CN106896128B - Method for identifying and evaluating cold resistance of small red beans - Google Patents
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Abstract
The invention discloses a method for identifying and evaluating cold resistance of small red beans. The method for identifying and evaluating cold resistance of small red beans provided by the invention specifically comprises the following steps: A) sowing the red beans to be detected, and moving the seedlings to the light intensity of 700-2Growing for 28-30 days at 10-13 deg.C, transferring the plant to light intensity of 1000-2Performing greening at the temperature of 20-25 ℃ in s; B) determination of H in plants over time for greening2O2The content, the chlorophyll content, the SOD enzyme activity, the APX enzyme activity or the CAT enzyme activity, thereby determining the cold resistance of the small red bean to be detected. The invention provides a set of accurate and rapid cold resistance identification and evaluation method of small red beans, which has important significance for the utilization of variety resources, the reasonable planning and layout of planting places and cold resistance breeding.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a method for identifying and evaluating cold resistance of small red beans.
Background
Low temperature is an important environmental factor affecting plant growth, development and biological yield. The low-temperature damage is a natural disaster frequently occurring in agricultural production, seriously affects the quality and yield of crops, and even causes the death of the crops in a large area. To date, there is no fundamental solution to the low temperature injury.
China is the country with the largest cultivation area and the largest export quantity of the small red beans. Small red beans are the main edible bean crops in China, but are the least cold-resistant low-temperature sensitive crops compared with other beans, so that the yield of the small red beans in cold regions is unstable. The cold resistance of the small red bean variety is a main factor for determining the planting and popularization of the small red bean variety, the cold resistance of different varieties is determined, and an accurate and rapid small red bean cold resistance identification and evaluation method is formulated, so that the method has important significance for the utilization of variety resources, the reasonable planning and layout of planting places and cold resistance breeding.
The cold damage of small red beans at low temperature occurs in the seedling emergence stage, the early growth stage and the flowering stage. The seedling emergence stage is at long-term low temperature and insufficient sunlight, so bowl-shaped symptoms of inward turning of leaves are caused and withered; the low-temperature cold damage encountered in the 4-5 leaf stage causes the growth disorder (growth point stopping phenomenon); the pod bearing disorder caused by pollen sterility caused by cold damage at the flowering stage is different from each other. The low-temperature obstacle of the seedling stage of the small red bean is caused by the fact that the growth of leaves is delayed due to low-temperature irradiation and the synthesis of primary chlorophyll, so that the photosynthesis of the leaves is abnormal, the nutrition is exhausted, and the leaves cannot be recovered and die finally. Researches show that in the growth process of plants, the phenomena of chlorophyll discoloration, leaf necrosis and the like can be caused by sudden temperature change. Chlorophyll and proplastid are photosensitizers that cause the generation of active oxygen, which can cause physiological disorders such as cell intima, protein damage, and photosynthesis inhibition. Once the chlorophyll is destroyed, the phenomena of chlorophyll discoloration, leaf necrosis and the like can be caused. To detoxify O2 -And H2O2In plant cells, an antioxidant system consisting of superoxide dismutase (SOD), Antithrombotic Peroxidase (APX), Catalase (CAT), and the like coexist. These antioxidases are closely related to the amount of active oxygen produced under stress conditions, antioxidase activity, and the like.
Disclosure of Invention
The invention aims to provide a method for identifying or assisting in identifying cold resistance of small red beans.
The method for identifying or assisting in identifying the cold resistance of the small red beans, provided by the invention, comprises the following steps of: sowing the red beans to be detected, and moving the seedlings to the light intensity of 700-2Growing for 28-30 days at 10-13 deg.C, transferring the plant to light intensity of 1000-2Performing greening at the temperature of 20-25 ℃ in s; measured during greening asDetermining the cold resistance of the small red bean to be detected according to any one of the following indexes: h in the plant2O2Content change, chlorophyll content change in the plant, superoxide dismutase activity change in the plant, ascorbic acid peroxidase activity change in the plant, and catalase activity change in the plant.
Specifically, the method for identifying or assisting in identifying the cold resistance of the small red beans provided by the invention can be any one of the following five methods:
the first method specifically comprises the following steps:
(A1) sowing the red beans to be detected, and moving the seedlings to the light intensity of 700-2Growing at 10-13 deg.C for 28-30 days (such as 28 days), and transferring to 1500 μmol/m with light intensity of 1000-2Performing greening at the temperature of 20-25 ℃ in s;
(A2) h in the plant is measured when the greening time is 0H2O2Content, and/or H in plants over time of greening2O2Determining the cold resistance of the small red bean to be detected according to the change condition of the content as follows: h in the plant when the greening time is 0H2O2The lower the content, the stronger the cold resistance of the red bean variety to be detected; and/or H in plants2O2The cold resistance of the red bean variety to be detected, the content of which is not obviously changed along with the greening time and is not 0, is stronger than that of H in the plant2O2The content of the red bean variety to be detected is reduced to 0 point along with the greening time.
In step (A2), "H in plant" in the plant2O2The content does not change significantly with the lapse of greening time' means that H in the plant measured at different measurement time points set in the greening time2O2The contents do not significantly vary from one another.
The second method specifically comprises the following steps:
(B1) sowing the red beans to be detected, and moving the seedlings to the light intensity of 700-2Growing at 10-13 deg.C for 28-30 days (such as 28 days), and transferring to 1500 μmol/m with light intensity of 1000-2The temperature is 20-25 ℃ per secondGreening is carried out in the environment;
(B2) measuring the change condition of the chlorophyll content in the plant along with the greening time, and determining the cold resistance of the small red bean to be detected according to the following steps: the cold resistance of the red bean variety to be detected with the increase of the chlorophyll content in the plant along with the lapse of the greening time is stronger than or the candidate is stronger than the cold resistance of the red bean variety to be detected with the decrease of the chlorophyll content in the plant along with the lapse of the greening time.
The third method specifically comprises the following steps:
(C1) sowing the red beans to be detected, and moving the seedlings to the light intensity of 700-2Growing at 10-13 deg.C for 28-30 days (such as 28 days), and transferring to 1500 μmol/m with light intensity of 1000-2Performing greening at the temperature of 20-25 ℃ in s;
(C2) measuring the change of superoxide dismutase (SOD) activity in plants along with the greening time, and determining the cold resistance of the red bean to be detected according to the following steps: the cold resistance of the red bean variety to be detected, which shows a fluctuation state of the superoxide dismutase (SOD) activity in the plant and is not 0, is stronger than or is candidate stronger than the cold resistance of the red bean variety to be detected, which shows the superoxide dismutase (SOD) activity in the plant to be detected reduced to 0 point along with the greening time.
The method IV specifically comprises the following steps:
(D1) sowing the red beans to be detected, and moving the seedlings to the light intensity of 700-2Growing at 10-13 deg.C for 28-30 days (such as 28 days), and transferring to 1500 μmol/m with light intensity of 1000-2Performing greening at the temperature of 20-25 ℃ in s;
(D2) measuring the change of the activity of the ascorbic Acid Peroxidase (APX) in the plants along with the greening time, and determining the cold resistance of the red bean to be detected according to the following steps: the cold resistance of the red bean variety to be tested, which shows a fluctuation state of the activity of the Ascorbate Peroxidase (APX) in the plant along with the greening time and is not 0, is stronger than or is candidate to be stronger than the cold resistance of the red bean variety to be tested, which reduces the activity of the Ascorbate Peroxidase (APX) in the plant along with the greening time to 0.
The method V specifically comprises the following steps:
(E1) sowing the red beans to be detected, and moving the seedlings to the light intensity of 700-2Growing at 10-13 deg.C for 28-30 days (such as 28 days), and transferring to 1500 μmol/m with light intensity of 1000-2Performing greening at the temperature of 20-25 ℃ in s;
(E2) measuring the change of Catalase (CAT) activity in the plants along with the greening time, and determining the cold resistance of the red bean to be detected according to the following steps: the cold resistance of the red bean variety to be detected which shows a fluctuation state of Catalase (CAT) activity in the plant along with the greening time and is not 0 is stronger than or is candidate stronger than the cold resistance of the red bean variety to be detected which firstly rises and then falls to 0 point along with the greening time.
In the above five methods, H in the plants is determined as the greening time goes on in the step (A2)2O2When the content changes, when the change of the chlorophyll content in the plant with the lapse of greening time is measured in the step (B2), when the change of the SOD enzyme activity in the plant with the lapse of greening time is measured in the step (C2), when the change of the APX enzyme activity in the plant with the lapse of greening time is measured in the step (D2), and when the change of the CAT enzyme activity in the plant with the lapse of greening time is measured in the step (E2), the measurement time points may be set within the range of greening time of 0-28h, specifically, the measurement time points are set as greening time 0h, 3h, 8h, and 28 h.
In the above five methods, step (A2), H in the plant2O2The content is H in plant leaves2O2Content (c); in the step (B2), the chlorophyll content in the plant is the chlorophyll content in the plant leaf; in the step (C2), the SOD enzyme activity in the plant is the SOD enzyme activity in the plant leaves; in the step (D2), the APX enzyme activity in the plant is APX enzyme activity in the plant leaf; in the step (E2), the CAT enzyme activity in the plant is the CAT enzyme activity in the plant leaf.
In the five methods, after the red beans to be tested are sown in the steps (A1), (B1), (C1), (D1) and (E1), the red beans are all put at the constant temperature of 25 ℃ and under the dark and dark conditions of 100 percent for emergence of seedlings.
In the five methods, the small red beans to be detected can be any variety of small red beans.
In one embodiment of the invention, the small red bean to be detected is a cold-resistant variety red-root large-Chinese-English and non-cold-resistant variety speckled small-grain line-1 of the small red bean.
The invention takes the cold-resistant and cold-resistant varieties of the small red beans as test materials, finds that the low-temperature shading treatment for 28 days is an important time point for identifying the cold resistance of the small red beans, performs greening treatment after the low-temperature shading treatment for 28 days, and detects H in plants2O2The cold resistance of different red bean varieties can be identified by the content, the chlorophyll, the SOD enzyme activity, the APX enzyme activity or the CAT enzyme activity. The invention provides a set of accurate and rapid cold resistance identification and evaluation method of small red beans, which has important significance for the utilization of variety resources, the reasonable planning and layout of planting places and cold resistance breeding.
Drawings
FIG. 1 shows the greening condition of red bean variety after short-term low-temperature treatment for 18 days. Wherein, A-D is the red root of the cold-resistant variety Danale. A is greening for 0 h; b, greening for 3 hours; c, greening for 8 hours; d is greening for 28 h. E-H is a non-cold-resistant variety speck small grain line-1. E, greening for 0 h; f, greening for 3 hours; g, greening for 8 hours; h is greening for 28H.
FIG. 2 shows the greening condition of small red bean variety after short-term low-temperature treatment for 28 days. Wherein, A-D is the red root of the cold-resistant variety Danale. A is greening for 0 h; b, greening for 3 hours; c, greening for 8 hours; d is greening for 28 h. E-H is a non-cold-resistant variety speck small grain line-1. E, greening for 0 h; f, greening for 3 hours; g, greening for 8 hours; h is greening for 28H.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The cold-resistant variety red bean red root major nano-grade and the non-cold-resistant variety speck small grain line-1 are described in the following documents: "Qianyei Yi Mei et al, differentiation and breeding of Japanese red bean variety, foreign agriculture-minor cereals crop, 1984, (02), 43-46." public available from the research institute of farming and cultivation of the academy of agricultural sciences of Heilongjiang province, and can only be used in the experiment of repeated inventions.
Example 1 by H2O2Content variation identification of cold resistance of small red beans
Method for processing small red beans
The most representative species of Hokkaido in Japan, namely the cold-resistant species in the seedling emergence stage, red-rooted salvia macrobiotic and the non-cold-resistant species in the seedling emergence stage, namely the speckled small grain line-1, are selected. Two varieties are respectively planted in nutrition pots, vermiculite is filled in the nutrition pots, and 30-40 small red bean seeds are sowed in each nutrition pot one at intervals of 2cm and are repeatedly planted for three times. After sowing, placing the seedlings under the conditions of constant temperature of 25 ℃ and 100% darkness for emergence, and after emergence, moving the seedlings to the condition that the light intensity is 700-2Each kind of small red bean is divided into two groups, the small red bean grows for 18 days and 28 days under the low temperature condition (10 ℃ to 13 ℃ around the clock), then each group is divided into three groups to move to a natural light room (1500 mu mol/m in sunny days with the light intensity of 1000-2And/s) greening treatment is carried out for 3h, 8h and 28h respectively. The automatic watering device of the artificial climate chamber waters regularly during the whole growth period. Collecting young and tender primary leaves, weighing, and quickly freezing at-80 deg.C with liquid nitrogen.
II, H2O2Determination of content
O present in plants2 -And H2O2Will generate OH with strong toxicity-，H2O2Induce cell membrane peroxidation, DNA chain breakage and protein damage, and cause damage to plant cells. Taking the leaves of the cold-resistant variety Chinabonglian and the non-cold-resistant variety Luma small grain line-1 tender leaves, respectively measuring the H of the leaves in the seedling stage of 0H, 3H, 8H and 28H after the short-term low-temperature treatment of 18d and the long-term low-temperature treatment of 28d2O2And (4) content. H2O2The content is determined according to Erich Water (Erich Water. George G, LatitesQuanentification of hydrogen peroxide in plant extracts by the chemical reaction with luminescence. HCytochemistry 1982, 21(4), 827-831). Each treatment set at least 3 replicates.
The results are shown in Table 1.
TABLE 1 content of hydrogen peroxide in seedling leaves of small red beans treated by greening under different low temperature conditions
Note: data are expressed using mean ± standard deviation; the upper and lower case letters represent significant differences at the 5% and 1% levels for the two varieties, respectively, for the different treatments; hydrogen peroxide unit: m mol/kg/fr · wt.
Whether the cold-resistant variety is subjected to short-term low-temperature treatment (18d) or long-term low-temperature treatment (28d), whether the H is subjected to pre-greening (0H) or post-greening (3H, 8H and 28H)2O2The influence of the content is not great, such as A-D in figure 1 and A-D in figure 2, and H is obtained after long-term low-temperature treatment2O2The content (0.27 mmol/kg/fr. wt.) was rather lower, and was 42% of the short-term low-temperature treatment (0.64 mmol/kg/fr. wt.).
The cold-resistant variety is treated at low temperature for 3H after greening (18d)2O2The content is remarkably increased compared with that before greening (0H), and H is increased along with the prolonging of greening time2O2The content gradually decreases to the level before greening; h before greening (0H) after long-term low-temperature treatment (28d)2O2The content reaches the maximum value (17.78m mol/kg/fr.wt), is 13 times of that before short-term low-temperature treatment for greening (1.35m mol/kg/fr.wt), and is H for greening for 3H (7.11m mol/kg/fr.wt)2O2The content is remarkably reduced compared with that before greening (17.78m mol/kg/fr.wt), and H is increased along with the prolonging of greening time2O2The content is still remarkably reduced until greening for 8H, the content is H2O2The content is reduced to 0.00m mol/kg/fr.wt, at this time, the leaf part is yellow and wilted, and the withering rate reaches 35% as shown by G in figure 2; by 28H of greening, the withering rate reaches 40% as shown by H in figure 2.
The above results show that: long term low temperature stress (28d) leading to pre-greening H in cold-intolerant varieties2O2The content is obviously increased (compared with the cold-resistant variety), and H is increased along with the prolonging of the greening time2O2The content is gradually reduced to 0, and most of the leaves are withered, yellow and wilted; and cold-resistant variety H2O2The content of the red bean seedlings is always kept at a normal level before and after greening, so that after the red bean seedlings are treated at low temperature for a long time (28d), H is treated by comparing greening treatment time (0H, 3H, 8H and 28H) of different varieties2O2The cold resistance of the small red bean variety can be identified by the content variation trend.
Namely, the cold resistance of the small red beans can be identified by measuring H in the plants when the greening time is 0H2O2Content and/or H in plants over time of greening2O2Determining the cold resistance of the small red bean to be detected according to the change condition of the content as follows: h in the plant when the greening time is 0H2O2The lower the content, the stronger the cold resistance of the red bean variety to be detected; h in the plant2O2The cold resistance of the red bean variety to be detected, the content of which is not obviously changed along with the greening time and is not 0, is stronger than that of H in the plant2O2The content of the red bean variety to be detected is reduced to 0 point along with the greening time.
Example 2 identification of Cold resistance of Small Red beans by changes in chlorophyll content
Method for processing small red beans
Same as example 1 step one.
Secondly, measuring chlorophyll content
Taking the leaves of the cold-resistant variety Chinabonglian and the non-cold-resistant variety Luma small grain line-1, and respectively measuring the chlorophyll content of the leaves in the seedling stage of 0h, 3h, 8h and 28h after the short-term low-temperature treatment for 18d and the long-term low-temperature treatment for 28 d. The chlorophyll content was measured by Katsumadi (photosynthesis research method, Tokyo, 1981, 40-41). Each treatment set at least 3 replicates.
The measurement results are shown in Table 2.
TABLE 2 chlorophyll content of small red bean leaves at seedling stage after greening treatment under different low temperature conditions
Note: data are expressed using mean ± standard deviation; the upper and lower case letters represent significant differences at the 5% and 1% levels for the two varieties, respectively, for the different treatments; chlorophyll unit: mu.g/g.
Compared with the method before greening (0h), after the cold-resistant variety is subjected to short-term low-temperature treatment for 18d for 8h, the chlorophyll content is remarkably improved and increased from 32.1 mu g/g to 46.8 mu g/g, the chlorophyll content gradually increases along with the prolonging of the greening time, the chlorophyll content reaches the highest after greening for 28h, and the chlorophyll content is remarkably increased compared with the method before greening; after the short-term low-temperature treatment of the cold-intolerant variety for 18d, although the chlorophyll content also increases along with the prolonging of the greening time, the chlorophyll content does not have a significant difference compared with that of the green variety in 28 h.
Compared with the method before greening (0h), after the cold-resistant variety is subjected to long-term low-temperature treatment for 28d and is greened for 3h, the chlorophyll content is remarkably improved and increased from 19.9 mu g/g to 32.6 mu g/g, the chlorophyll content is always maintained at a certain level along with the prolonging of the greening time, the chlorophyll content reaches the highest value after greening for 8h, and the chlorophyll content has no obvious difference compared with greening for 3 h; after the cold-resistant variety is treated at low temperature for 28d for a long time, the chlorophyll content is remarkably reduced to 0.0 mu g/g after greening for 8h compared with that before greening (greening for 0h), and most leaves are withered, yellow and wilted because the leaves of the cold-resistant variety can not recover to a normal state due to overlong greening time.
After the cold-resistant variety is subjected to low-temperature shading for 18d and 28d and re-greening, primary leaves are quickly greened; after the variety without cold resistance is treated for 18d, the greening speed is delayed, and the withering rate before the greening treatment for 28d is 20 percent, as shown in E in figure 2; the withered number of plants is gradually increased along with the prolonging of the greening treatment time, and the withered rate of 3h of greening treatment is 30 percent, as shown in F in figure 2; the withering rate of greening treatment for 8h is 35%, as shown in G in figure 2; the withering rate of greening treatment for 28H was 40%, as shown by H in FIG. 2.
The above results show that: the chlorophyll content of the cold-resistant variety is remarkably reduced after long-term low-temperature stress (28d), the chlorophyll content of the cold-resistant variety is remarkably improved after 3 hours of greening treatment, and the chlorophyll content of the cold-resistant variety is reduced to 0 after 8 hours. Therefore, after the small red bean seedlings (28d) are treated at low temperature for a long time, the varieties with the chlorophyll content increasing along with the greening time have stronger cold resistance than the varieties with the chlorophyll content decreasing along with the greening time.
Example 3 Activity measurement of antioxidant enzyme
Method for processing small red beans
Same as example 1 step one.
Second, determination of antioxidase Activity
Under adverse conditions, free radicals generated in plants can cause the defatting and peroxidation of plasma membranes and destroy the structure and function of biological membranes, and antioxidant enzymes in plants include superoxide dismutase (SOD), ascorbic Acid Peroxidase (APX), Catalase (CAT) and the like, which can eliminate the adverse effects of active oxygen and peroxide to a certain extent and maintain the stability of cell membranes.
SOD is a kind of metal enzyme commonly existing in aerobic organisms, which is cooperated with peroxidase, catalase, etc. to prevent active oxygen or other peroxide free radicals from damaging cell membranes, and can catalyze disproportionation reaction of oxygen free radicals to generate hydrogen peroxide, which can be converted into oxygen molecules and water by catalase. Therefore, the SOD activity is closely related to the stress resistance, and is an important research object of the plant stress physiology.
Plant cells produce a large number of oxygen radicals under stress conditions, causing a series of adverse physiological changes, and plants themselves form a set of clearance mechanisms in order to respond and defend against oxidative stress. Ascorbic Acid Peroxidase (APX) is an important key enzyme for eliminating peroxides in plants, and the main function is to decompose H through the ascorbic acid-glutathione cycle (AsA-GSH)2O2Due to APX to H2O2Has higher affinity, so that H in chloroplast2O2Cleared primarily by APX. In addition, APX is also directly involved in the redox metabolism of ascorbic acid. Thus, APX is considered to be closely related to the cold resistance of plants.
Catalase (CAT) is an enzyme widely existing in animals, plants and microorganisms, is one of key enzymes of a biological defense system, has a biological function of catalyzing decomposition of hydrogen peroxide in cells to prevent peroxidation, and is mainly involved in physiological processes such as stress resistance and oxidative aging in plants.
Taking the cold-resistant variety of the red-root large-leaf allelopathy and the non-cold-resistant variety of the speck small grain line-1 tender leaves, and respectively measuring the activities of SOD enzyme, APX enzyme and CAT enzyme of the leaves in seedling stage at 0h, 3h, 8h and 28h after short-term low-temperature treatment for 18d and long-term low-temperature treatment for 28 d.
1. Preparation of enzyme solution
Frozen leaves (0.3g) were weighed into a mortar and ground with liquid nitrogen. 3.5ml of an extract [0.4mM EDTA, 1mM ascorbic acid, 2% (W/V) PVP containing 25mM potassium phosphate buffer (pH7.0) ] was added to each 1g of the pulverized leaf, and the mixture was stirred uniformly and centrifuged at 14000rpm for 25min at 4 ℃ to obtain a supernatant as an enzyme solution.
2. Determination of enzyme solution protein
The enzyme solution protein was measured according to the method of Shenlixin (method of testing the science of weeds, method of measuring the antioxidant activity of plants, published by the Japanese society for weeds, Tokyo. 2001296-298).
3. Determination of antioxidase (SOD, APX, CAT Activity)
SOD activity, APX activity and CAT activity were measured by the method of Shenlixin (weed science experimental method, plant antioxidant activity measurement method, published by Japan weed society, Tokyo. 2001296-. Each treatment set at least 3 replicates.
4. Results
(1) SOD enzyme Activity measurement results
The SOD enzyme activity measurement results are shown in Table 3.
TABLE 3 Activity of superoxide dismutase (SOD) in seedling stage of small red bean treated by greening under different low temperature conditions
Note: data are expressed using mean ± standard deviation; the upper and lower case letters represent significant differences at the 5% and 1% levels for the two varieties, respectively, for the different treatments; SOD unit: unit/mg protein.
The SOD activity of the cold-resistant variety is obviously improved after 3h greening compared with that before (0h) greening whether the cold-resistant variety is treated at the short-term low temperature (18d) or treated at the long-term low temperature (28d), the greening time reaches the maximum value after 8h greening, the SOD activity is gradually reduced along with the prolonging of time, and the SOD activity (0.020Unit/mg protein) is close to that before (0.013Unit/mg protein) greening after 28h greening after the long-term low temperature treatment.
Treating the cold-resistant variety at low temperature for a short time (18d), keeping the activity of the SOD enzyme after greening at the level before greening, obviously reducing the activity of the SOD enzyme after 8h of greening, and gradually increasing the activity of the SOD enzyme (0.017Unit/mg protein) at 28h of greening to be close to the level before greening (0.014Unit/mg protein); the SOD activity reaches the highest value (0.063Unit/mg protein) before greening (0H) for long-term low-temperature treatment (28d), the SOD activity gradually decreases with the beginning of greening, the SOD activity remarkably decreases to 0.016Unit/mg protein 3H after greening, the SOD activity decreases to 0.000Unit/mg protein 8H after greening, the SOD activity is kept at 0.000Unit/mg protein all the time along with the prolonging of greening time and is not recovered, as shown in E-H in figure 2, the leaves gradually wither and yellow and wither along with the prolonging of greening time in the whole greening process, and the SOD activity is in a decreasing state all the time.
The above results show that: the SOD enzyme activity of cold-resistant varieties of different small red beans is in a fluctuation state and is not 0 under the condition of long-term low-temperature treatment; and the SOD enzyme activity of the cold-resistant variety is reduced to 0 point. Therefore, when the small red bean seedlings (28d) are treated at low temperature for a long time, the cold resistance of the red bean variety to be tested, which shows a fluctuation state of SOD enzyme activity in the plant along with the lapse of greening time and is not 0, is stronger than that of the red bean variety to be tested, which shows that the SOD enzyme activity in the plant drops to 0 point along with the lapse of greening time.
(2) Result of APX enzyme Activity measurement
The results of the APX enzyme activity measurement are shown in Table 4.
TABLE 4 Activity of ascorbic Acid Peroxidase (APX) in seedling stage of small red bean treated by greening under different low temperature conditions
Note: data are expressed using mean ± standard deviation; the upper and lower case letters represent significant differences at the 5% and 1% levels for the two varieties, respectively, for the different treatments; APX units: μ mol/min/mg protein.
After the cold-resistant variety is treated for short-term low temperature (18d) and greened (3h, 8h), the APX enzyme activity gradually rises until the enzyme activity reaches the highest value of 0.21 mu mol/min/mg protein after 8h of greening but has no obvious difference with the enzyme activity before (0h) of greening, the APX enzyme activity gradually decreases along with the prolonging of the greening time, and the enzyme activity remarkably decreases to the lowest value of 0.11 mu mol/min/mg protein after 28h of greening; the long-term low-temperature treatment (28d) is similar to the short-term low-temperature treatment, the APX enzyme activity gradually increases after greening (3h and 8h) until the APX enzyme activity remarkably increases to 0.20 mu mol/min/mg protein after greening for 8h, the APX enzyme activity gradually decreases along with the prolonging of greening time, and the APX enzyme activity decreases to 0.14 mu mol/min/mg protein after greening for 28 h.
Short-term low-temperature treatment (18d) is carried out on the cold-resistant variety, the activity of the APX enzyme activity is continuously reduced along with the prolonging of the greening time after greening (3h, 8h and 28h) compared with that before greening (0h), and the APX enzyme activity is obviously reduced to 0.08 mu mol/min/mgprotein when greening for 28 h; after long-term low-temperature treatment (28d), compared with the time before greening (0h), the APX enzyme activity has no obvious difference and is maintained at a certain level, and the APX enzyme activity is obviously reduced to 0.00 mu mol/min/mgprotein at 8h of greening along with the prolonging of greening time and is not recovered until greening for 28 h.
The above results show that: the APX enzyme activity of cold-resistant varieties of small red beans is in a fluctuation state and is not 0 under the condition of long-term low-temperature treatment; and the APX enzyme activity of the cold-resistant variety is reduced to 0 point. Therefore, after the small red bean seedlings (28d) are treated at low temperature for a long time, the cold resistance of the small red bean variety to be detected, of which the APX enzyme activity in the plant is in a fluctuation state and is not 0, is stronger than that of the small red bean variety to be detected, of which the APX enzyme activity in the plant is reduced to 0 point in the greening time.
(3) Result of CAT enzyme Activity measurement
The results of CAT enzyme activity measurement are shown in Table 5.
TABLE 5 Catalase (CAT) Activity of small red bean at seedling stage after greening treatment under different low temperature conditions
Note: data are expressed using mean ± standard deviation; the upper and lower case letters represent significant differences at the 5% and 1% levels for the two varieties, respectively, for the different treatments; CAT unit: μ mol/min/mg protein.
After the cold-resistant variety is treated for short-term low temperature (18d) and greened (3h, 8h), the CAT enzyme activity is gradually increased until the enzyme activity reaches the maximum value of 1.87 mu mol/min/mg protein after 8h of greening, the CAT enzyme activity is obviously increased compared with that before (0h) of greening, the CAT enzyme activity is gradually reduced along with the prolongation of greening time, and the CAT enzyme activity is obviously reduced to 0.93 mu mol/min/mg protein after 28h of greening; the long-term low-temperature treatment (28d) is similar to the short-term low-temperature treatment, the CAT enzyme activity gradually increases after greening (3h and 8h) until the CAT enzyme activity remarkably increases to 1.19 mu mol/min/mg protein after greening for 8h, the CAT enzyme activity gradually decreases along with the prolonging of greening time, and the CAT enzyme activity decreases to 0.81 mu mol/min/mg protein after greening for 28 h.
Performing short-term low-temperature treatment (18d) on the cold-resistant variety, wherein the CAT enzyme activity remarkably rises to the highest value of 1.95 mu mol/min/mg protein after 3h after greening compared with that before greening (0h), the CAT enzyme activity gradually decreases along with the prolonging of greening time, and the CAT enzyme activity decreases to 1.03 mu mol/min/mg protein after greening for 28 h; after long-term low-temperature treatment (28d), compared with the condition before greening (0h), the CAT enzyme activity is remarkably increased to the maximum value of 1.59 mu mol/min/mg protein after greening (3h), the CAT enzyme activity is remarkably reduced to 0.00 mu mol/min/mg protein after greening for 8h along with the prolonging of greening time, and most leaves are withered, yellow and wilted because the leaves of cold-resistant varieties can not recover to the normal state due to overlong greening time.
The above results show that: the CAT enzyme activity of cold-resistant varieties of different small red beans is in a fluctuation state and is not 0 under the condition of long-term low-temperature treatment (28d), and the CAT enzyme activity of the cold-resistant varieties is increased firstly and then is reduced to 0 point. Therefore, after the small red bean seedlings (28d) are treated at low temperature for a long time, the cold resistance of the small red bean variety to be detected, which has the CAT enzyme activity in a fluctuation state and is not 0, in the plant along with the greening time is stronger than the cold resistance of the small red bean variety to be detected, which has the CAT enzyme activity in the plant firstly increased and then decreased to 0 point along with the greening time.
The test results of all the above examples show that: after long-term low-temperature treatment (28D), greening (0h, 3h, 8h and 28h), the leaves of the cold-resistant variety (A-D in figure 2) can be quickly recovered from the etiolation state, and the leaves are in a green and extended normal growth state when the leaves reach 28 h; leaves of the cold-intolerant species (E-H in FIG. 2) were unable to recover from the etiolation state and partially withered and dead. In conclusion, by comparing different varieties H2O2The content, chlorophyll content, SOD enzyme activity, APX enzyme activity and CAT enzyme activity are important indexes and methods for identifying cold resistance of small red beans.
Claims (5)
1. A method for identifying or assisting in identifying cold resistance of small red beans comprises the following steps: sowing the red beans to be detected, and moving the seedlings to the light intensity of 700-2Growing for 28-30 days at 10-13 deg.C, transferring the plant to light intensity of 1000-2Performing greening at the temperature of 20-25 ℃ in s; determining the cold resistance of the small red bean to be detected according to any one of the following indexes measured in the greening process: h in the plant2O2Content change, chlorophyll content change in the plant, superoxide dismutase activity change in the plant, ascorbic acid peroxidase activity change in the plant, and catalase activity change in the plant;
(A1) sowing the red beans to be detected, and moving the seedlings to the light intensity of 700-2Growing for 28-30 days at 10-13 deg.C, transferring the plant to light intensity of 1000-2Performing greening at the temperature of 20-25 ℃ in s;
(A2) h in the plant is measured when the greening time is 0H2O2Content, and/or H in plants over time of greening2O2Determining the cold resistance of the small red bean to be detected according to the change condition of the content as follows: h in the plant when the greening time is 0H2O2The lower the content, the stronger the cold resistance of the red bean variety to be detected; and/or H in plants2O2The content of the fertilizer does not change obviously with the lapse of greening timeThe cold resistance of the red bean variety to be detected which is changed to be not 0 is stronger than that of H in the plant2O2The content of the red bean variety to be detected is reduced to 0 point along with the greening time;
(B1) sowing the red beans to be detected, and moving the seedlings to the light intensity of 700-2Growing for 28-30 days at 10-13 deg.C, transferring the plant to light intensity of 1000-2Performing greening at the temperature of 20-25 ℃ in s;
(B2) measuring the change condition of the chlorophyll content in the plant along with the greening time, and determining the cold resistance of the small red bean to be detected according to the following steps: the cold resistance of the red bean variety to be detected with the rising of the chlorophyll content in the plant along with the lapse of the greening time is stronger than or the candidate is stronger than the cold resistance of the red bean variety to be detected with the falling of the chlorophyll content in the plant along with the lapse of the greening time;
(C1) sowing the red beans to be detected, and moving the seedlings to the light intensity of 700-2Growing for 28-30 days at 10-13 deg.C, transferring the plant to light intensity of 1000-2Performing greening at the temperature of 20-25 ℃ in s;
(C2) determining the change condition of the activity of superoxide dismutase in the plant along with the greening time, and determining the cold resistance of the red bean to be detected according to the following steps: the cold resistance of the red bean variety to be detected, which shows a fluctuation state of the superoxide dismutase activity in the plant and is not 0, is stronger than or candidate stronger than the cold resistance of the red bean variety to be detected, which reduces the superoxide dismutase activity to 0 point in the plant along with the greening time;
(D1) sowing the red beans to be detected, and moving the seedlings to the light intensity of 700-2Growing for 28-30 days at 10-13 deg.C, transferring the plant to light intensity of 1000-2Performing greening at the temperature of 20-25 ℃ in s;
(D2) measuring the change of the activity of the ascorbate peroxidase in the plants along with the greening time, and determining the cold resistance of the red bean to be detected according to the following steps: the cold resistance of the red bean variety to be detected which shows a fluctuation state of the ascorbic acid peroxidase activity in the plant along with the lapse of the greening time and is not 0 is stronger than or candidate stronger than the cold resistance of the red bean variety to be detected which reduces the ascorbic acid peroxidase activity in the plant to 0 point along with the lapse of the greening time;
(E1) sowing the red beans to be detected, and moving the seedlings to the light intensity of 700-2Growing for 28-30 days at 10-13 deg.C, transferring the plant to light intensity of 1000-2Performing greening at the temperature of 20-25 ℃ in s;
(E2) measuring the change of catalase activity in the plants along with the greening time, and determining the cold resistance of the red bean to be detected according to the following steps: the cold resistance of the red bean variety to be detected which shows a fluctuation state of catalase activity in the plant along with the greening time and is not 0 is stronger than or is candidate stronger than the cold resistance of the red bean variety to be detected which firstly rises and then falls to 0 point along with the greening time.
2. The method of claim 1, wherein: step (A2) of determining H in the plants over the course of greening time2O2The measurement time points were set within the range of 0 to 28 hours for the greening time, when the change in the chlorophyll content in the plant with the lapse of greening time was measured in the step (B2), when the change in the superoxide dismutase activity in the plant with the lapse of greening time was measured in the step (C2), when the change in the ascorbate peroxidase activity in the plant with the lapse of greening time was measured in the step (D2), and when the change in the catalase activity in the plant with the lapse of greening time was measured in the step (E2).
3. The method of claim 2, wherein: step (A2) of determining H in the plants over the course of greening time2O2Measuring chlorophyll in the plant with the lapse of greening time in the step (B2) when the content is changedThe measurement time points were set to greening times of 0h, 3h, 8h and 28h, respectively, when the change in the content was measured, the change in the superoxide dismutase activity in the plant with the lapse of greening time was measured in the step (C2), the change in the ascorbate peroxidase activity in the plant with the lapse of greening time was measured in the step (D2), and the change in the catalase activity in the plant with the lapse of greening time was measured in the step (E2).
4. A method according to any one of claims 1 to 3, wherein: in the step (A2), the H2O2The content is H in plant leaves2O2Content (c);
in the step (B2), the chlorophyll content in the plant is the chlorophyll content in the plant leaf;
in the step (C2), the activity of the superoxide dismutase in the plant is the activity of the superoxide dismutase in the plant leaves;
in step (D2), the ascorbate peroxidase activity in the plant is ascorbate peroxidase activity in a leaf of the plant;
in the step (E2), the catalase activity in the plant is catalase activity in plant leaves.
5. A method according to any one of claims 1 to 3, wherein: in the steps (A1), (B1), (C1), (D1) and (E1), after the red beans to be detected are sown, the red beans are all put at a constant temperature of 25 ℃ and under 100% dark and no light conditions for emergence of seedlings.
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