CN103477749B - A kind of Course of Corn Seed Germination phase drought resistance appraisement method based on spontaneous luminescence - Google Patents

Abstract

The present invention discloses a method for evaluating the drought resistance of corn seeds based on spontaneous luminescence. The PEG-6000 solution of -0.3MPa is used to stress the germinated corn seeds to water, and the spontaneous luminescence of the germinated seeds is measured every 24 hours during the germination process. The drought resistance of maize seeds during germination can be evaluated by the relative change rate of spontaneous luminescence after 24 hours of water stress. The present invention is based on the spontaneous luminescence drought resistance evaluation method of corn seeds in the germination stage, without the need for field experiments, only need to carry out water stress on germinated seeds, directly measure the spontaneous luminescence of seeds during the germination process, and realize the evaluation of corn seeds through the relative change rate of spontaneous luminescence. The non-destructive and rapid evaluation of the drought resistance in the germination stage is simple and practical, and the evaluation effect is reliable.

Description

A method for evaluating drought resistance of maize seeds during germination based on spontaneous luminescence

technical field

The invention belongs to the technical field of agriculture, and relates to a method for evaluating the stress resistance of crops, in particular to a method for evaluating the drought resistance of corn seeds in germination stage based on spontaneous luminescence.

Background technique

In the arid and semi-arid areas of China, corn often encounters drought during the germination period after sowing, and the germinated corn seeds are very sensitive to drought, which often greatly affects seed germination and seedling emergence. Corn with strong drought resistance in the germination period is used Seeds are of great significance to food production in arid and semi-arid regions of my country.

In order to breed maize seeds with strong drought resistance during germination, it is necessary to establish a set of methods and techniques that can accurately identify and evaluate the drought resistance of maize seeds during germination.

For a long time, the drought resistance of maize seeds during germination has been evaluated according to the biological changes of maize seeds under drought stress. The evaluation indexes include biological drought resistance indexes and physiological drought resistance indexes. The biological drought resistance index is based on the direct damage caused by drought stress to the growth and development of the variety. The evaluation indexes include seed germination drought resistance index (GDRI), storage material transfer rate, relative germination rate, radicle number, main radicle length, and coleoptile length, plant height, etc. Physiological drought resistance indicators evaluate the drought resistance of crop varieties based on the changes in physiological and biochemical reactions under drought stress. According to incomplete statistics, physiological drought resistance indicators include respiration rate, plasma membrane permeability, proline content, malondialdehyde (MDA ) content, superoxide dismutase (SOD) activity, catalase (CAT) activity, peroxidase (POD) activity, nitrate reductase activity, ATPase activity, proteolytic enzyme activity, VC content, K+ content wait. Since the research found that the drought resistance of crops cannot be accurately evaluated by relying on a single physiological index, quantitative analysis methods such as the subordination function method, cluster analysis, and gray relational analysis for the comprehensive evaluation of drought resistance of multiple indexes, as well as the screening and analysis of drought resistance genes have been studied in recent years. QTL (quantitativetraitlocus) positioning and other deeper aspects of development.

However, in the current drought resistance evaluation method, the determination of biological drought resistance indicators is complicated, the workload is large and the cycle is long, and it needs to be evaluated after the appearance of macroscopic traits, and early diagnosis cannot be made; most of the physiological drought resistance indicators are obtained through destructive measurement of test tube experiments However, non-destructive testing cannot be achieved; the multi-indicator quantitative analysis method is based on the one-sided results of test tube experiments, and the indicators to be measured are many and complicated, and the workload is heavy; molecular biology techniques need to destroy biological tissues, and are inseparable from cumbersome Specific sample preparation procedures and complex detection procedures lead to high measurement costs.

More importantly, crop drought resistance is the embodiment of the self-regulation ability of crop cells under drought stress. The characteristic of these experiments is that in vitro operations are required, that is, cells must be broken, purified and enriched, and real-time positioning and measurement of living cells cannot be carried out, resulting in the inconsistency of interaction information between components and levels in cells. Lost, the measurement results cannot reflect the real operating state of biological metabolism or life. Therefore, although a lot of progress has been made in the application of biological methods to study crop drought resistance, the research results cannot accurately reflect the ability of cell self-coordination and crop drought resistance under drought stress. A new method for evaluating drought resistance of crops that can achieve non-destructive and rapid measurement of overall cell metabolism changes.

Biophotonics research shows that crop seeds in the dark have a weak luminescence phenomenon, which is produced by seeds in the life activities of cells, called spontaneous luminescence. Studies have found that the spontaneous luminescence of germinated seeds is closely related to the cellular respiration metabolism and DNA synthesis in germinated seeds, and is a kind of life information that reflects the physiological metabolism of seed cells. Since drought inhibits the physiological metabolism of germinated seeds, it will inevitably lead to the reduction of spontaneous luminescence. Then, the strength of drought resistance of maize seeds during germination can be judged according to the change degree of spontaneous luminescence of germinating maize under drought stress. Since this evaluation method is realized by measuring the luminescence of seeds, it can reflect the change of the overall metabolism of cells under drought stress, so it has the advantage of accurate judgment; and because the measurement of luminescence of seeds does not need to destroy the seeds, the measurement is rapid and non-destructive. and fast features.

Contents of the invention

The purpose of the present invention is to provide a method for evaluating the drought resistance of corn seeds in germination stage based on spontaneous luminescence, which solves the problems of long cycle, time-consuming and laborious, and inaccurate destructive measurement and evaluation of existing detection methods. The present invention has determined the conditions, measurement methods and evaluation indexes for evaluating the drought resistance of corn seeds through experiments, and the drought resistance data obtained according to the method proposed by the present invention can reflect the drought resistance of corn seeds in a non-destructive, fast and accurate manner. weak.

The technical scheme adopted in the present invention is a method for evaluating the drought resistance of corn seeds in the germination stage based on spontaneous luminescence, which is specifically implemented according to the following steps:

Step 1: Select seeds with the same plumpness and size among the corn seeds to be evaluated, clean and disinfect;

Step 2: placing the treated corn seeds obtained in step 1 evenly in a petri dish covered with two layers of filter paper, adding PEG-6000 solution for water stress;

Step 3: Put the corn seeds obtained in Step 2 into an incubator, culture at a constant temperature and dark at 25°C, and replace the PEG-6000 solution every 24 hours;

Step 4: Measure the spontaneous luminescence of maize seeds at the beginning of water stress, denoted as Thereafter, the spontaneous luminescence of corn seeds was measured every 24 hours, which was recorded as I _SL ;

Step 5: Calculate the relative change rate of spontaneous luminescence every 24 hours during the germination process of corn seeds under water stress according to the following formula:

${\mathrm{<span\; class="notranslate">\; R</span>}}_{\mathrm{<span\; class="notranslate">\; SL</span>}}<span\; class="notranslate">\; =</span>\frac{{\mathrm{<span\; class="notranslate">\; I</span>}}_{\mathrm{<span\; class="notranslate">\; SL</span>}}}{{\mathrm{<span\; class="notranslate">\; I</span>}}_{\mathrm{<span\; class="notranslate">\; SL</span>}}^{\mathrm{<span\; class="notranslate">\; 0</span>}}}<span\; class="notranslate">\; -</span>\mathrm{<span\; class="notranslate">\; 1</span>}<span\; class="notranslate">\; ,</span>$

Among them, R _SL is the relative change rate of spontaneous luminescence, I _SL is the current spontaneous luminescence, Spontaneous luminescence for the onset of stress;

Step 6: Evaluate the drought resistance of maize seed germination according to the size of R _SL . The larger the R _SL value, the stronger the drought resistance, and the smaller the R _SL value, the weaker the drought resistance.

The present invention is also characterized in that,

The osmotic potential of the PEG-6000 solution in step 2 is -0.3Mpa, and the amount of the PEG-6000 solution does not submerge the seeds.

The measurement of the spontaneous luminescence of the corn seeds in step 4 is carried out in a dark room, or the measurement is performed after putting the seeds into the dark room from the light for 10 minutes.

The beneficial effect of the present invention is that no field experiment is required, only the water stress of the germinated seeds is required, the spontaneous luminescence of the germinated seeds is directly measured, and the non-destructive and rapid evaluation of the drought resistance of the germinated seeds is realized through the relative change rate of the spontaneous luminescence. Simple and practical, the evaluation effect is reliable.

Description of drawings

Fig. 1 is the change and difference of the spontaneous luminescence R _SL value of two maize varieties under water stress in the embodiment of the present invention.

Detailed ways

The principle of the invention is: the spontaneous luminescence in the seed germination process comes from the oxidative metabolism and DNA synthesis reaction in the seed cells. In the process of seed germination, cells begin to repair and activate after absorbing water, the defects of the inner mitochondrial membrane are restored to integrity, electron transferases are synthesized or activated and embedded in the membrane, the oxidative phosphorylation process gradually returns to normal, and the recovery of respiratory metabolism makes ATP Supply accelerates, DNA synthesis gradually begins, and these physiological events allow for a gradual increase in spontaneous luminescence during germination. Excessive free radicals under water stress will directly attack cell membrane lipids and thiol-containing proteins, causing degradation of cell membrane components and loss of certain enzyme functions, resulting in cell membrane damage, oxidative phosphorylation uncoupling, decreased respiration rate and decreased ATP production , DNA synthesis is hindered, and these changes will inevitably lead to a decrease in spontaneous luminescence. Therefore, the decrease of spontaneous luminescence under water stress provides the information of suppressed cellular metabolism and hindered DNA synthesis. Crop seeds with different drought resistance have different adaptability to water stress. Under the same stress degree, cell metabolism is inhibited and DNA synthesis is hindered to different degrees. This difference can be sensitively reflected by the change of spontaneous luminescence.

The present invention is based on a method for evaluating the drought resistance of corn seeds in the germination stage of spontaneous luminescence, which is specifically implemented according to the following steps:

The present invention adopts the hyperosmotic solution method, that is, the osmotic potential of -0.3MPa-0.3MPa polyethylene glycol-6000 (PEG-6000) aqueous solution is used to water-stress the germinated corn seeds, and the PEG-6000 of -0.3MPa can obviously inhibit the growth of corn seeds. sprout. Specifically follow the steps below:

Step 1: Select seeds with the same plumpness and size among the corn seeds to be evaluated, clean and disinfect them, and kill surface microorganisms; since microorganisms have spontaneous luminescence, if they are not killed, the accuracy of the evaluation will be affected.

Step 2: Place the cleaned and sterilized corn seeds evenly in a petri dish covered with two layers of filter paper, add -0.3MPa PEG-6000 solution for water stress, and the amount of PEG-6000 solution should not submerge the seeds.

Step 3: Put the seeds in step 2 into an incubator, culture at a constant temperature and dark at 25°C, and replace the PEG-6000 solution every 24 hours. In order to evaluate truly and objectively, the stress culture of the present invention was repeated 4 times, that is, divided into 4 groups under the same conditions.

Step 4: Measure the spontaneous luminescence of maize seeds at the beginning of water stress, denoted as Thereafter, the seeds in step 3 were taken out every 24 hours, and their spontaneous luminescence was measured, which was recorded as I _SL . The measurement of seed spontaneous luminescence should be carried out in a dark room, or after the seeds are put into the dark room from the light for 10 minutes, the measuring instrument can use various high-sensitivity low-light detection instruments.

Step 5: Calculate the relative change rate of spontaneous luminescence every 24 hours during the germination process of seeds under water stress according to the following formula:

${\mathrm{<span\; class="notranslate">\; R</span>}}_{\mathrm{<span\; class="notranslate">\; SL</span>}}<span\; class="notranslate">\; =</span>\frac{{\mathrm{<span\; class="notranslate">\; I</span>}}_{\mathrm{<span\; class="notranslate">\; SL</span>}}}{{\mathrm{<span\; class="notranslate">\; I</span>}}_{\mathrm{<span\; class="notranslate">\; SL</span>}}^{\mathrm{<span\; class="notranslate">\; 0</span>}}}<span\; class="notranslate">\; -</span>\mathrm{<span\; class="notranslate">\; 1</span>}<span\; class="notranslate">\; -</span><span\; class="notranslate">\; -</span><span\; class="notranslate">\; -</span><span\; class="notranslate">\; (</span>\mathrm{<span\; class="notranslate">\; 1</span>}<span\; class="notranslate">\; )</span>$

Among them, R _SL is the relative change rate of spontaneous luminescence, I _SL is the current spontaneous luminescence, Spontaneous luminescence for initial value (when stress starts). Each replicate was measured individually and then averaged.

Step 6: Evaluate the drought resistance of maize seed germination according to the R _SL value after 24 hours of stress. The greater the R _SL value, the stronger the drought resistance.

The spontaneous luminescence of germinated seeds reflects the intensity of cell metabolism and DNA synthesis during germination. The relative change rate of spontaneous luminescence R _SL of maize seeds during germination under water stress is used as a sign of drought resistance during seed germination. R _SL The larger the value, the stronger the cell metabolism and DNA synthesis during the germination process, the less the inhibition, the stronger the drought resistance; on the contrary, the smaller the R _SL value, the more the cell metabolism and DNA synthesis are inhibited during the germination process. The larger, the weaker the drought resistance. This method, which uses the relative rate of change of spontaneous luminescence R _SL as the evaluation index, has nothing to do with crop varieties and is universal.

Example

To judge the drought resistance of two maize seeds, Wanrui 168 and Yandan 8, two plump seeds with the same size and appearance were selected, washed with distilled water, sterilized by adding 0.2% HgCl ₂ , washed, and evenly Place them in petri dishes covered with two layers of filter paper, place 120 corn seeds in each petri dish, add PEG-6000 solution with an osmotic potential of -0.3MPa, and culture at a constant temperature and dark at 25°C, the PEG-6000 solution The amount added is subject to not submerging the seeds. Considering the effect of seed water absorption on osmotic potential, the PEG-6000 solution was replaced every 24 hours. Another group of seeds was taken as a control, and the conditions of the control group were the same as those of the treatment group except that they were cultured with distilled water. There were 4 replicates in each treatment group and control group.

Record the germination number of the corn seeds of the control group and the treatment group every 24 hours, calculate the seed germination rate, and get the average. The seed germination rate is equal to the number of germinated seeds/total number of seeds, and dew white seeds are defined as germinated seeds. Calculate the seed germination drought resistance index (GDRI) according to the conventional method, and the calculation method is: seed germination drought resistance index (GDRI) = seed germination index under water stress / control seed germination index

Seed Germination Index (PI)=(1.00)nd2+(0.75)nd4+(0.50)nd6+(0.25)nd8(2) Among them, nd2, nd4, nd6 and nd8 are 2 days, 4 days, 6 days and 8 days after germination The germination rate, 1.00, 0.75, 0.50 and 0.25 are the drought resistance coefficients given by the corresponding germination days respectively.

Put all the seeds in each group of corn seeds after measuring the germination rate of the seeds (including the grown part after cultivation and ungerminated seeds) in an oven, bake at 80°C until constant weight, and weigh the dry weight (including root weight, bud weight, Grain weight), according to conventional methods to measure the transfer rate of germinated seed storage substances, the calculation method is:

Storage material transfer rate (%) = (bud + root) dry weight / (bud + root + grain) dry weight × 100% (3)

The spontaneous luminescence I _SL of germinated seeds was measured by a BPCL weak luminescence measuring instrument, and the unit of I _SL was counts/s.

Table 1 shows the seed germination drought resistance index and storage material transfer rate of two maize varieties calculated from the experimental results. It can be seen from Table 1 that under the water stress of -0.3MPa PEG-6000 solution, Wanrui 168 maize The drought resistance index of seed germination and the transfer rate of storage materials were both lower than those of Yandan 8 maize, which indicated that the drought resistance of Yandan 8 maize was stronger than that of Wanrui 168 during seed germination.

Table 1 Seed germination drought resistance index and storage material transfer rate of two maize varieties under water stress

Figure 1 shows the changes and differences in the relative change rate R _SL of spontaneous luminescence of maize seeds of Wanrui 168 and Yandan 8 under the water stress of -0.3MPa PEG. It can be seen from Figure 1 that the R _SL value of Wanrui 168 maize with weaker drought resistance germinated under water stress was much lower than that of Yandan 8 maize with strong drought resistance from 24 hours after germination. The strength of drought resistance of varieties during seed germination can be distinguished by the value of R _SL .

It can be seen from this example that the method for evaluating the drought resistance of corn seeds at the germination stage proposed by the present invention does not require field experiments, and only needs to apply a hypertonic solution to the germinated seeds to form water stress, and directly measure the spontaneous luminescence of the germinated seeds, and the corn seed germination stage can be realized. The non-destructive and rapid evaluation of drought resistance is consistent with conventional methods such as seed germination drought resistance index (GDRI) and storage material transfer rate (see Table 1), which shows that the evaluation method proposed by the present invention is reliable.

In particular, the dynamic change process of spontaneous luminescence R _SL during maize seed germination under drought stress can sensitively reflect the occurrence time and degree of seed cell metabolism and DNA synthesis blockage. For example, in Figure 1, under the osmotic stress of -0.3MPa, the R _SL value of spontaneous luminescence of Yandan No. 8 corn increased rapidly at the beginning of germination, an inflection point appeared at 24 hours, and then increased slowly, indicating that at 24 hours _Drought stress began to affect cell metabolism and DNA synthesis. Afterwards, the cells were adjusted and gradually adapted to the drought environment, and DNA synthesis gradually recovered; It shows that the damage and influence of drought stress on maize cells of Wanrui 168 is greater than that of Yandan 8. Since then, the R _SL value of Yandan 8 has been significantly greater than that of Wanrui 168, indicating that Yandan 8 has a greater effect on drought stress. The degree of adaptation and recovery is much higher than that of Wanrui 168. Therefore, the evaluation method proposed by the present invention has the characteristics of sensitivity and accuracy.

Claims (1)

1. A method for evaluating drought resistance during germination of corn seeds based on spontaneous luminescence, characterized in that, it is specifically implemented according to the following steps: Step 1: Select seeds with the same plumpness and size among the corn seeds to be evaluated, clean and disinfect; Step 2: Place the treated corn seeds obtained in step 1 evenly in a petri dish covered with two layers of filter paper, add PEG-6000 solution for water stress; the osmotic potential of the PEG-6000 solution is -0.3Mpa, PEG The amount of -6000 solution does not submerge the seeds; Step 3: Put the corn seeds obtained in Step 2 into an incubator, culture at a constant temperature and dark at 25°C, and replace the PEG-6000 solution every 24 hours; Step 4: Measure the spontaneous luminescence of maize seeds at the beginning of water stress, denoted as Thereafter, the spontaneous luminescence of corn seeds was measured every 24 hours, denoted as _ISL ; the measurement of the spontaneous luminescence of corn seeds was carried out in a darkroom, or the seeds were put into the darkroom from the light and then measured after 10 minutes; Step 5: Calculate the relative change rate of spontaneous luminescence every 24 hours during the germination process of corn seeds under water stress according to the following formula: ${\mathrm{<span\; class="notranslate">\; R</span>}}_{\mathrm{<span\; class="notranslate">\; SL</span>}}<span\; class="notranslate">\; =</span>\frac{{\mathrm{<span\; class="notranslate">\; I</span>}}_{\mathrm{<span\; class="notranslate">\; SL</span>}}}{{\mathrm{<span\; class="notranslate">\; I</span>}}_{\mathrm{<span\; class="notranslate">\; SL</span>}}^{\mathrm{<span\; class="notranslate">\; 0</span>}}}<span\; class="notranslate">\; -</span>\mathrm{<span\; class="notranslate">\; 1</span>}<span\; class="notranslate">\; ,</span>$ Among them, R _SL is the relative change rate of spontaneous luminescence, I _SL is the current spontaneous luminescence, Spontaneous luminescence for the onset of stress; Step 6: Evaluate the drought resistance of maize seed germination according to the size of R _SL . The larger the R _SL value, the stronger the drought resistance, and the smaller the R _SL value, the weaker the drought resistance.