CN113176235A - Hickory nut high temperature resistance evaluation method based on rapid fluorescence induction kinetics - Google Patents
Hickory nut high temperature resistance evaluation method based on rapid fluorescence induction kinetics Download PDFInfo
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
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- G—PHYSICS
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Abstract
The invention discloses a hickory nut high temperature resistance evaluation method based on rapid fluorescence induction kinetics, which comprises the steps of collecting and culturing a sample, then carrying out heat treatment on the sample, determining the sample by adopting a multifunctional plant efficiency analyzer to obtain a rapid fluorescence induction kinetics curve (OJIP curve) and parameters, and finally carrying out comparison analysis according to the change of the OJIP curve and the difference of the parameters of each group of samples at a certain temperature to obtain an evaluation result of hickory nut heat resistance. The invention provides a method for evaluating the heat resistance of pecans grafted on different stocks in a non-destructive manner by utilizing a rapid chlorophyll fluorescence rising kinetic technology, and the heat resistance of the pecans can be analyzed through the change of rapid chlorophyll fluorescence of different varieties of pecans at different temperatures, so that the method is scientific, effective, non-destructive and accurate.
Description
Technical Field
The invention relates to the technical field of hickory nut high temperature resistance evaluation, in particular to a hickory nut high temperature resistance evaluation method based on rapid fluorescence induction kinetics.
Background
The Carya cathayensis is a deciduous tree, and is a plant of Carya of Juglandaceae, named as Carya cathayensis and Rhus verniciflua Stokes. Mainly produced in Tianmu mountain areas, Changbai areas and cross country areas of Zhejiang and Wan junctions, and is one of important economic crops in China.
Temperature is the main environmental factor affecting plant growth. The pecan seedlings are required to grow in a cool environment. In summer, in high-temperature seasons or in dry and rainy months, leaves of the hickory nut are often wilted or necrotic to different degrees, and the hickory nut shows poor heat resistance. High temperature can affect the growth and metabolism of plants, so that the plants are damaged and even die in severe cases. Therefore, the study of heat resistance is the focus and focus of the research of physiological resistance of economic plants.
The rapid chlorophyll fluorescence dynamics OJIP rising curve and JIP-test analysis are widely and successfully applied to the research of the physiological state of the plant stress by the characteristics of no damage, accuracy and rapidness. At present, people have more and more complicated heat resistance research methods for the pecans, which are destructive and need sampling analysis, so that the methods are complicated to operate and are not visual enough, and particularly, the heat resistance of the pecans is tested and evaluated systematically and harmlessly by utilizing rapid chlorophyll fluorescence dynamics, so that the heat resistance of different pecan seedlings is screened.
Disclosure of Invention
Technical problem to be solved
The invention establishes a method for evaluating the heat resistance of pecans grafted on different stocks by utilizing a rapid chlorophyll fluorescence rising kinetic technology, and can solve the problems that the conventional pecan heat resistance research method is more and more complicated, destructive, complex in operation, not visual enough, especially the method lacks the rapid chlorophyll fluorescence kinetic, and the heat resistance of the pecans is systematically tested and evaluated without damage.
(II) technical scheme
In order to achieve the purpose, the invention adopts the following technical scheme, and the hickory nut high temperature resistance evaluation method based on rapid fluorescence induction kinetics comprises the following specific steps:
s1: sample collection and culture: selecting different varieties of pecan seedlings to be planted in the culture bowls, and placing the pecan seedlings as samples in a nursery for seedling revival and maintenance;
s2: sample heat treatment: setting a control group and an experimental group of seedlings, respectively placing the seedlings in a heat treatment environment, setting the same heat treatment environment factors, and setting different stress temperatures;
s3: obtain ojis curves and parameters: measuring an OJIP curve of a sample leaf and a related chlorophyll fluorescence parameter;
s4: analysis and evaluation: the obtained ojis curve was subjected to a jis-test analysis, data was processed and plotted, one-way anova (one-way anova) and Duncan multiple comparative analysis (α ═ 0.05) were performed, and the analysis results were statistically collated to obtain evaluation results.
As a preferred embodiment of the present invention, in S3, the specific steps of measuring the ojis curve of the sample blade include:
s301: dark adaptation of the sample for 20min before measurement, and continuous irradiation of dark adapted leaves with 1s saturated pulse red light (650nm, 3500. mu. mol. m)-2·s-1)；
S302: selecting the 4 th to 5 th mature leaves of the plant from top to bottom as measuring leaves, measuring a rapid chlorophyll fluorescence induction kinetic curve, namely an OJIP curve, of the sample leaves by using a multifunctional plant efficiency analyzer, and obtaining the following chlorophyll fluorescence parameters including initial fluorescence Fo (50 mu s), Reaction Centers (RCs), fluorescence levels Fk of K points (300 mu s) and fluorescence levels F of J points (2ms)JFluorescence level FI at Point I (30ms) and maximum fluorescence intensity F at Point P (0.3-2s)M。
As a preferred technical scheme of the invention, the rapid chlorophyll fluorescence induction kinetic curve of a sample is measured every day, 6 carya cathayensis seedlings of each variety are selected, each plant is measured twice, and after each measurement, the plant is put back to a growth chamber and continuously subjected to heat treatment.
In a preferred embodiment of the present invention, in S4, the formula in the parameter comparison table of the obtained ojis curve is calculated and converted into the jis-test parameters selected in the present invention, and the reference table is:
TABLE 1 formula description of OJIP curves and JIP-test parameters
As a preferred technical solution of the present invention, in S4, the specific steps of performing the one-way anova include:
analyzing the performance of OJIP fluorescence induction kinetic curves of each group of samples at different stress temperatures, analyzing the performance of OJIP curves K phase and J phase of OJIP curves of each group of samples at different high temperatures, and analyzing, wherein the OJIP curves of fluorescence induction kinetics have correlation with fluorescence intensity and temperature, so that the difference of damage degree of different sample leaves under different high temperature stresses and the difference of relative variable fluorescence (delta Vt) of different samples can be analyzed.
As a preferred embodiment of the present invention, in S4, the specific steps of performing Duncan multiple comparison analysis (α ═ 0.05) include:
through WK＝W300μs＝(F300μs-FO)/(Fj-FO) Calculating to obtain WK and delta WK, and analyzing WKAnd Δ WKAnalyzing the changes of ABS/RC and DIo/RC of different samples under the high temperature stress of different temperatures;
analyzing the photosynthetic performance of each group of sample leaves at different high temperatures: Fv/Fm reflects the maximum light energy conversion efficiency of PS II, and each sample is observed at different temperatures
Variation of the value according to the PI of each sampleABSRanking the heat resistance of the plants;
and analyzing the PSI and PSII electron transfer changes of each group of sample leaves at different high temperatures, wherein the lower the electron transfer connectivity of the sample after high temperature stress is, which indicates that the sample is more sensitive to the temperature.
As a preferred technical solution of the present invention, in S2, the heat treatment environment is specifically set as follows: the illumination intensity is 520--2·s-1The relative humidity of air is 65% -75%, soil moisture is supplemented at regular time and quantity every day, the relative water content of soil is kept at 75% -85%, and the stress temperature is set as follows: the control group (CK) was set at a light high temperature of 14h/25 ℃ overnight for 10h at 30 ℃ day, a medium high temperature of 35 ℃ day for 14h/25 ℃ overnight for 10h, a severe high temperature of 40 ℃ day for 14h/35 ℃ overnight for 10h, and a temperature of 43 ℃ day for 14h/38 ℃ overnight for 10h, and at 25 ℃ day for 14h/20 ℃ overnight for 10 h.
(III) advantageous effects
1. The pecan high temperature resistance evaluation method based on rapid fluorescence induction kinetics provided by the invention has the advantage that the change of the OJIP curve mainly depends on the PSII (Q)A) The influence of a primary electron acceptor, the change of an OJIP curve reflects the transmission of an electron transfer chain for photosynthesis, an OJIP test reflects the behavior of PSII functions, including energy absorption, capture and electron transmission, and the connectivity degree of electron transmission of the hickory nut in a photosystem can be known through the OJIP curve, so that the high temperature resistance of the hickory nut of the variety can be conveniently evaluated;
2. the hickory nut high temperature resistance evaluation method based on rapid fluorescence induction kinetics provided by the invention has the advantages that all treated leaves are dark-adapted for 20min before measurement, and 1s of saturated pulse red light (650nm, 3500 mu mol. m) is continuously irradiated to the dark-adapted leaves-2·s-1) The rapid fluorescence curve is induced to rise, so that the curve change is convenient to observe;
3. the pecan high-temperature resistance evaluation method based on rapid fluorescence induction kinetics provided by the invention performs difference analysis on the relative variable fluorescence (delta Vt) among all groups of samples, can obviously reflect the change of the leaf oxygen release complex and the PSII complex unit, and is convenient to be used as a reference for analyzing the high-temperature resistance of all groups of pecans.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are required to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a schematic block flow diagram of the present invention;
FIG. 2 is a schematic diagram showing the variation of the rapid fluorescence induction curve of the hickory leaf under high temperature stress according to the present invention;
FIG. 3 is a graph showing the effect of high temperature stress on the relative variable fluorescence (. DELTA.Vt) of the leaves of a hickory nut according to the present invention;
FIG. 4 is a graph showing groups F of samples of the zone of curve L in example 1 of the present inventiont-FOAt FK-FOA plot of variable fluorescence rate over;
FIG. 5 shows groups F of samples of the curve K band in example 1 of the present inventiont-FOAt FJ-FOA plot of variable fluorescence rate over;
FIG. 6 shows that the high temperature stress of the present invention is applied to V in OJIP of hickory leafK、VJ、WK、ΔWKSchematic diagram of the effects of (1);
FIG. 7 is a schematic diagram showing the effect of high temperature stress on the energy flux of PSII reaction center of the hickory leaf according to the present invention
FIG. 8 is a schematic view showing the effect of high temperature stress on the photosynthesis index of the hickory leaf according to the present invention;
FIG. 9 is a schematic diagram showing the effect of high temperature stress on the quantum yield and flux ratio of the hickory leaf.
The implementation, functional features and advantages of the objects of the present invention will be further explained with reference to the accompanying drawings.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be described clearly and completely with reference to the accompanying drawings of the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
Thus, the following detailed description of the embodiments of the present invention, presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
It should be noted that: like reference numbers and letters refer to like items in the following figures, and thus, once an item is defined in one figure, it need not be further defined and explained in subsequent figures.
In the description of the present invention, it is to be understood that the terms "longitudinal", "upper", "lower", "left", "right", "top", "bottom", "inner", "outer", and the like, indicate orientations or positional relationships based on the orientations or positional relationships shown in the drawings, are used only for convenience in describing the present invention and for simplicity in description, and do not indicate or imply that the referenced devices or elements must have a particular orientation, be constructed and operated in a particular orientation, and thus, are not to be construed as limiting the present invention.
Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of the present invention, "a plurality" means two or more unless specifically defined otherwise.
As shown in fig. 1-9, the hickory nut high temperature resistance evaluation method based on rapid fluorescence induction kinetics specifically comprises the following steps:
s1: sample collection and culture: the test varieties are respectively as follows: hunan rootstock hickory, American rootstock hickory, Lin ' an rootstock hickory and seedling (hereinafter abbreviated as ' HN ', ' MG ', ' LA ', ' SS '). Seedlings with good growth vigor and consistent growth are planted in a polyethylene culture pot (the upper caliber is about 24cm, the height is 26cm, and the lower caliber is 19.7cm) and are used as samples to be placed in a nursery for seedling revival and maintenance;
s2: sample heat treatment: respectively placing the seedlings in an intelligent illumination incubator with illumination intensity of 600 mu mol.m-2·s-1The relative humidity of the air is about 70%, the soil moisture is supplemented according to the quantity every day, the relative moisture content of the soil is kept at 80%, and the stress temperature is set as follows: a mild high temperature of 30 ℃/25 ℃ (day 14 h/night 10h), a moderate high temperature of 35 ℃/25 ℃ (day 14 h/night 10h), a severe high temperature of 40 ℃/35 ℃ (day 14 h/night 10h) and 43 ℃/38 ℃ (day 14 h/night 10h), and a control group (CK) is set at 25 ℃/20 ℃;
s3: obtain ojis curves and parameters: measuring a rapid chlorophyll fluorescence induction kinetic curve, namely an OJIP curve, of a sample leaf by using a multifunctional plant efficiency analyzer, measuring the rapid chlorophyll fluorescence induction kinetic curve of the sample every day, selecting 6 seedlings of each variety of pecan, measuring each plant twice, and after each measurement, putting the plant back to a growth chamber and continuing heat treatment;
s4: analysis and evaluation: the obtained ojis curve was subjected to a jis-test analysis with reference to the formula description of the table, data was processed and plotted using Origin2017 software, one-way anova (one-way anova) and Duncan multiple comparison analysis (α is 0.05) were performed using span 17.0, and the analysis results were statistically collated to obtain evaluation results.
S301: dark adaptation of the sample for 20min before measurement, and continuous irradiation of dark adapted leaves with 1s saturated pulse red light (650nm, 3500. mu. mol. m)-2·s-1)；
S302: selecting the 4 th to 5 th mature leaves of the plant from top to bottom as the measured leaves, measuring the rapid chlorophyll fluorescence induction kinetic curve of the sample leaves by using a multifunctional plant efficiency analyzer, namely an OJIP curve, and obtaining the following chlorophyll fluorescence parameters including initial fluorescence Fo (50 mu s), Reaction Centers (RCs) and fluorescence level F of K points (300 mu s)kJ Point (2ms) fluorescence level FJFluorescence level F at Point I (30ms)IAnd maximum fluorescence intensity F at the P point (0.3-2s)MAnd the PSII under high-temperature stress can be conveniently and accurately recordedRCsEnergy capture and changes in electron transfer on the donor and acceptor sides of the PSII.
The formula in the parameter comparison table of the obtained OJIP curve is calculated and converted into the JIP-test parameters selected in the invention, and the reference table is as follows:
TABLE 1 formula description of OJIP curves and JIP-test parameters
The specific steps for performing one-way anova and Duncan multiple comparison analysis (α ═ 0.05) using span 17.0 are:
the fluorescence induction kinetic curves of the 4 hickory nut control groups (at 25 ℃) are typical O-J-I-P curves. The OJIP curves of 4 kinds of hickory nuts have no obvious difference compared with a control group under the high-temperature treatment at 30 ℃. As the stress temperature increased (from 35 ℃ to 43 ℃), the OJIP curves of 4 pecans appeared with the "K" phase around 300ms, gradually changing into O-K-J-I-P curves. The ojis curves of 'LA' and 'SS' start to change at 35 ℃, respectively, and the change of the ojis curve becomes more significant as the processing temperature is higher. The ojis curves of 'LA', 'SS' are more pronounced than the ojis curves of 'HN' and 'MG' as the temperature increases. When the stress temperature is higher than 35 ℃, the chlorophyll fluorescence intensity and the P point fluorescence level of the leaves are sharply reduced (see figures 2C-E). Under the high-temperature stress of 35-43 ℃, the K point of the 'SS' is obviously increased compared with the K point of 25 ℃, meanwhile, the I point and the P point are sharply reduced, and the P point is obviously lower than that of the other three varieties. In the case of treatment at 43 ℃, the K-spot fluorescence level of ` SS ` exceeded that of the other three species (see FIG. 2E). In contrast, the chlorophyll fluorescence intensity of the 'HN' and 'MG' leaves is reduced to a smaller extent under the high-temperature stress of 40 ℃ or 43 ℃, and the K point level is also lower.In addition, F was subjected to heat stress at 40 ℃ and 43 ℃OAnd FMThere are also different degrees of increase and decrease in the value. Compared with the other three pecans, F of' SSOValue sum FMThe change in value is evident (see fig. 2F).
The relative variable fluorescence (Δ Vt) between 4 pecans was analyzed differentially. As shown in FIG. 3, under the high temperature treatment of 35 deg.C, the delta K and delta J of 4 Carya cathayensis seedlings are both >0, wherein the delta K and delta J of 'HN' and 'MG' are lower than those of 'LA' and 'SS'. The chlorophyll fluorescence yield at the K point appears to be obviously increased (delta K is more than 0) at about 300 mu s after treatment at 40 ℃ and 43 ℃. Wherein Δ K of 'LA' and 'SS' is significantly increased compared to 'HN' and 'MG'. The Δ J values at 2ms also showed the same rising tendency, indicating that the activities of the oxygen evolution complexes of 'HN', 'LA', and 'SS' were inhibited when treated at 35 ℃. The degree of inhibition was ` LA ` SS `.
Plotting the normalized OJIP induction curves of 4 hickory nuts between O (50 mus) -K (300 mus), i.e., Wok＝(Ft-Fo)/(Fk-Fo) And kinetic differences between high temperature treatment and CK (Δ Vok ═ Vok treatment-Vok control). An extra L-band of about 150 mus hidden between the O-point and the K-point can be observed by the difference. The L-band is an indicator of PSII specific energy connectivity, which is higher when connectivity is lower. Therefore, ` LA `, ` SS ` under mild high temperature stress (35 ℃ C.), Δ VokIncreasing with temperature change indicates that the energy connectivity of the L-band decreases significantly with temperature change. Under the same treatment temperature, the energy connectivity of 'MG' is higher than that of other varieties, and the damage degree is as follows: 'SS'>‘LA’>‘HN’>'MG'. Therefore, 'LA', 'SS' that lose the PSII energy connection are more sensitive to high temperatures. FIG. 5 shows that K-phase of 4 pecans was gradually excited under heat stress at 35 ℃ and 40 ℃ or 43 ℃, but could not be excited at K-point at 30 ℃. VOJ(Wk) Reflects the condition of the oxygen evolving complex and is temperature dependent. VOJThe larger the size, the more serious the oxygen evolving complex is damaged. This trend is the same as described above. Different high temperature stresses lead to 'LA', 'SS' Δ VOJThe strength of the K phase is obviously higher than that of 'HN' and 'MG', which indicates that OEC centers of 'LA' and 'SS' are easier to lose activity.
4 kinds of hickory leaf OJIP curve K phase and J phase WKAnd Δ WKValues (fig. 6C-D) increased more rapidly as stress temperature increased from 35 ℃ to 43 ℃ relative to "HN" and "MG", "LA" and "SS", with 'MG' being minimally affected by high temperature stress.
The energy activity parameter of the unit reaction center can more accurately reflect the absorption, conversion and dissipation of the photosynthetic organ to the light energy. ABS/RC is the light energy absorbed per reaction center; high temperature stress on the blade results in an increase in ABS/RC. Referring to fig. 7, the 'LA' and 'SS' ABS/RC were significantly increased (P <0.05) under the influence of high temperature stress. After heat stress at 43 ℃, the value increased by 29%, much higher than 'HN', 'MG'. TRo/RC is the light energy captured per reaction center, with flux increasing significantly from 30 ℃ for 'LA' and 'SS'; whereas 'HN' and 'MG' occur after 40 ℃. ETo/RC is the light energy per reaction center for electron transport. Before the high temperature treatment, 'LA' and 'SS' decreased by 15.4% and 14.3% compared to the control group, and 'HN' and 'MG' began to decrease after heat stress at 40 ℃. Under the same conditions, the amplification of 'LA' and 'SS' DIo/RC is significantly higher than that of 'HN' and 'MG', and the increase starts to be significant under the high temperature stress of 35 ℃ (P < 0.05).
Fv/Fm reflects the maximum light energy conversion efficiency of PS II. Referring to FIG. 8, 4 kinds of hickory leaves at 30 deg.C and 35 deg.C
The value was kept at 0.816 ± 0.007. Above 35 ℃ of ' LA ' and ' SS
A significant drop in the initial value. In addition, of ' LA ' and ' SS
Is more sensitive to high temperatures above 40 ℃ than 'HN' and 'MG'. The 'LA' is reduced by 6.21% compared with 25 deg.C and the 'SS' is reduced7.5% decrease, ` HN ` 5.34% decrease, ` MG ` 4.5% decrease.
PIABSIs the most sensitive of all fluorescence parameters, is a performance index based on absorbed light energy, and is used to express PSII overall photosynthesis activity, so that it can be ranked according to the heat tolerance of plants. PI when Fv/Fm and many other parameters have not changedABSSignificant changes have occurred. PI for 'HN' and 'MG' at 30 ℃ABSPI with slight increase in 'LA' and 'SS' valuesABSThe value begins to decrease, indicating that it begins to be affected by high temperature stress. For 'HN' and 'MG', PI at 35 deg.C or higherABSWill be significantly reduced. Wherein the reduction of 'LA' and 'SS' is significantly greater than that of the other two varieties. PI of four hickory leaves at 43 deg.C compared to control (25 deg.C)ABSThe reduction was about 44.2%, 37.9%, 57.9% and 54.9%.
Referring to FIG. 9, psi Eo and psi were observed when 4 hickory leaves were under mild stress (30 ℃ C.)
No significant change occurred. Psi Eo and of ` HN ` and ` MG ` under moderate stress (35 ℃ C.)
Slightly increased by psi Eo and psi of ' LA ' and ' SS
The value starts to decrease. Psi Eo and psi at stress temperatures of 40 ℃ and 43 ℃
Sharply decreases in which of psi Eo and of' LA
The reduction is 22.2 percent and 21.4 percent respectively compared with the control group.
The parameter δ Ro represents the efficiency of electron transfer to the PSI end. At 35 deg.C, 4 kinds of hickory nutsThe leaf delta Ro starts to increase and the delta Ro value increases further when the stress temperature rises to 40 ℃ and 43 ℃. At 30 ℃ for 'LA' and 'SS',
obviously increased, when the stress temperature is increased to more than 35 ℃,
begin to fall sharply; 'HN' and 'MG' at 30 ℃
No obvious change, when the stress temperature is increased to 35 ℃,
begins to rise significantly and then falls significantly. Many researchers found that moderate temperature stress did increase the activity of PSI. However, changes occurred when the 'LA', 'SS', and 'HN', and 'MG' stress temperatures were increased by 35 ℃ and 40 ℃ respectively,
starts to drop significantly and is minimized when the stress temperature reaches 43 ℃.
Photosynthesis is one of the most sensitive physiological processes of plants to high temperature stress, and chlorophyll fluorescence kinetic technology can reflect the intrinsic changes of photosynthetic organs under adversity stress. A large number of researches show that high-temperature stress can cause OEC inactivation, electron transfer capacity reduction and PSII activity reduction. Following high temperature stress, the OJIP curves J-I and I-P phases of the hickory leaf begin to drop substantially at 35 ℃ with both at the lowest level at 43 ℃. Wherein 'LA' and 'SS' decrease more rapidly than 'MG' and 'HN'. FIG. 2 shows that under the high temperature stress of 35 ℃, the Δ K and Δ J values of 'LA' and 'SS' are significantly increased compared with 'MG' and 'HN', which indicates that the photosynthetic performance of leaves of the varieties 'LA' and 'SS' is damaged, OEC is destroyed, and electron transfer is inhibited when the leaves are treated at 35 ℃; the Δ K values of the varieties 'LA' and 'SS' were significantly increased compared to the other two varieties when treated at 43 ℃, indicating that the 'LA' and 'SS' leaves had a large amount of QA-accumulation, the electron transfer between QA and QB was severely inhibited, the PSII receptor side suffered more severe injury, and the 'MG' and 'HN' were able to maintain the stability of PSII (oec) under high temperature stress. The L band can represent the aggregation among different components of PSII or the energy transfer connectivity of an antenna pigment and the RCs of the PSII active reaction center, and is higher after the plants are stressed. The variety ` LA ` leads to a significant decrease in the energy connectivity of the L band even under mild heat stress (30 ℃). Under the same stress temperature, connectivity of 'LA' is much lower than the other three varieties. The L band amplitude of 'LA' increases more rapidly after high temperature stress, and thus 'LA' losing more PSII energy connection is more sensitive to temperature.
In summary, the hickory nuts of 'LA' and 'SS' have a lower high temperature resistance than 'MG' and 'HN', wherein the 'MG', i.e., the american rootstock hickory nuts, have the most excellent high temperature resistance.
In conclusion, the invention can utilize the OJIP curve to solve the connectivity degree of the hickory nut in the electron transmission of an optical system, is convenient to evaluate the high temperature resistance of the hickory nut, and utilizes the quick chlorophyll fluorescence dynamics OJIP rising curve and JIP-test analysis in the process for screening the heat resistance difference between varieties, thereby scientifically and effectively evaluating the high temperature resistance of the hickory nut by the characteristics of no damage, accuracy and quickness.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (7)
1. The hickory nut high temperature resistance evaluation method based on rapid fluorescence induction kinetics is characterized by comprising the following specific steps of:
s1: sample collection and culture: selecting different varieties of pecan seedlings to be planted in the culture bowls, and placing the pecan seedlings as samples in a nursery for seedling revival and maintenance;
s2: sample heat treatment: setting a control group and an experimental group of seedlings, respectively placing the seedlings in a heat treatment environment, setting the same heat treatment environment factors, and setting different stress temperatures;
s3: ojis curves and parameters: measuring an OJIP curve of a sample leaf and a related chlorophyll fluorescence parameter;
s4: analysis and evaluation: the obtained ojis curve was subjected to a jis-test analysis, data was processed and plotted, one-way anova (one-way anova) and Duncan multiple comparative analysis (α ═ 0.05) were performed, and the analysis results were statistically collated to obtain evaluation results.
2. The hickory nut high temperature resistance evaluation method based on rapid fluorescence induction kinetics of claim 1, wherein in S3, the specific steps of determining the ojis p curve of the sample leaf are as follows:
s301: dark adaptation of the sample for 20min before measurement, and continuous irradiation of dark adapted leaves with 1s saturated pulse red light (650nm, 3500. mu. mol. m)-2·s-1)；
S302: selecting the 4 th to 5 th mature leaves of the plant from top to bottom as measuring leaves, measuring a rapid chlorophyll fluorescence induction kinetic curve, namely an OJIP curve, of the sample leaves by using a multifunctional plant efficiency analyzer, and obtaining the following chlorophyll fluorescence parameters, including initial fluorescence Fo (50 mu s), Reaction Centers (RCs), a fluorescence level Fk of a K point (300 mu s), a fluorescence level FJ of a J point (2ms), a fluorescence level FI of a I point (30ms) and a maximum fluorescence intensity FM at the P point (0.3-2 s).
3. The method for evaluating the high temperature resistance of the hickory nut based on the rapid fluorescence induction kinetics of claim 2, wherein in the steps of S2 and S3, the rapid chlorophyll fluorescence induction kinetics curve of the sample is measured every day, 6 seedlings of the hickory nut of each variety are selected, each plant is measured twice, and after each measurement, the plants are continuously heat-treated.
4. The method for evaluating the high temperature resistance of pecan nuts based on the rapid fluorescence induction kinetics of claim 2, wherein in step S4, the formula in the parameter comparison table of the obtained ojis curve is calculated and converted into the jis-test parameters selected in the present invention, and the reference table is:
TABLE 1 formula description of OJIP curves and JIP-test parameters
5. The hickory nut high temperature resistance evaluation method based on rapid fluorescence induction kinetics of claim 4, wherein in S4, the specific steps of performing one-way anova are as follows:
analyzing the performance of OJIP fluorescence induction kinetic curves of each group of samples at different stress temperatures, analyzing the performance of OJIP curves K phase and J phase of OJIP curves of each group of samples at different high temperatures, and analyzing, wherein the OJIP curves of fluorescence induction kinetics have correlation with fluorescence intensity and temperature, so that the difference of damage degree of different sample leaves under different high temperature stresses and the difference of relative variable fluorescence (delta Vt) of different samples can be analyzed.
6. The method for evaluating the high temperature resistance of the hickory nut based on the rapid fluorescence induction kinetics of claim 4, wherein in the step S4, the specific steps for performing Duncan multiple comparison analysis (α ═ 0.05) are as follows:
through WK＝W300μs＝(F300μs-FO)/(Fj-FO) Calculate to obtain WKAnd Δ WKAnd analyzing WKAnd Δ WKAnalyzing the change of different samples under high temperature stress at different temperatures according to ABS/RC and DA change in Io/RC;
analyzing the photosynthetic performance of each group of sample leaves at different high temperatures: Fv/Fm reflects the maximum light energy conversion efficiency of PS II, and each sample is observed at different temperatures
Variation of the value according to the PI of each sampleABSRanking the heat resistance of the plants;
and analyzing the PSI and PSII electron transfer changes of each group of sample leaves at different high temperatures, wherein the lower the electron transfer connectivity of the sample after high temperature stress is, which indicates that the sample is more sensitive to the temperature.
7. The hickory nut high temperature resistance evaluation method based on rapid fluorescence induction kinetics of claim 1, wherein in S2, the heat treatment environment is specifically set as follows: the illumination intensity is 520--2·s-1The relative humidity of air is 65% -75%, soil moisture is supplemented at regular time and quantity every day, the relative water content of soil is kept at 75% -85%, and the stress temperature is set as follows: the control group (CK) was set at a light high temperature of 14h/25 ℃ overnight for 10h at 30 ℃ day, a medium high temperature of 35 ℃ day for 14h/25 ℃ overnight for 10h, a severe high temperature of 40 ℃ day for 14h/35 ℃ overnight for 10h, and a temperature of 43 ℃ day for 14h/38 ℃ overnight for 10h, and at 25 ℃ day for 14h/20 ℃ overnight for 10 h.
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