植物抗旱性鉴定（Plant drought resistance identification）

1、植物抗旱性鉴定（Plant drought resistance identification）Experiment 17 identification of plant drought resistanceWater deficit is one of the most common effect of plant productivity and environmental stress, although vegetable crops are generally in the cultivation area of adequate water, but usually vegetab

2、le water, and almost the whole growth period of water are more; fruit mostly cultivated in hilly land, more vulnerable to water deficit. Therefore, it is very important to study the drought resistance of plants and to carry out drought resistance breeding.Drought resistant breeding success depends l

3、argely on how much resources with resistance and research level, therefore, the drought resistance of germplasm resources identification, evaluation and selection is a key link in drought resistant breeding, breeding by researchers around the world attention. There are many methods for drought resis

4、tance identification, including direct field identification method, dry shed method, artificial climate chamber method, potted method and indoor simulated drought condition method. These methods have their own advantages and disadvantages, and are suitable for the identification and study of drought

5、 resistance in different periods and different purposes.In this experiment, we will introduce the main methods and steps of drought resistance identification of common wheat varieties with different drought resistance.Materials and utensilsWheat seedlings, germination box, filter paper, Petri dishes

6、, punch, balance, dryer, conductivity meter, 20ml plug with a graduated tube, double blade, thermostatic bath, thermometer, glass rod, mortar, filter funnel, volumetric flask (50ml), pipettes (2ml, 5ml, 10ml), high-speed centrifuge, spectrophotometer, micro injector, fluorescent lamp (reaction tube

7、at the light intensity of 4000lx); scale Straw, G3 sintered glass funnel.Two, method steps(1) direct identification in the field;When soil drought comes, especially when drought occurs at the booting stage to the filling stage, the plants wither gradually because of water loss, and the leaves become

8、 yellow and dry. After the highest sunshine and the highest temperature in the afternoon, 5 degrees were recorded according to the degree of wheat leaf wilting. The smaller the series, the stronger the drought resistance.1 symptoms without symptoms;A small part of 2 leaves atrophy and lose luster, w

9、ith fewer leaves rolled into needles;Most of the leaves of 3 stage were atrophied and more leaves were rolled into needles;4 severe leaf curling, normal color color significantly in the deep variety, the lower leaves begin to turn yellow;5 stems and leaves apparently atrophy, and the lower leaves tu

10、rn yellow to dry.The above is based on the drought resistance of wilting qualitative identification of varieties, also can put various varieties were planted in Dryland (stress) and water (non stress), determination of dryland yield and water yield, the drought resistance of varieties of quantitativ

11、e evaluation of the following formula.Drought resistance coefficient (DC) =) average yield under non stress () average yield under duress (PDYYDrought resistance index (DI) = average value of all varieties) dry land yield () drought resistance coefficient (DDCYYD *)The greater the drought resistance

12、 coefficient or drought resistance index, the stronger the drought resistance.(two) germination test identificationThe method was applied to simulate drought conditions in the laboratory and identify drought resistance during the wheat bud stage.1. of the tested seeds in 0.1% mercuric chloride solut

13、ion, sterilization and disinfection of 10 15min;2. place 4 qualitative filter paper in the diameter 10cm culture dish and add 15%PEG (polyethylene glycol) solution 6ml or 17.6%30ml sucrose solution, 1 dishes per dish, evenly placed in good health, 30 grains, repeat 34 times;3. put the dishes into th

14、e germination box and germinate at 25 7d;4. the germination potential and germination rate of seeds were measured at third and seventh days after germination, and the drought resistance of the cultivars was evaluated. The length and root length of coleoptile can also be measured to reflect the droug

15、ht resistance of varieties.(three) determination of water holding capacity of detached leavesThe specific steps are as follows:1. weighing 510 pieces of wheat flag leaves with 1/10000 electronic scales (or taking the seedlings to carry out a few pieces of parietal leaves, respectively called the fre

16、sh weight), and numbering each sample, repeating it 34 times;2., the leaves weighing fresh weight should be placed in the dryer at 25 DEG C to 30 DEG C, drying 2 6h in dark condition, and then weighing the weight of the leaves after dehydration;3. calculate the water loss rate of each sample:Water l

17、oss rate (%) =%100 * fresh weight, fresh weight - after dehydrationThe average water loss rate of each leaf was calculated and compared. In general drought resistant varieties, the water holding capacity of leaves is higher than that of drought resistant varieties.(four) determination of free prolin

18、e contentThe extraction of proline from plants by using salicylic acid not only greatly reduces the interference of other amino acids, but also is fast, easy to operate and free from sample condition (dry or fresh sample). Under acidic conditions, proline reacts with the three keto ketone to form a

19、stable red condensate. After extraction with toluene, the condensation has a maximum absorption peak at 520nm. The concentration of proline is proportional to its degree of extinction in a certain range.2.5%, acid, three ketones color liquid: glacial acetic acid and 6mol/L phosphoric acid, mixed wit

20、h 3:2, as solvent, prepared, heated at 70 degrees, dissolved, cooled, rear Brown reagent bottle, stored at 4 degrees reserve, stable within two days.1. draw the standard curve of proline(1) take 10mg proline, distilled water is dissolved to 100ml, and its concentration is 100 mum g/ml.(2) from 0, 0.

21、5, 1.25, from 2.5, 5, 7.5 and 10.0ml respectively into 7 50ml volumetric flask, adding distilled water volume to 50ml, with a series of 0, 1, 2.5, was 5, 10, 15, 20 g/ml.(3) do not take the above solution 2ml, add 7 numbered test tubes, and then add 2ml glacial acetic acid, 4ml acid, three ketone re

22、agents, 2ml sulfo salicylic acid solution;(4) shake well, cover with a glass tube, and react with 2h in boiling water bath;(5) take out the tube, cool it to room temperature, and then add 4ml toluene to the test tube. After the shock, it is placed about 10min, and the red reaction product is extract

23、ed to the toluene layer;(6) suck the red toluene extract with a dropper in the cuvette, and measure the absorbance at the 520nm wavelength of the spectrophotometer;(7) draw the standard curve with the content of proline as abscissa and absorbance as ordinate.2. extraction of free prolineWeigh 0.3g l

24、eaf fresh (from different materials by drought treatment and control), cut into a stoppered test tube, add 5ml 3% sulfosalicylic acid was extracted after 10min, plugging in a boiling water bath and filtered liquid to be measured.3. determination of free prolineTake the 2ml of the extract in a stoppe

25、red test tube, add 2ml distilled water, 2ml acetic acid and 4ml acid indene ketone three reagent, shake after heating in a boiling water bath for 2h coloration, removed after cooling to room temperature, add 4ml of toluene, the product to fully shake the red death. Stand aside for 10min, absorb the

26、toluene layer and measure the absorbance at the 520nm wavelength of spectrophotometer.4. calculate the content of proline in the sampleProline content (g/g) = (C * V/A) /WorProline content (%) = (C * V/A) /W * 10-4Formula: C - the proline g number is found by the standard solution;V - total volume o

27、f extraction liquid (ML);A - Determination of total liquid volume (ML);W sample weight (g); 1g = 106 G.Studies on many plants have shown that a large accumulation of free proline occurs when water stress occurs, so many researchers suggest that proline accumulation may be used as an indicator of pla

28、nt drought resistance. However, some studies have shown that a variety of plants (including wheat varieties) under water stress, proline accumulation has great difference, proline in mild drought stress in some drought resistant varieties did not increase, and some drought resistant varieties, the i

29、nternal organs water potential decreased fast, free proline accumulation is also fast. Therefore, when proline accumulation is used as a drought resistance identification index, it should be evaluated in combination with other drought resistant identification indexes.(five) determination of betaine

30、contentBetaine was first found in the plant of Lycium barbarum. It is known that there are 12 kinds of betaine. At present, betaine (Glycine), or betaine, is widely studied. It is widely found in various organs of flowering plants. The accumulation of betaine in cells can improve the osmotic regulat

31、ion ability of cells,Stabilize the structure and function of intracellular macromolecules, proteins and biological membranes, and also protect the activities of enzymes required for many important metabolic activities in cells. As a non toxic osmotic regulator, betaine plays a very important role in

32、 plant stress resistance physiology. Studies have shown that the accumulation of betaine is proportional to plant resistance to stress.1. solution preparationRay salt solution: precision called 1.5g ray salt, add water to 100ml, with hydrochloric acid tune pH value of 1, room temperature mixing 45mi

33、n, must be prepared before use.Betaine extract: methanol: chloroform: water, =12:5:3 (V/V).Betaine standard solution: the precise name is betaine 100mg, which is distilled into the 100ml volumetric flask with distilled water. After being dissolved completely, the solution is diluted to the standard,

34、 then the standard liquid is 1mg/ml.2. standard curve drawingDraw standard curve, the standard solution containing 0.2 per ml, 0.4, 0.6, 0.8, 1.0mg solution, 3.0ml solution from above the water bath, put 3h, G3 sintered glass funnel, 3 x 3ml ether wash the precipitate, after the ether was dried. Usi

35、ng 3 * 5ml 70% acetone solution and precipitation transfer to a 25ml volumetric flask, with 70% acetone added to the scale, with 70% acetone blank, the absorbance of the 525nm in.3. extraction of betaineWeigh about 1g (from wheat leaves with different materials of drought treatment and control), add

36、ing 10ml betaine extract, grinding, grinding fluid in the warm water bath to keep 10min, after cooling in 20oC centrifugal 10m minutes, collect the aqueous phase, the lower chloroform phase adding 10ml extracts (chloroform: methanol: water =12:5:3 (V/V); and then centrifuged from the upper aqueous phase, lower added 4ml50% methanol, centrifugation, and then the upper water with PH5 7, 70oC dry, then 5ml dissolved in water.4. det