CN110278738B

CN110278738B - Based on Ca2+Method for measuring activity of corn seeds by flow velocity

Info

Publication number: CN110278738B
Application number: CN201910701325.5A
Authority: CN
Inventors: 李岩; 朱鹏宇; 宋雪皎; 毛玉泉; 张春庆
Original assignee: Shandong Agricultural University
Current assignee: Shandong Agricultural University
Priority date: 2019-07-31
Filing date: 2019-07-31
Publication date: 2020-07-28
Anticipated expiration: 2039-07-31
Also published as: CN110278738A

Abstract

The invention discloses a Ca-based material²⁺The invention relates to a method for measuring the activity of corn seeds by using a flow velocity, which utilizes a non-damage micrometering system to measure the Ca at the radicle end of the embryo of the corn seeds²⁺Flow rate according to Ca²⁺Flow Rate to assess corn seed vigorThe flow rate and the seedling dry weight vitality index have extremely obvious positive correlation. The method can realize accurate determination of the activity of the corn seeds, and compared with the existing detection method, the method has the advantages of less time consumption and higher efficiency.

Description

Based on Ca2+Method for measuring activity of corn seeds by flow velocity

Technical Field

The invention relates to the technical field of seed vitality detection, in particular to a method based on Ca²⁺Flow rate corn seed vigor determination method.

Background

The seed vigor is an important index for measuring the sowing quality of the seeds. The high-activity seeds have quick and regular emergence of seedlings, strong seedlings and strong stress resistance, and have obvious growth advantages and production potential; although the low-activity seeds can germinate under proper conditions, the seedlings emerge irregularly even without emergence under adverse conditions, and the yield is greatly influenced. The selection of high-activity seeds is an important guarantee for realizing full seedling and strong seedling in one-time sowing.

The seed vitality measuring method has more than 30 kinds, and is generally divided into a direct method and an indirect method. The direct method is to measure the rate of emergence or the growth rate and the robustness of seedlings by simulating poor conditions in the field under laboratory conditions, such as a low-temperature treatment test, a brick and gravel test, an extended radicle counting method and the like; the indirect method is to measure some physiological and biochemical indexes and physical characteristics related to the seed activity, such as enzyme activity, soaking liquid conductivity, respiration intensity and the like, in a laboratory. The extended radicle counting method is taken as a method suitable for measuring the vitality of the corn seeds in the international seed inspection regulation issued by ISTA. The elongation radicle counting method adopts 20 +/-1 ℃ or 13 +/-1 ℃ as the germination temperature, counts the percentage of the seeds with radicles breaking through the seed coat by more than 2mm at 66h +/-15 min and 144 +/-1 h respectively, and takes the percentage as the index for evaluating the seed vitality.

There are several Ca pools in plant cells, mainly including the central large vacuole, endoplasmic reticulum, mitochondria and chloroplasts, and in addition, a large amount of Ca is also stored in the cell wall. Ca when the plant receives external stimulus²⁺Flows from the Ca reservoir into the cytoplasm, and in the resting state, Ca²⁺And from the cytoplasm into the Ca reservoir. Exogenous application of Ca²⁺Can relieve the inhibition effect of abiotic stress such as low temperature, drought, salt stress and the like on seed germination (honest and clean and the like 2015; xudaojia and the like 2017; jade anyhow and the like 2019).

Non-invasive Micro-test Technology (NMT) is a technique for studying the physiological function of living materials. The technology utilizes the selective electrode, can detect the three-dimensional flow velocity information of molecules/ions entering and exiting a biological living body under the conditions of ensuring the integrity of a detected sample and approximating to an actual physiological environment, and is widely applied to the field of physiological function research. Consumption of O by seeds during germination through respiration₂For providing energy for itself, Xin et al (2013) determined O of imbibition seeds of soybean, wheat and rape by NMT technique₂Flow rate to judge the seed viability, provides for the use of imbibition seed O₂Flow rate method of evaluating seed viability. However, Ca-based has not been found yet²⁺Flow rate evaluation of maize seed viability.

Disclosure of Invention

In view of the above prior art, the object of the present invention is to provide a Ca-based alloy²⁺Flow rate corn seed vigor determination method. The method can realize accurate determination of the activity of the corn seeds, and compared with the existing detection method, the method of the invention has the advantages of less time consumption and higher efficiency.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect of the invention, there is provided a Ca-rich fraction of the radicle end of a maize seed embryo²⁺Use of a flow rate in a maize seed viability assay.

When the corn seeds germinate, the radicle firstly breaks through the seed coat, so that the cells at the radicle end are more vigorously metabolized than other parts in the process of seed imbibition, and Ca is generated²⁺The flow rate measurement value is larger and is easier to measure. The invention selects the Ca at the radicle end of the embryo of the maize seed embryo²⁺The flow velocity is used for determining the activity of the corn seeds, and compared with other parts of the corn seed embryos, the determination result is more accurate and reliable.

In a second aspect of the present invention, there is provided a Ca-based²⁺Corn seeds of flow velocityA method for measuring seed viability comprising the steps of:

determination of corn embryo radicle Ca by non-damage micrometering system²⁺Flow rate according to Ca²⁺Flow rate to evaluate the viability of the corn seeds.

Preferably, the corn seed embryo is obtained by the following treatment:

and (3) imbibing the corn seeds to be detected for 24h at 25 ℃, cutting off endosperm of the corn seeds, and stripping out complete corn embryo.

We have examined whole corn seeds in the research, and found that Ca is the object of examination of whole corn seeds²⁺The flow rate stability is poor and the value is low. In seeds of gramineous plants including corn, embryo is living tissue and endosperm and pericarp are dead tissue, only living tissue can respond to external environmental signals, and interference of dead tissue may be Ca measured by whole seeds as test objects²⁺The main reason for poor flow rate stability. Thus, the present invention selects corn embryos as the detection target, Ca of which²⁺The flow rate is stable, the determination value is high, and the evaluation on the activity of the corn seeds is facilitated.

Preferably, Ca²⁺The flow rate is measured by the following method: fixing the corn embryo with the back facing downwards in a culture dish, adding a test solution, and balancing for 5-10 min; placing the culture dish on an objective table of a non-damage micrometering system, utilizing a microscope and a three-dimensional motion displacement platform to clearly display the electrode and the corn embryo in the same visual field, enabling the tip of the electrode to be close to the root end of the corn embryo, operating software, and carrying out Ca treatment on the root end of the corn embryo²⁺The flow rate is measured.

For non-invasive microassay systems, the determination of different ions or molecules requires the use of different test solutions, preferably having a composition for the subject to be determined according to the present invention: KCl (0.1mM), CaCl₂(0.1mM)、MgCl₂(0.1mM)、NaCl(0.5mM)、Na₂SO₄(0.2mM), MES (0.3mM), pH 6.0.

More preferably, the distance between the tip of the electrode and the radicle end of the corn seed embryo is 2-5 μm.

More excellentOptionally, Ca is added to the root of the embryo²⁺The flow rate was continuously measured for 10 min.

Preferably, the seedling dry weight vitality index is used as a Y value, and the corn embryo radicle end Ca is used²⁺The flow rate is X value, and the seedling dry weight activity index and Ca are obtained by fitting²⁺Linear curve of flow rate, and the activity of the corn seeds is evaluated through the linear curve.

More preferably, the linear curve is Y ═ 0.0006218X +0.1454, R²＝0.8604。

The invention has the beneficial effects that:

the invention utilizes a non-damage micrometering system to carry out Ca treatment on the germ root end of the maize embryo²⁺Flow rate was measured according to Ca²⁺Flow rate to evaluate the viability of the corn seeds. The method can be used for quickly detecting the corn seeds, and has high accuracy and less seed consumption.

Drawings

FIG. 1: detecting sites of the corn embryos under a normal visual field and a microscope; wherein, A: normal field of view; b field under microscope; wherein the test site is the position marked by the arrow.

FIG. 2: embryo Ca of corn seeds after 24h of imbibition²⁺Dynamic changes in the influx rate, different broken lines representing 4 maize varieties including hybrid Zea mays 335(XY335) and Zhengdan 958(ZD958) and its parent species PH6WC and Zheng 58(Z58), a blank control (B L ANK) was measured in a test solution without samples placed, the values of which fluctuate around 0.

FIG. 3: seed vigor index and embryo Ca among different varieties²⁺A flow rate; in the figure, (A) four maize varieties Ca²⁺Flow rate value, (B) plant dry weight viability index. The four corn varieties are hybrid corn 335(XY335) and Zhengdan 958(ZD958) and female parent thereof is PH6WC and Zheng 58 (Z58).

FIG. 4: different treatment of seed embryo Ca²⁺The flow rate is dynamically changed; (A) yu 335, Zheng 958 (B). Wherein CK means no treatment; SA represents artificial aging treatment, namely continuously aging for 96 hours at the temperature of 45 ℃ and the humidity of 90%; SP means "aging + initiation"The treatment, i.e. the initiation of the aging, is carried out, B L ANK is measured in a test solution without samples placed, and each sample is continuously examined for 10 min.

FIG. 5: different treatment of seed vigor and embryo Ca²⁺A flow rate; (A) first treating Ca with jade 335²⁺Flow rate, (B) first, treating seedlings with different dry weight vitality indexes by Yu 335, (C) Zheng 958 treating with different Ca²⁺Flow rate, (D) zheng 958 dry weight viability index of the different treated seedlings. CK means no treatment; SA represents artificial aging treatment; SP stands for "aging + initiation" treatment.

FIG. 6: corn seed vigor and embryo Ca²⁺The flow rates are significantly positively correlated.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

As described in the background art, with the popularization of the single-seed sowing technology of corn, the index of seed vigor is more and more emphasized, and the accurate determination of seed vigor is very important for corn production. The seed vitality measuring method is more, but most methods are longer in use and low in efficiency.

Based on the method, the invention provides a novel method for measuring the vitality of the corn seeds, and the invention utilizes a non-damage micrometering system to measure Ca at the radicle end of the embryo of the corn seeds²⁺Flow rate was measured according to Ca²⁺Flow rate to evaluate the viability of the corn seeds. The method can be used for rapidly detecting the vitality of the corn seeds, and has the advantages of high accuracy and less seed consumption.

In one embodiment of the invention, the Ca at the radicular end of the embryo of a maize seed is given²⁺The flow rate measurement method specifically comprises the following steps:

placing 20-30 corn seeds to be detected in a paper bed (between papers) and carrying out imbibition for 24 hours at the temperature of 25 ℃; cutting the endosperm of corn seed with a scalpel, peeling off a whole embryo with the back facing downwards, fixing it on the culture medium with filter paper strip and quartzAdding 5ml of test solution into a culture dish (35mM), wherein the test solution comprises KCl (0.1mM) and CaCl₂(0.1mM)、 MgCl₂(0.1mM)、NaCl(0.5mM)、Na₂SO₄(0.2mM), MES (0.3mM), pH 6.0; balancing for 5-10 min; placing the culture dish on the objective table of the non-damage micrometering system, and utilizing a microscope and a three-dimensional motion displacement platform to clearly display the electrode and the sample in the same visual field, wherein the tip of the electrode is close to the radicle end of the sample (figure 1); gradually approaching the microelectrode to the sample by adjusting a computer motion module until the distance between the tip of the microelectrode and the sample is kept between 2 and 5 mu m, and both the microelectrode and the sample are clearly imaged in the same visual field; operating the software, Ca for the sample²⁺The flow rate was continuously measured for 10 min.

Cytoplasmic free Ca²⁺Is an important second messenger, Ca, in plants²⁺The signal is produced by cytoplasmic Ca²⁺Increase, decrease, and fluctuation. Ca in extracellular calcium stores (cell walls) or/and intracellular calcium stores (e.g., vacuoles) when cells are subjected to external stimuli²⁺Seed imbibition allows the seed to enter a metabolically active state from a relatively quiescent state during which extracellular Ca is present²⁺The active systemic of (A) probably plays an important role in the (A), the systemic rate of the metabolically active high-activity seeds is high, and the systemic rate of the metabolically weaker low-activity seeds is low. No Ca is found in the seed imbibition process²⁺And (4) an efflux phenomenon. Therefore, Ca detected by the present invention²⁺The flow rate is the rate of imbibition within the seed.

Setting the distance between the electrode tip and the sample to be 2-5 μm, and selectively applying the electrode to Ca to be measured²⁺Two-point measurement is carried out in the concentration gradient at a known distance dx to obtain a voltage V₁And V₂The difference dc between the two points can be determined by V₁、V₂And calculating the known voltage concentration correction curve of the electrode and Nernst equation, and substituting the known voltage concentration correction curve and Nernst equation into Fick first diffusion law formula J₀Ca can be calculated as-D (dc/dx) (where D is the diffusion constant)²⁺The velocity and direction of flow across the membrane.

In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific embodiments.

The test materials used in the examples of the present invention are those which are conventional in the art and are commercially available unless otherwise specified.

Example 1: harvesting corn seed vitality and embryo Ca in the same year²⁺Flow rate of flow

Using corn hybrid firstly Yu 335, Zhengdan 958 and its female parent PH6WC, Zheng 58 as research material, utilizing NMT technology to detect embryo Ca of above 4 corn varieties respectively²⁺Flow rate. As can be seen from FIG. 2, Ca is present between different maize varieties²⁺The flow rates are different, wherein Ca of Yu 335 and Zheng 958²⁺The flow rate was high and close, Ca at pH6WC²⁺Second order of flow rate, Ca of Zheng 58²⁺The flow rate value is the lowest. Seedling dry weight vitality index difference rule of 4 materials and Ca²⁺The flow rates were the same (fig. 3).

Example 2: the vitality and embryo Ca of the seeds obtained by the artificial accelerated aging and 'aging + initiation' treatment of the seeds harvested in the current year²⁺Flow rate of flow

Carrying out artificial aging treatment and 'aging + priming' treatment on the seeds of corn hybrid seeds firstly Yu 335 and Zhengdan 958 (after aging, priming treatment is carried out on the seeds), and respectively detecting Ca for treating seed embryos by utilizing NMT technology²⁺Flow rate (fig. 4). The first jade 335 seed after different treatments and its embryo Ca²⁺The flow rate was changed as shown by the seed embryo Ca without any treatment²⁺The largest flow rate value, the second time of the aging + priming treatment, the aging treatment of the seed embryo Ca²⁺The lowest flow rate value (fig. 5A); the trend of the change of the dry weight viability index of the corresponding seed seedlings was the same (fig. 5B). While Zhengdan 958 'aging + initiating' treatment of embryo Ca²⁺The flow rate was not significantly different from the aged seeds (due to poor effect of priming treatment) (fig. 5C), and the corresponding seedling dry weight viability index difference was also smaller (fig. 5D).

The dry weight vitality index of the seedling and the corresponding Ca²⁺8 sets of flow rate valuesSubstitution of data into SPSS with Ca²⁺And (4) performing data correlation analysis by taking the flow rate value as an independent variable and the seedling dry weight activity index as a dependent variable. Obtaining the seedling dry weight activity index and Ca²⁺The flow rate was very significantly positively correlated (p 0.0009) and the dry weight viability index of the shoots was fitted to Ca using SPSS²⁺Linear regression of flow rate, Y-0.0006218X +0.1454 (R)²0.8604), wherein Y represents the shoot dry weight viability index and X represents the embryo Ca²⁺Flow rate value of (unit: pmol cm)^-2S^-1) (FIG. 6).

The method for calculating the seedling dry weight vitality index comprises the following steps:

standard germination tests were first performed. The sand bed prepared by building sand with the grain diameter of 0.05-0.8mm is used for a germination test, the water content of the sand bed is 60% of the saturated water content, 50 seeds are repeated for 3 times, the sand bed is placed into a plant growth chamber with the temperature of 25 ℃, the relative humidity of 60% and the illumination of 16 h/dark 8h for culture, the cover of the germination box is opened after 3 days, the number of seedlings (normal seedlings) is recorded every day, meanwhile, lost water is supplemented properly, and the requirement of the seedlings on water in the normal growth process is guaranteed. And (4) taking out the seedlings after the last counting on the 7 th day, washing the seedlings, removing residual seed tissues, separating the seedlings on the roots and the overground parts, drying and weighing the seedlings on the overground parts, and calculating the dry weight vitality index of the seedlings.

The formula is that the dry weight vitality index VI of the seedling is GI × S,

GI ∑ (Gt/Dt), where Dt is the number of germinating days, Gt is the number of germinating seeds per day corresponding to Dt,

s is the dry weight of the seedling on the single plant.

The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. Corn embryo radicle Ca²⁺The use of flow rate in the determination of maize seed vigor;

the corn seed embryo is obtained by the following treatment:

imbibing the corn seeds to be detected for 24h at 25 ℃, cutting off endosperm of the corn seeds, and stripping out complete corn embryos;

ca detected²⁺The flow rate is the rate of imbibition in the seeds;

taking the seedling dry weight vitality index as a Y value and taking the corn embryo radicle Ca²⁺The flow rate is X value, and the seedling dry weight activity index and Ca are obtained by fitting²⁺Linear curve of flow rate, and evaluating the vitality of corn seeds, the seedling dry weight vitality index and Ca through the linear curve²⁺The flow rates are in a very significant positive correlation.

2. Based on Ca²⁺The method for measuring the activity of the corn seeds at the flow rate is characterized by comprising the following steps of:

determination of corn embryo radicle Ca by non-damage micrometering system²⁺Flow rate according to Ca²⁺Flow rate to evaluate the viability of the corn seeds;

the corn seed embryo is obtained by the following treatment:

Ca²⁺the flow rate is measured by the following method: fixing the corn embryo with the back facing downwards in a culture dish, adding a test solution, and balancing for 5-10 min; placing the culture dish on an objective table of a non-damage micrometering system, utilizing a microscope and a three-dimensional motion displacement platform to clearly display the electrode and the corn embryo in the same visual field, enabling the tip of the electrode to be close to the root end of the corn embryo, operating software, and carrying out Ca treatment on the root end of the corn embryo²⁺Detecting the flow rate;

the test solution comprises the following components:

KCl 0.1mM、CaCl₂0.1mM、MgCl₂0.1mM、NaCl 0.5mM、Na₂SO₄0.2mM, MES 0.3mM, pH 6.0.

3. The method for determining the viability of a maize seed according to claim 2 wherein the distance between the tip of the electrode and the radicle end of the maize embryo is 2-5 μm.

4. The method for determining the viability of maize seeds according to claim 2, wherein Ca is added to the radicle end of maize embryo²⁺The flow rate was continuously measured for 10 min.

5. The method for determining the vitality of corn seeds according to claim 2, wherein the vitality index of the dry weight of the seedling is used as the Y value, and the Ca value of the embryonic root end of the corn seed embryo is used²⁺The flow rate is X value, and the seedling dry weight activity index and Ca are obtained by fitting²⁺Linear curve of flow rate, and the activity of the corn seeds is evaluated through the linear curve.

6. The method for determining the viability of corn seeds according to claim 5, wherein said linear curve is Y-0.0006218X +0.1454, R²＝0.8604。