technology carry out germination period or seedling stage root system of plant K
+The flow direction of ion and flow speed data detect, with root system of plant under the salt stress to K
+The foundation estimated as brine tolerance of the ability of possessing, and to select instantaneous salt stress scope be 50-150mMNaCl, realized to plant salt endurance fast (from the seed germination to the detection, only need 3-6 days) Non-Destructive Testing, when saving kind and material, the error that causes because of individual difference has been eliminated in plant recycling after can also realizing detecting, has improved the accuracy that detects.
Above embodiment only is used to illustrate the present invention; and be not limitation of the present invention; the those of ordinary skill in relevant technologies field; under the situation that does not break away from the spirit and scope of the present invention; can also make various variations and modification; therefore all technical schemes that are equal to also belong to category of the present invention, and scope of patent protection of the present invention should be defined by the claims.
Claims (8)
1. the plant salt endurance method for quick based on SIET is characterized in that, may further comprise the steps:
S1, the sample of root system of plant of choosing germination period or seedling stage place nutrient solution to utilize the non-damage scan ion to select electrode technology SIET that this sample is detected in sample as detected object, obtain the K of sample
+The flow direction of ion and flow velocity;
S2 after the certain hour, adds salt solusion in nutrient solution, treat that NaCl reaches certain final concentration in the nutrient solution after, continue to detect and to obtain described K
+The flow direction of ion and flow velocity, according to the detected flow direction and flow velocity judge plant under condition of salt stress to described K
+The ability of possessing of ion, and then the foundation of the brine tolerance of plant is estimated in conduct.
2. the plant salt endurance method for quick based on SIET as claimed in claim 1 is characterized in that the index that is detected is the root system K under instantaneous NaCl induces
+Ion flow, certain final concentration that NaCl reached is 50-150mM.
3. the plant salt endurance method for quick based on SIET as claimed in claim 1 is characterized in that, in step S1 or S2, detects and obtains described K
+The flow direction of ion and the method for flow velocity are specially: utilize the voltage difference between 2 on the SIET test sample, and the ion concentration that this voltage difference is converted between these 2 is poor, then this ion concentration differential conversion is become the flow direction and flow velocity between these 2.
4. the plant salt endurance method for quick based on SIET as claimed in claim 1 is characterized in that the composition of described nutrient solution comprises 0.5mM KCl and 0.1mM CaCl
2
5. the plant salt endurance method for quick based on SIET as claimed in claim 4 is characterized in that used microelectrode is K in described SIET
+The ion selectivity glass microelectrode.
6. the plant salt endurance method for quick based on SIET as claimed in claim 1 is characterized in that described certain hour is 10 minutes.
7. the plant salt endurance method for quick based on SIET as claimed in claim 1 is characterized in that described SIET is the SIET in the non-damage micrometering system.
8. as each described plant salt endurance method for quick of claim 1～7, it is characterized in that described plant comprises monocotyledon and dicotyledon based on SIET; Described monocotyledon comprises wheat, corn and paddy rice, and described dicotyledon comprises tomato and soybean.
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| CN102507701A (en) * | 2011-11-16 | 2012-06-20 | 北京农业智能装备技术研究中心 | Method for screening out drought tolerant paddy rice through dynamic ion flow based on non-invasive micro-test technology (NMT) |
| CN102520046A (en) * | 2011-11-16 | 2012-06-27 | 北京农业智能装备技术研究中心 | Method for detecting paddy fungal rhizoctonia solani based on microscopic dynamic ion flow technology |
| CN102577852A (en) * | 2012-02-14 | 2012-07-18 | 山东棉花研究中心 | Method for identifying salt tolerance of cotton |
| CN102687612A (en) * | 2012-04-27 | 2012-09-26 | 北京农业智能装备技术研究中心 | Method for evaluating banded sclerotial blight resistance |
| CN105612851A (en) * | 2014-11-04 | 2016-06-01 | 中国农业大学 | Potassium-ion (K+)-current-based wheat salt resistance evaluation method or salt-resistant wheat screening method |
| WO2019113997A1 (en) * | 2017-12-14 | 2019-06-20 | 江苏大学 | Micro-ct-based method for measuring flow velocity of assimilation products of rosaceae crop |
| CN111983002A (en) * | 2020-08-31 | 2020-11-24 | 国际竹藤中心 | Real-time detection of plant root system Ca under low-temperature stress2+Streaming method |
| CN112119857A (en) * | 2020-09-09 | 2020-12-25 | 中国农业科学院棉花研究所 | Method for rapidly identifying salt tolerance of cotton seeds in germination period |
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| CN102520046A (en) * | 2011-11-16 | 2012-06-27 | 北京农业智能装备技术研究中心 | Method for detecting paddy fungal rhizoctonia solani based on microscopic dynamic ion flow technology |
| CN102507701B (en) * | 2011-11-16 | 2014-05-07 | 北京农业智能装备技术研究中心 | Method for screening out drought tolerant paddy rice through dynamic ion flow based on non-invasive micro-test technology (NMT) |
| CN102577852A (en) * | 2012-02-14 | 2012-07-18 | 山东棉花研究中心 | Method for identifying salt tolerance of cotton |
| CN102687612A (en) * | 2012-04-27 | 2012-09-26 | 北京农业智能装备技术研究中心 | Method for evaluating banded sclerotial blight resistance |
| CN102687612B (en) * | 2012-04-27 | 2014-01-29 | 北京农业智能装备技术研究中心 | Method for evaluating banded sclerotial blight resistance |
| CN105612851A (en) * | 2014-11-04 | 2016-06-01 | 中国农业大学 | Potassium-ion (K+)-current-based wheat salt resistance evaluation method or salt-resistant wheat screening method |
| CN105612851B (en) * | 2014-11-04 | 2018-03-13 | 中国农业大学 | One kind carries out Evaluation of Salt Tolerance or screening Salt-tolerant Wheat method based on potassium current to wheat |
| WO2019113997A1 (en) * | 2017-12-14 | 2019-06-20 | 江苏大学 | Micro-ct-based method for measuring flow velocity of assimilation products of rosaceae crop |
| CN111983002A (en) * | 2020-08-31 | 2020-11-24 | 国际竹藤中心 | Real-time detection of plant root system Ca under low-temperature stress2+Streaming method |
| CN111983002B (en) * | 2020-08-31 | 2021-10-15 | 国际竹藤中心 | Real-time detection of plant root system Ca under low-temperature stress2+Streaming method |
| CN112119857A (en) * | 2020-09-09 | 2020-12-25 | 中国农业科学院棉花研究所 | Method for rapidly identifying salt tolerance of cotton seeds in germination period |
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Application publication date: 20101110 |