CN108739127B - Method for rapidly comparing high-temperature drought resistance characteristics of tomato varieties - Google Patents
Method for rapidly comparing high-temperature drought resistance characteristics of tomato varieties Download PDFInfo
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Abstract
The invention discloses a method for rapidly comparing high-temperature drought resistance characteristics of tomato seedlings, which comprises the following steps: sowing, transferring the seedlings to a climatic chamber for adaptation 18 to 23 days after sowing, and moving the seedlings to the climatic chamber for adaptation every timePouring nutrient solution in the morning to adapt to the 7 th day, taking the 1 st completely unfolded leaf, immediately weighing fresh weight, naturally standing at room temperature for 1 hr and 20 hr, weighing fresh weight of the leaf again, and calculating WLR1 h-controlAnd WLR20 h-control(ii) a Weighing at 40 deg.C, stopping irrigation, taking 1 st completely unfolded leaf after 48 hr, immediately weighing fresh weight, naturally standing at room temperature for 1 hr and 20 hr, weighing fresh weight of leaf again, and calculating WLR1 h-stressAnd WLR20 h-stress(ii) a According to (| WLR)1 h-stress‑WLR1 h-control|+|WLR20 h-stress‑WLR20 h-controlThe value of |))/2 is compared with the high temperature drought resistance of tomato varieties.
Description
Technical Field
The invention belongs to the field of stress tolerance comparison biotechnology of tomatoes, and particularly relates to a method for rapidly comparing high temperature and drought resistance of tomato varieties.
Background
Tomatoes (Solanum lycopersicum L.) are important vegetables throughout the world and are widely used in open field and facility cultivation. However, the abiotic stress caused by the abiotic environmental factors can have an adverse effect on the cultivation and planting of tomatoes. On the one hand, as global climate warmth increases, high temperature stress has a growing impact on tomato production, while in the over-summer cultivation of tomatoes, high temperature stress also occurs frequently. On the other hand, drought stress caused by insufficient rainfall or soil water deficiency can also cause damage to tomato production. High temperature and drought stress severely limit world crop yield, causing significant losses to the agricultural economy. It is noteworthy that under natural conditions, multiple abiotic stresses often occur simultaneously, especially high temperature and drought, with high temperature drought combined stresses often being more harmful to tomatoes than a single stress. The inventor discovers that the high temperature is in the flank through previous researchForced, chlorophyll fluorescence parameter Fv/FmCan compare tolerance differences of different tomato varieties[1]. Under drought stress, the Water Loss Rate (WLR) of the plant leaves is kept unchanged, which shows that the plant has better water retention and stronger drought resistance[2]. Different tomato varieties have different sensitivity degrees to high-temperature drought composite stress[3]. However, a method for rapidly and effectively comparing high-temperature drought resistance characteristics of tomato seedlings is lacked at present[3]。
Disclosure of Invention
The inventor researches and discovers that for tomatoes, under high-temperature drought composite stress, the drought stress plays a leading role, namely under the high-temperature drought composite stress, the better the water retention of tomato leaves is, the stronger the high-temperature drought resistance is, and the response of the tomatoes is more obvious under the treatment at 40 ℃ than 36 ℃, so that the invention adopts the extreme high-temperature treatment at 40 ℃; meanwhile, in order to prevent the recovery caused by repeated irrigation from influencing the experimental result, the irrigation stopping method is adopted for drought stress, and different tomato varieties show differences after high-temperature drought composite stress is carried out for 48 hours. Therefore, the invention aims to provide a method for rapidly comparing high-temperature drought resistance of tomato seedlings, which is used for judging the degree of damage of the tomatoes caused by high-temperature drought stress by using the water loss rate of tomato leaves under the high-temperature drought stress so as to solve the problem that a method for rapidly and effectively comparing the tolerance of tomato varieties under the high-temperature drought stress is lacked in the prior art and lay a foundation for rapid comparison and phenotypic typing of the high-temperature drought resistance of the tomato seedlings.
In order to achieve the purpose, the invention provides the following technical scheme:
a method for rapidly comparing high-temperature drought resistance characteristics of tomato varieties comprises the following steps:
putting the substrate into a hole tray, thoroughly watering the substrate before sowing, sowing the tomato seeds to be compared into a hole basin, sowing 2 seeds in each hole, and covering a layer of substrate after sowing; after 7-14 days of sowing, removing redundant seedlings, and reserving one seedling in each hole;
step (2), after sowing for 18-23 days, putting the seedlings into an artificial climate box for adaptive culture, and immersing the hole tray by using the Japanese garden test formula nutrient solution at 8:00 a day in the morning;
step (3), adaptively culturing the seedlings in an artificial climate box for 7 days, taking the 1 st fully-unfolded leaf as a control leaf, and immediately weighing the fresh weight FW of the control leaf0 h-controlAs a control, naturally standing at room temperature for 1 hr and 20 hr, respectively weighing fresh weight FW of leaves1 h-control、FW20 h-control；
Step (4), weighing the control leaves, continuously treating the control leaves at the temperature of 40 ℃ for 48h at 19:00 evening, and stopping irrigation to enable the seedlings to be subjected to severe high-temperature drought synergistic stress in a short time, so as to detect the tolerance of the seedlings to the composite stress;
step (5), after high-temperature drought treatment, taking the 1 st fully-unfolded leaf, immediately weighing the fresh weight FW of the stressed leaf0 h-stressNaturally standing at room temperature for 1 hr and 20 hr, and weighing fresh weight FW of leaves1 h-stress、FW20 h-stress；
And (6) determining the water loss conditions of the leaves in a short time (1h) and a long time (20h), and calculating the water loss rate of the control leaves and the water loss rate of the stress-treated leaves:
WLR1 h-control＝[(FW1 h-control-FW0 h-control)/FW0 h-control]*100，
WLR20 h-control＝[(FW20 h-control-FW0 h-control)/FW0 h-control]*100，
WLR1 h-stress＝[(FW1 h-stress-FW0 h-stress)/FW0 h-stress]*100，
WLR20 h-stress＝[(FW20 h-stress-FW0 h-stress)/FW0 h-stress]*100；
Calculating absolute value | WLR of difference value of water loss rate of 1h control blade and 20h stress treatment blade1 h-stress-WLR1 h-control|、|WLR20 h-stress-WLR20 h-controlAnd obtaining (| WLR) through further calculation1 h-stress-WLR1 h-control|+|WLR20 h-stress-WLR20 h-controlI.)/2 for evaluationHigh temperature drought characteristics of eggplant varieties; if (| WLR)1 h-stress-WLR1 h-control|+|WLR20 h-stress-WLR20 h-controlThe smaller the value of |))/2 is, the more resistant the tomato variety is to high-temperature drought composite stress; (| WLR)1 h-stress-WLR1 h-control|+|WLR20 h-stress-WLR20 h-controlThe larger the value of I)/2 is, the more sensitive the tomato variety is to high-temperature drought composite stress.
In the step (1), the substrate is a Ping's organic substrate.
The length of the plug tray is 54cm, the width of the plug tray is 27cm, and the plug tray has 50 holes.
The temperature, light, moisture and humidity conditions for the growth of the tomato seedlings prior to moving to the climatic chamber are suitable and well known to those skilled in the art.
In the step (2), the model of the artificial climate box is RDN-560E-4, the artificial climate box is from Ningbo southeast instruments Co., Ltd, and has 4 layers, and each layer can be provided with 2 hole trays.
Before high-temperature drought treatment, the temperature in the artificial climate box is set to be 26/18 ℃, the day/night is 14h/10h, the day is 5:00-19:00, the night is 19: 00-the next day is 5:00, the relative humidity is 60%, and the illumination intensity is 25000 lux.
The plug tray was submerged using 500-.
The invention adopts a balance to measure the fresh weight of the blade, and the weight is accurate to four digits after decimal point. The blades are placed in the same place.
In the present invention, three replicates per tomato variety are set up.
In the step (6), preferably, the evaluation of the high-temperature drought characteristic standard of the tomato variety is as follows: (| WLR)1 h-stress-WLR1 h-control|+|WLR20 h-stress-WLR20 h-controlThe value of |)/2 is less than or equal to 0.8, the high temperature drought resistance of the tomato genotype is strong; (| WLR)1 h-stress-WLR1 h-control|+|WLR20 h-stress-WLR20 h-controlIf the value of I)/is more than or equal to 22.0, the tomato genotype is sensitive to high-temperature drought composite stress.
Compared with the prior art, the invention has the beneficial effects that:
according to the method, the high-temperature drought resistance characteristics of the tomato seedlings can be rapidly and accurately compared in a short time (1h) by using the change of the leaf Water Loss Rate (WLR) after the control and high-temperature drought combined stress treatment for 48h after the short time (1h) and the long time (20h), the accuracy rate of the evaluation on the high-temperature drought resistance combined stress characteristics of the tomato can reach 100%, an effective method is provided for the evaluation on the high-temperature drought resistance combined stress characteristics of crops and the material screening, and the method is favorable for the stress tolerant breeding of the crops. Meanwhile, each artificial climate box used by the method can be used for placing 8 plug trays at most, and each plug tray can be used for sowing 16 tomato varieties at most, namely, 128 tomato varieties can be compared at most in one batch.
Detailed Description
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making creative efforts based on the embodiments of the present invention, belong to the protection scope of the present invention.
Example 1
A method for rapidly comparing high-temperature drought resistance characteristics of tomato seedlings comprises the following steps:
sowing tomato seeds into a plug tray filled with Pindstrup organic matrix (Denmark), wherein the length of the plug tray is 54cm, the width of the plug tray is 27cm, and 50 holes are formed in total; 2 seeds are sowed in each hole, and 3 holes are sowed in each tomato variety; placing the hole tray in a greenhouse or a shed, and culturing under appropriate conditions of external temperature, illumination, moisture, humidity and the like; after sowing for 14 days, if 2 seedlings are produced in one hole, only one seedling is reserved;
step (2), after seeding for 23 days, placing the hole tray in an artificial climate box for adaptation, setting the temperature of the artificial climate box to be 26/18 ℃ (14h/10h, day/night), setting the relative humidity to be 60%, and setting the illumination intensity to be 25000 lux; the artificial climate box has 4 layers, and each layer is provided with 2 hole trays; before high-temperature drought treatment, soaking a hole tray by using 500mL of Japanese garden trial formula nutrient solution 8:00 a day in the morning;
step (3), on the 30 th day after sowing, taking the 1 st fully-unfolded leaf as a control leaf, immediately weighing fresh weight (namely FW) by balance0 h-controlFour digits after decimal point precisionCounting, same as below), naturally standing at room temperature for 1 hr and 20 hr, and respectively weighing fresh weight of leaves (FW) with the same balance1 h-control、FW20 h-control)。
Step (4), weighing the control leaves, starting high-temperature treatment at 40 ℃ for 48h (14h/10h, day/night) at 19:00 night on the day, controlling the relative humidity to be 60% and the illumination intensity to be 25000lux, and stopping irrigation;
after high-temperature drought treatment for 48h, taking the 1 st fully-unfolded leaf as a stress treatment leaf, and immediately weighing the fresh weight (namely FW) by using a balance same as the balance in the step (3)0 h-stress) Naturally standing at room temperature for 1 hr and 20 hr, and respectively weighing Fresh Weight (FW) of leaves with the same balance1 h-stress、FW20 h-stress)。
And (6) calculating the water loss rate of the blade, namely:
WLR1 h-control＝[(FW1 h-control-FW0 h-control)/FW0 h-control]*100，
WLR20 h-control＝[(FW20 h-control-FW0 h-control)/FW0 h-control]*100，
WLR1 h-stress＝[(FW1 h-stress-FW0 h-stress)/FW0 h-stress]*100，
WLR20 h-stress＝[(FW20 h-stress-FW0 h-stress)/FW0 h-stress]*100；
Comparing the water loss rate changes of different tomato varieties under control and stress, namely calculating the difference (WLR) between the water loss rates of the control and the stress leaves at 1h and 20h respectively1 h-stress-WLR1 h-controlAnd WLR20 h-stress-WLR20 h-control). Three repeats per variety, solve (| WLR)1 h-stress-WLR1 h-control|+|WLR20 h-stress-WLR20 h-controlThe value of |))/2 is used for evaluating the high-temperature drought characteristics of the variety.
Step (7), when the high temperature and drought resistant characteristics of a plurality of tomato varieties are compared, if | WLR1 h-stress-WLR1 h-controlI and I WLR20 h-stress-WLR20 h-controlThe smaller the average value of |, theThe tomato variety is more resistant to high-temperature drought composite stress; i WLR1 h-stress-WLR1 h-controlI and I WLR20 h-stress-WLR20 h-controlThe larger the average value of | is, the more sensitive the tomato variety is to high-temperature drought complex stress.
The high temperature drought resistance of 25 different tomato varieties was compared, with 3 replicates per variety (table 1). As shown in table 1, (| WLR) nos. 17 and 22 after high temperature drought combined stress treatment1 h-stress-WLR1 h-control|+|WLR20 h-stress-WLR20 h-controlThe value of |)/2 is minimum (0.8); number 19 (| WLR)1 h-stress-WLR1 h-control|+|WLR20 h-stress-WLR20 h-controlThe value of | 2 is the largest (22.0). The water loss rates of leaves in different periods of time after high-temperature drought treatment are combined to obtain that, of the 25 tomato varieties, the varieties 17 and 22 have certain tolerance to high-temperature drought stress, and the variety 19 is sensitive to high-temperature drought stress.
TABLE 1 Water loss rates of seedlings of different tomato varieties
Note: the No. 1-8, No. 11-12 and No. 20-22 tomato varieties are obtained by introducing foreign excellent tomato germplasm resources, purifying through multi-generation selfing, and hybridizing with the domestic existing excellent tomato materials. Number 9 is tomb dream tomato variety, number 10 is tomb pink tomato variety, number 13 is suma 12 tomato variety, number 14 is suma 14 tomato variety, number 15 is tomb beautiful tomato variety, number 16-19 is wild currant tomato, number 23 is European shell tomato variety, number 24 is tomb blackish jab tomato variety, number 25 is ali tomato variety.
Comparative example 1
Evaluation of high-temperature drought resistance of tomato seedlings by seedling growth amount by predecessors[3,4]. External growth reaction of tomato plant and seedling growth amount under high-temperature drought stressThe responses are substantially uniform[4]The tomato which is resistant to high temperature and drought has less stress external symptoms, and meanwhile, the reduction value of the dry weight is less.
After 4 days of high temperature drought treatment, the tolerance of the tomato to high temperature drought is judged by a method of observing the plant phenotype and the seedling growth amount by naked eyes, 2 tomato varieties (No. 19 tomato sensitive to high temperature drought; No. 22 tomato resistant to high temperature drought) in example 1 are compared, and the specific steps are as follows:
(1) the preparation of the material is the same as the steps (1) - (2) of the method in the embodiment 1, the seedlings are adaptively cultured in a climatic chamber for 7 days, the dry and fresh weight of the overground part (above cotyledonary node) of the seedlings is weighed as a contrast before treatment, and each variety is repeated three times;
(2) carrying out high-temperature treatment at 38/26 ℃ (14h/10h, day/night) in a climatic chamber, and stopping irrigation;
(3) and carrying out visual observation on the plants after high-temperature drought treatment for 4 days, weighing the dry and fresh weight of the overground part (above cotyledonary node) of the seedling, and calculating the dry and fresh weight growth amount, namely the fresh weight growth amount (the treated fresh weight-the fresh weight before treatment)/the fresh weight before treatment, the dry weight growth amount (the treated dry weight-the dry weight before treatment)/the dry weight before treatment, wherein each variety is repeated three times.
The comparison result is: at the early stage of the high-temperature drying treatment at 38/26 ℃ (after 2 days), no wilting symptom appears on the No. 22 tomato seedling, and wilting symptom appears on the middle upper leaf of the No. 19 tomato; at the later stage of the high-temperature drying treatment at 38/26 ℃ (after 4 days), 19 # whole plant shows symptoms of drought, withering and even death, and 22 # tomato shows a light wilting symptom. Meanwhile, after high-temperature drought treatment, the fresh weights of No. 19 and No. 22 seedlings are respectively reduced by 68.9 percent and 60.2 percent; the dry weight of No. 19 and No. 22 seedlings is reduced by 30.5 percent and 23.9 percent respectively, and the dry weight reduction value of No. 22 with certain tolerance to high-temperature drought stress is obviously less than that of No. 19. In conclusion, the high temperature drought resistance of the No. 22 tomato is superior to that of the No. 19 tomato.
Reference documents:
[1]Rong Zhou,Xiaqing Yu,Katrine H.Kjaer,Eva Rosenqvist,Carl-OttoOttosen,Zhen Wu.Screening and validation of tomato genotypes under heatstress using Fv/Fmto reveal the physiological mechanism of heat tolerance[J].Environmental and Experimental Botany,2015,118:1-11.
[2] yan Hui Ru, cotton GhWRKY17 transcription factor participates in ABA signal pathway and drought and high salt stress reaction [ D ]. Shandong agricultural university.2013.
[3] Zhang Benyuan, Jiangsan, the difference in resistance of different tomato varieties to high temperature drought [ J ]. Changjiang vegetables, 2010,17:49-50.
[4] Influence of high-temperature drought treatment on growth of seedlings of different varieties of tomatoes [ J ] North horticulture, 2007,1:1-5.
Claims (7)
1. A method for rapidly comparing high-temperature drought resistance characteristics of tomato varieties is characterized by comprising the following steps: the method comprises the following steps:
putting the substrate into a hole tray, thoroughly watering the substrate before sowing, sowing the tomato seeds to be compared into a hole basin, sowing 2 seeds in each hole, and covering a layer of substrate after sowing; after 7-14 days of sowing, removing redundant seedlings, and reserving one seedling in each hole;
step (2), after sowing for 18-23 days, putting the seedlings into an artificial climate box for adaptive culture, and immersing the hole tray by using the Japanese garden test formula nutrient solution at 8:00 a day in the morning;
step (3), adaptively culturing the seedlings in an artificial climate box for 7 days, taking the 1 st fully-unfolded leaf as a control leaf, and immediately weighing the fresh weight FW of the control leaf0 h-controlNaturally standing at room temperature for 1 hr and 20 hr, respectively weighing fresh weight FW of leaves1 h-control、FW20 h-control；
Step (4), weighing the control leaves, starting high-temperature treatment at 40 ℃ for 48h at 19:00 evening of the day, and stopping irrigation;
step (5), after high-temperature drought treatment, taking the 1 st fully-unfolded leaf, immediately weighing the fresh weight FW of the stressed leaf0 h-stressNaturally standing at room temperature for 1 hr and 20 hr, respectively weighing fresh weight FW of leaves1 h-stress、FW20 h-stress；
Step (6), calculating the water loss rate of the control blade:
WLR1 h-control＝[(FW1 h-control-FW0 h-control)/FW0 h-control]*100，
WLR20 h-control＝[(FW20 h-control-FW0 h-control)/FW0 h-control]*100；
Calculating the water loss rate of the stress treatment blade:
WLR1 h-stress＝[(FW1 h-stress-FW0 h-stress)/FW0 h-stress]*100，
WLR20 h-stress＝[(FW20 h-stress-FW0 h-stress)/FW0 h-stress]*100；
Calculating absolute values of the difference between the water loss rates of the 1h control leaves and the stress-treated leaves for 20h respectively according to the absolute value of the water loss rate of the stress-treated leaves1 h-stress-WLR1 h-controlI and I WLR20 h-stress-WLR20 h-controlAverage value of |, evaluating the high temperature drought characteristics of different tomato varieties:
(|WLR1 h-stress-WLR1 h-control|+|WLR20 h-stress-WLR20 h-controlThe value of |)/2 is less than or equal to 0.8, the high temperature drought resistance of the tomato genotype is strong; (| WLR)1 h-stress-WLR1 h-control|+|WLR20 h-stress-WLR20 h-controlIf the value of |)/2 is more than or equal to 22.0, the tomato genotype is more sensitive to high-temperature drought composite stress.
2. The method for rapidly comparing the high temperature and drought resistance of tomato varieties according to claim 1, wherein the method comprises the following steps: in the step (1), the substrate is a Ping's organic substrate.
3. The method for rapidly comparing the high temperature and drought resistance of tomato varieties according to claim 1, wherein the method comprises the following steps: in the step (1), the length of the plug tray is 54cm, the width of the plug tray is 27cm, and 50 holes are formed in total.
4. The method for rapidly comparing the high temperature and drought resistance of tomato varieties according to claim 1, wherein the method comprises the following steps: in the step (2), before high-temperature drought treatment, the temperature in the artificial climate box is set to be 26/18 ℃, the day/night time is 14h/10h, the relative humidity is 60%, and the illumination intensity is 25000 lux.
5. The method for rapidly comparing the high temperature and drought resistance of tomato varieties according to claim 1, wherein the method comprises the following steps: in step (2), the well tray is immersed in 500-.
6. The method for rapidly comparing the high temperature and drought resistance of tomato varieties according to claim 1, wherein the method comprises the following steps: the artificial climate box has 4 layers, and each layer is provided with 2 hole trays.
7. The method for rapidly comparing the high temperature and drought resistance of tomato varieties according to claim 1, wherein the method comprises the following steps: three replicates were set for each tomato variety.
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