CN114720252A - Method for rapidly identifying and evaluating low-temperature stress tolerance of camellia oleifera - Google Patents
Method for rapidly identifying and evaluating low-temperature stress tolerance of camellia oleifera Download PDFInfo
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Abstract
The invention discloses a method for quickly identifying and evaluating low-temperature stress tolerance of oil tea, belonging to the field of plant physiology. The method takes the camellia oleifera leaves subjected to cold acclimation at the temperature of above zero and below zero (about 4 ℃) in autumn and winter as experimental materials, the leaves are placed in a low-temperature environment at the temperature of-10 ℃, the relative conductivity is detected by sampling for multiple times according to time gradient, the change rate of the relative conductivity is calculated based on the obtained relative conductivity, the relative conductivity and the size and the change trend of the change rate of the relative conductivity in the low-temperature treatment process of different camellia oleifera leaves are compared by combining variance analysis, and the strength of the low-temperature stress tolerance of the camellia oleifera can be preliminarily deduced. The method has the advantages of convenient treatment, simple operation and reliable result, can quickly and conveniently judge the tolerance of the oil tea to low-temperature stress, and provides a foundation for the breeding of improved varieties of the oil tea with the low-temperature stress.
Description
Technical Field
The invention relates to the technical field of plant physiology, in particular to a method for quickly identifying and evaluating low-temperature stress tolerance of camellia oleifera.
Background
Low temperature stress is one of the common abiotic stresses and is an important environmental factor affecting the growth and development of plants and limiting their geographical distribution. Low temperature stress can be generally divided into cold stress (0-20 ℃) and freeze stress (<0 ℃). In order to cope with low-temperature stress, plants have evolved a series of complex regulatory mechanisms, one of which is cold acclimation. Cold acclimation refers to the process by which plants acquire greater tolerance to freezing stress after they are exposed to a subzero (non-lethal) low temperature for a period of time. Tea-oil tree (Camellia oleifera) is the first major woody oil crop developed in China, and the cultivation area and the annual yield of the tea-oil tree are the top of the woody oil crops in China. The camellia oleifera is widely distributed in Yangtze river basin of China and subtropical evergreen broad-leaved forest areas in the south of the Yangtze river. One of the representative species of subtropical evergreen broadleaf forest is camellia, which has a special phenology, usually blooms and fruits in autumn and winter, and is easily damaged when exposed to low-temperature climate, resulting in severe yield reduction. Therefore, the breeding of the low temperature stress resistant oil tea variety has important significance.
At present, in most of methods for evaluating the low-temperature stress tolerance of the camellia oleifera, in-vitro leaves are used as experimental materials, a plurality of physiological indexes of the leaves are measured after the leaves are subjected to low-temperature treatment, and the low-temperature stress tolerance is evaluated through comprehensive analysis. The previous research results show that the selection of leaves in a proper period and the experimental method are very important, and the leaves of the camellia oleifera not subjected to cold acclimation are directly subjected to subzero low temperature treatment, so that the leaves hardly have the resistance to the cold stress, the cells of the leaves of the camellia oleifera of different varieties die rapidly under the low temperature treatment, and the difference of the low temperature stress tolerance among the varieties cannot be displayed (as shown in fig. 2). In autumn and winter, when the camellia oleifera subjected to the cold acclimation at the subzero low temperature is subjected to freezing stress, the low-temperature stress tolerance of different varieties can generate obvious difference under the influence of genetic difference. Therefore, the method is suitable for identifying and evaluating the low-temperature stress tolerance of the camellia oleifera leaves subjected to the above-zero low-temperature cold acclimation. The existing research generally sets temperature gradient to process the camellia oleifera leaves, and cannot show the difference of low-temperature stress tolerance among different camellia oleifera varieties in a short time. According to the method, proper low temperature is selected, constant low-temperature treatment is carried out according to time gradient, and compared with gradient cooling, the difference of low-temperature stress tolerance among different oil tea varieties can be distinguished more quickly.
In addition, the process of measuring multiple physiological indexes is complicated, the efficiency is low, and the rapid detection in large batch is not convenient. Therefore, a representative physiological index is required to reflect the low temperature stress tolerance of leaves. Research shows that the relative conductivity of the leaves during low-temperature treatment has obvious correlation with various physiological indexes for evaluating the low-temperature stress tolerance, such as malondialdehyde, proline, soluble sugar and the like, can reflect the overall condition of a plant cell membrane system, is a key factor in evaluating the low-temperature stress tolerance, and has better representativeness. The method uses the relative conductivity of the leaves as a single index to evaluate the low-temperature stress tolerance of the camellia oleifera.
Disclosure of Invention
The invention aims to provide a method for rapidly identifying and evaluating low-temperature stress tolerance of camellia oleifera, which is characterized in that camellia oleifera leaves subjected to cold acclimation at a temperature of above zero (about 4 ℃) in autumn and winter are used as experimental materials, the leaves are placed in a low-temperature environment at the temperature of-10 ℃, the relative conductivity of the leaves is detected by sampling in a set time gradient in a sub-step manner, the change rate of the relative conductivity is calculated based on the obtained relative conductivity, and the relative conductivity and the change rate and the change trend of the relative conductivity of different camellia oleifera leaves in the low-temperature treatment process are compared by combining variance analysis, so that the strength of the low-temperature stress tolerance of the camellia oleifera is preliminarily deduced.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for rapidly identifying and evaluating low-temperature stress tolerance of oil tea comprises the following steps:
s1, selecting a proper oil tea tree from an oil tea forest to be detected, and carrying out sample collection and classification on leaves of the oil tea tree;
s2, preprocessing the collected tea-oil tree leaf sample, and taking part of leaves after the processing to measure the relative conductivity of the leaves as the relative conductivity of the tea-oil tree leaf sample before low-temperature processing;
s3, carrying out low-temperature treatment on the pretreated camellia oleifera leaf sample according to a certain time gradient;
s4, carrying out relative conductivity detection on the camellia oleifera leaf samples subjected to low-temperature treatment in the step S3 to obtain the relative conductivity of the camellia oleifera leaf samples subjected to low-temperature treatment for different durations;
step S5, recording the relative conductivity of the camellia oleifera leaf sample obtained in the step S2 before low-temperature treatment as REC0And recording the relative conductivity of the camellia oleifera leaf sample obtained in the step S4 after being processed at low temperature for different time lengths as RECTCalculating the relative conductivity change rate V of the camellia oleifera leaf sample after low-temperature treatment for different time periodsT；
S6, carrying out low-temperature treatment on the oil-tea tree leaf sample obtained in the step S5 for different time periods to obtain the relative conductivity RECTAnd relative rate of change of conductivity VTAnd respectively carrying out variance analysis, and making corresponding line graphs so as to evaluate the low-temperature stress tolerance of the camellia oleifera variety to be tested.
Specifically, in step S1, a suitable camellia oleifera tree is selected from the camellia oleifera forest to be tested, and the selection criteria of the camellia oleifera tree are as follows: selecting oil tea trees with good growth vigor and similar tree ages after cold acclimation at a low temperature of about-4 ℃ in autumn and winter; the method for collecting the camellia oleifera leaf sample comprises the following steps: the method comprises the steps of collecting enough fresh and undamaged healthy leaves of annual branch tips, selecting at least 3 oil-tea trees for biological repetition in each oil-tea tree population or variety, taking down the branches by using a branch shear when collecting leaf samples in order to ensure the freshness of the leaves, and carrying the branches back to a laboratory for immediate treatment.
Specifically, in the step S2, the collected camellia oleifera tree leaf sample is preprocessed, and the preprocessing process includes:
taking the leaves with the leaf stalks left down from the branches, washing the surfaces of the leaves with deionized water, wiping the leaves with absorbent paper, dividing the leaves into a plurality of groups according to a set time gradient, respectively filling the groups into plastic self-sealing bags with the sizes of 4 mm and 120mm multiplied by 85mm, marking the self-sealing bags, putting the self-sealing bags into a 4 ℃ incubator for treatment for 12 hours, taking partial leaves after the treatment, measuring the relative conductivity of the partial leaves, and marking the relative conductivity as the relative conductivity of the leaves before the low-temperature treatment of the camellia oleifera leaves.
Specifically, in step S3, the pre-processed camellia oleifera leaf sample is subjected to low-temperature processing according to a certain time gradient, where the low-temperature processing process is as follows:
transferring the pretreated camellia oleifera leaves from a thermostat at 4 ℃ to a low-temperature environment at-10 ℃, sequentially taking out samples according to time gradient, unfreezing the taken samples in the thermostat at 0 ℃ for 12h, and detecting the relative conductivity of the samples again.
Specifically, the method for detecting the relative conductivity of the camellia oleifera leaf sample in the step S2 and the step S4 includes: the method comprises the following steps of detecting the relative conductivity of a sample of the leaves of the camellia oleifera by adopting a soaking method, wherein the detection process comprises the following steps:
keeping away from main veins of the leaves of the camellia oleifera tree, using perforating pliers to punch 5 round pieces with the diameter of 5mm on the leaves of the camellia oleifera tree, putting the round pieces into a clean 50ml centrifugal tube, adding 20ml deionized water into the centrifugal tube, slightly shaking the centrifugal tube, and then placing the centrifugal tube at room temperature for 24 hours; then, a conductivity meter is used for detecting the conductivity of the solution in the centrifugal tube, and the conductivity is recorded as R1(ii) a Boiling the solution in the centrifuge tube for 20min, cooling, shaking, detecting the conductivity, and recording as R2(ii) a The conductivity of the deionized water is recorded as R0。
Specifically, in step S5, the relative conductivity REC of the camellia oleifera leaf sample after being processed at low temperature for different time periodsTThe calculation formula of (a) is as follows:
relative conductivity change rate V of tea-oil tree leaf sampleTThe calculation formula of (a) is as follows:
further, the method of the present invention can also be used for comparing low temperature stress tolerance of different varieties of camellia oleifera trees:
REC by comparing different varieties of oil teaTAnd VTREC of different varieties of Camellia oleiferaTAnd VTSignificant differences exist, which indicate that the low-temperature stress tolerance of different camellia varieties is different; if RECTAnd VTNo significant difference exists between the varieties, which shows that the low temperature stress tolerance of different oil tea varieties is similar.
The invention has the beneficial effects that:
the method is convenient to process, simple to operate and reliable in result, can quickly and conveniently judge the low-temperature stress tolerance of the oil tea, and provides basic support for the breeding of improved varieties of the low-temperature stress-resistant oil tea.
Drawings
FIG. 1 is a flow chart of a rapid identification and evaluation method for low-temperature stress tolerance of camellia oleifera according to the invention;
FIG. 2 shows the relative conductivities (REC) of the leaves of Camellia Luminalia and the leaves of Camellia Ganseng No. 1, which have not undergone cold acclimation at-10 deg.C for different durations in the example of the present inventionT) A trend graph of the change;
FIG. 3 shows the relative conductivities of the leaves of the Lushan oil Camellia and the Gannao No 1 oil Camellia in the examples of the present invention (REC) at-10 deg.C for different durations of timeT) A trend graph of (1 month sample 2020);
FIG. 4 shows the relative conductivities of the leaves of the Lushan oil tea and the Jiangxiang No 1 oil tea of the present invention at-10 deg.C for different durations (REC)T) Trend graph of (1 month sample 2021);
FIG. 5 shows the relative conductivity change rates (V) of the leaves of Lushan oil tea and Gannan No 1 oil tea of the present invention at-10 deg.C for different durationsT) A trend graph of (1 month sample 2020);
FIG. 6 shows that the leaves of Lushan oil tea and Gannan No 1 oil tea of the present invention are treated at-10 deg.CLong time relative conductivity rate of change (V)T) Trend graph of (1 month sample of 2021).
Detailed Description
In order to make those skilled in the art better understand the technical solutions in the present specification, the technical solutions in the embodiments of the present specification will be clearly and completely described below with reference to the drawings in the embodiments of the present specification, and it is obvious that the described embodiments are only a part of the embodiments of the present application, and not all of the embodiments.
The embodiment is as follows: see fig. 1-6.
In this embodiment, the effectiveness of the method for rapidly identifying and evaluating the low temperature stress tolerance of camellia oleifera provided by the invention is verified by the following experimental method, which mainly comprises the following steps:
step S1. sample collection
The samples of the camellia oleifera leaves collected in the experiment are from the scientific research institute of forestry in Jiangxi province and the Lushan high altitude area (about 987 m) in Jiujiang City in Jiangxi province, are divided into two years in 2020 and 2021, and are collected in 1 month, and the camellia oleifera has undergone cold acclimation at subzero low temperature.
Ganxianwu tea-oil tree is a fine variety of tea-oil tree bred by Jiangxi province academy of forestry sciences in 2007, and is favored to live in low hilly areas with the altitude of less than 100 m; the air temperature in the high-altitude area of Lushan mountain is low, and the lowest temperature in winter is usually lower than-10 ℃ and even lower than-30 ℃. Therefore, compared with the Gangwu No 1 oil tea, the oil tea distributed in the high altitude area of the Lushan mountain has stronger low-temperature stress tolerance.
In 2020 and 1 month in 2021, 3 leaves of Ganxiang No. 1 tea oil and Lushan camellia oil with good growth vigor are respectively collected from Lushan high altitude areas of forestry scientific research institute in Jiangxi province and Jiujiang city in Jiujiang province, each tea oil tree collects sufficient fresh and undamaged leaves (no less than 14 leaves) of annual branch tips of the tea oil trees, the freshness of the leaves is ensured, and branches are taken down together by using branch shears.
Step S2, pretreatment
Taking down collected leaf samples of the Lushan oil tea and the Jiangxi No. 1 oil tea from branches, cleaning the surfaces of leaves by using deionized water, wiping the leaves dry by using absorbent paper, dividing the leaves into a plurality of groups according to a set time gradient, respectively filling the groups into No. 4 (120mm multiplied by 85mm) plastic self-sealing bags with small holes, and marking the self-sealing bags. And (3) placing the leaves into a 4 ℃ thermostat for treatment for 12h, taking part of the leaves after the treatment, and measuring the relative conductivity of the leaves, wherein the relative conductivity is recorded as the relative conductivity of the leaves before the low-temperature treatment of the camellia oleifera leaves.
Step S3, low-temperature treatment
Sample 1 month 2020: and transferring the pretreated residual leaves from a thermostat at 4 ℃ to a climatic chamber, setting the temperature in the climatic chamber to be fixed at-10 ℃, taking out samples after placing for 1h, 3h, 6h, 16h and 32h respectively, unfreezing the taken samples in the thermostated chamber at 0 ℃ for 12h, and detecting the relative conductivity of the samples.
Year 2021 month sample: and transferring the pretreated residual leaves from a thermostat at 4 ℃ to a climatic chamber, setting the temperature in the climatic chamber to be fixed at-10 ℃, taking out samples after placing for 1h, 3h, 6h, 16h, 32h and 48h respectively, unfreezing the taken samples in the thermostat at 0 ℃ for 12h, and detecting the relative conductivity of the samples.
S4, detecting the relative conductivity of the camellia oleifera leaves
The relative conductivity of the leaves was measured using the soaking method. Keeping away from main veins, using perforating pliers to punch 5 round pieces with the diameter of 5mm on the camellia oleifera leaves, putting the round pieces into a clean 50ml centrifugal tube, adding 20ml deionized water into the centrifugal tube, slightly shaking, and then placing the centrifugal tube at room temperature for 24 hours; the conductivity of the solution in the centrifuge tube was measured using a conductivity meter and recorded as R1(ii) a Boiling the solution in the centrifuge tube for 20min, cooling, shaking, detecting the conductivity, and recording as R2(ii) a The conductivity of the deionized water is recorded as R0Finally, the relative conductivity (REC) of the camellia oleifera leaves processed at low temperature for different time durations is calculated according to a formulaT)。
S5, calculating the change rate of the relative conductivity of the camellia oleifera leaves
The relative conductivity of the camellia oleifera leaves before low-temperature treatment is recorded as REC0The relative conductivities after different durations of the low-temperature treatment are recorded as RECT. Respectively calculating the change rate (V) of the relative conductivity of the camellia oleifera leaves subjected to low-temperature treatment for different time periods according to a formula IIT)。
Step S6, evaluating low-temperature stress tolerance
Respectively measuring the relative conductivity (REC) of leaves of Camellia Lushan and Camellia Ganseng No. 1 at-10 deg.C for different durationsT) And relative rate of change of conductivity (V)T) For measured RECTAnd VTAnalysis of variance was performed and a line graph was created.
Low-temperature treatment of RECs with different time lengths for samples of 1 month in 2020T、VTAs shown in fig. 3 and 5, samples of 1 month in 2021 were processed at low temperature for RECs of different durationsT、VTThe change trend of (c) is shown in fig. 4 and 6.
Evaluation results were as follows:
relative conductivity (REC) of Lushan oil tea and Jiangxiang No 1 oil tea leaves measured for two years continuouslyT) The variation trends are similar, and the experimental repeatability is better (fig. 3 and 4). During low temperature treatment at-10 deg.C, REC of Gannan non 1 oil tea leaf and Lushan oil tea leafTThe REC measured under the low-temperature treatment for 1-6 h shows an increasing trend along with the increase of the duration of the low-temperature treatmentTNo significant difference (p > 0.05) was detected and the rise was small; along with the duration of low-temperature treatment reaching 32 hours, the REC of the Jiangxiang tea-oil tree leaf without 1TREC of camellia oleifera leaf capable of rapidly breaking through 50%TUp to about 35%, REC in betweenTExhibit a very significant difference (p)<0.01), and finally, REC of Jiangxiang No 1 camellia oleifera leafTThe rising amplitude is far larger than the Lushan oil tea leaves. The results of two-factor anova show that the time length of the camellia oleifera variety and low-temperature treatment is opposite to RECTAll have obvious influence, and the REC of the Jiangxiang tea-oil tree leafTSignificantly higher than Lushan oilTea leaves (2020: F-25.287, p)<0.01; 2021F 9.948, p<0.01)。
Relative conductivity change rate (V) of Lushan oil tea and Jiangxiang No 1 oil tea leaves measured for two years continuouslyT) The variation trends are similar, and the experimental repeatability is better (fig. 5 and 6). During the low-temperature treatment process at minus 10 ℃, the Ganxingwu 1 camellia oleifera leaf blade VTV of Lushan Camellia oleifera leaves shows a rising trend as the duration of the low-temperature treatment increasesTThe stability is kept relatively; treating the two at low temperature for 1-6 hTHas no obvious difference (p is more than 0.05), and the V of the Gannan 1 tea-oil tree leaves is increased along with the increase of the duration of the low-temperature treatmentTQuickly rises, and after 16 hours, the Jiangxiang does not have 1 camellia oleifera leaf VTSignificantly higher than Lushan oil tea leaf (p)<0.01). The results of two-factor anova show that the length of the camellia oleifera variety and the low-temperature treatment is VTAll have obvious influence, Jiangxiang has no V of 1 camellia oleifera leafTIs obviously higher than the leaves of the oil tea of cottonshan (202 years: F is 31.564, p)<0.01; 2021F 12.406, p<0.01)。
From this, it can be judged that: the low-temperature stress tolerance of the camellia oleifera at the Lushan mountain and the camellia at the Gannan province is remarkably different from that of the camellia at the Gannan province, and the low-temperature stress tolerance of the camellia oleifera at the Lushan mountain is stronger than that of the camellia at the Gannan province.
The experimental results and the analysis results of two years are consistent with those of the earlier conjecture, and therefore, when the method is applied to quickly identifying and evaluating the low-temperature stress tolerance of the camellia oleifera, the stability, the reliability and the convenience of the results are very high.
While the preferred embodiments of the present invention have been illustrated and described, it will be understood by those skilled in the art that the present invention is not limited to the details of the embodiments shown and described, but is capable of numerous equivalents and substitutions without departing from the spirit of the invention as set forth in the claims appended hereto.
Claims (6)
1. A method for rapidly identifying and evaluating low-temperature stress tolerance of oil tea is characterized by comprising the following steps:
step S1, sample collection: selecting a proper oil tea tree from an oil tea forest to be detected, and carrying out sample collection and classification on leaves of the oil tea tree;
s2, pretreatment: pretreating the collected tea-oil tree leaf sample, taking part of leaves after the pretreatment, and measuring the relative conductivity of the leaves to obtain the relative conductivity of the leaves before the tea-oil tree leaf sample is subjected to low-temperature treatment;
s3, low-temperature treatment: carrying out low-temperature treatment on the pretreated camellia oleifera leaf sample according to a certain time gradient;
s4, detecting the relative conductivity of the camellia oleifera leaves: performing relative conductivity detection on the camellia oleifera leaf samples subjected to low-temperature treatment in the step S3 to obtain the relative conductivity of the camellia oleifera leaf samples subjected to low-temperature treatment for different durations;
s5, calculating the relative conductivity change rate of the camellia oleifera leaves: recording the relative conductivity of the camellia oleifera leaf sample obtained in the step S2 before low-temperature treatment as REC0And recording the relative conductivity of the camellia oleifera leaf sample obtained in the step S4 after being processed at low temperature for different durations as RECTCalculating the relative conductivity change rate V of the camellia oleifera leaf sample after low-temperature treatment for different time periodsT；
Step S6, evaluating low-temperature stress tolerance: performing low-temperature treatment on the camellia oleifera leaf sample obtained in the step S5 for different time periods to obtain the relative conductivity RECTAnd relative rate of change of conductivity VTAnd respectively carrying out variance analysis, and making corresponding line graphs so as to evaluate the low-temperature stress tolerance of the camellia oleifera variety to be tested.
2. The method for rapidly identifying and evaluating the low temperature stress tolerance of the camellia oleifera according to claim 1, wherein in the step S1, a proper camellia oleifera is selected from the camellia oleifera forest to be tested, and the selection criteria of the camellia oleifera are as follows: selecting oil tea trees with good growth vigor and similar tree ages after cold acclimation at a low temperature of about-4 ℃ in autumn and winter; the method for collecting the camellia oleifera leaf sample comprises the following steps: the method comprises the steps of collecting enough fresh and undamaged healthy leaves of annual branch tips, selecting at least 3 oil-tea trees for biological repetition in each oil-tea tree population or variety, taking down the branches by using a branch shear when collecting leaf samples in order to ensure the freshness of the leaves, and carrying the branches back to a laboratory for immediate treatment.
3. The method for rapidly identifying and evaluating the low temperature stress tolerance of the camellia oleifera according to claim 1, wherein the collected camellia oleifera leaf samples are pretreated in the step S2, and the pretreatment process comprises the following steps:
taking the leaves with the leaf stalks left down from the branches, cleaning the surfaces of the leaves with deionized water, wiping the leaves with absorbent paper, dividing the leaves into a plurality of groups according to a set time gradient, respectively filling the groups into a number 4 plastic self-sealing bag with small holes, the number 4 plastic self-sealing bag is 120mm multiplied by 85mm, marking the self-sealing bag, putting the self-sealing bag into a 4 ℃ incubator for treatment for 12 hours, taking part of the leaves after the treatment is finished, measuring the relative conductivity of the leaves, and marking the relative conductivity as the relative conductivity of the leaves before the oil tea leaves are subjected to low-temperature treatment.
4. The method for rapidly identifying and evaluating the low temperature stress tolerance of the camellia oleifera according to claim 1, wherein the pre-treated camellia oleifera leaf samples are subjected to low temperature treatment according to a certain time gradient in the step S3, and the low temperature treatment process comprises the following steps:
transferring the pretreated camellia oleifera leaves from a thermostat at 4 ℃ to a low-temperature environment at-10 ℃, sequentially taking out samples according to time gradient, unfreezing the taken samples in the thermostat at 0 ℃ for 12h, and detecting the relative conductivity of the samples again.
5. The method for rapidly identifying and evaluating the low temperature stress tolerance of the camellia oleifera according to claim 1, wherein the method for detecting the relative conductivity of the camellia oleifera leaf sample in the steps S2 and S4 comprises the following steps: the method comprises the following steps of (1) detecting the relative conductivity of a tea-oil tree leaf sample by adopting a soaking method, wherein the detection process comprises the following steps:
keeping away from main veins of the leaves of the camellia oleifera tree, using perforating pliers to punch 5 round pieces with the diameter of 5mm on the leaves of the camellia oleifera tree, putting the round pieces into a clean 50ml centrifugal tube, adding 20ml deionized water into the centrifugal tube, slightly shaking the centrifugal tube, and then placing the centrifugal tube at room temperature for 24 hours; then, a conductivity meter is used for detecting the conductivity of the solution in the centrifugal tube, and the conductivity is recorded as R1(ii) a Then the cell is separatedBoiling the solution in the heart tube for 20min, cooling, shaking, and measuring the conductivity as R2(ii) a The conductivity of the deionized water is recorded as R0。
6. The method for rapidly identifying and evaluating low temperature stress tolerance of camellia oleifera according to claim 1, wherein the relative conductivity REC of the camellia oleifera leaf samples subjected to low temperature treatment for different time periods in step S5TThe calculation formula of (c) is as follows:
relative conductivity change rate V of tea-oil tree leaf sampleTThe calculation formula of (a) is as follows:
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