CN106508460A - Method for rapid and indoor identification of broomrape resistance level of sunflowers - Google Patents

Abstract

The invention relates to a method for rapidly inducing the germination of sunflower gentang under indoor conditions, that is, the petri dish filter paper method, and its application in the identification of sunflower resistance to ligantang. Induce the germination of sunflower seeds on a petri dish indoors, keep the filter paper moist, and observe the germination of the seeds directly under a microscope. After continuing to cultivate for 3 weeks, the parasitic tumor-like structure of sunflower sunflower can be observed with the naked eye. Compared with the nutrient pot method, the steps of sunflower planting and fine root counting in the later stage are omitted, which is not only easy to operate and easy to observe, but also greatly saves time. The establishment of this system can be used for rapid screening and identification of resistance levels of different sunflower varieties, and it is the premise and basis of sunflower resistance breeding. The identification results of the petri dish filter paper method and the field test results have a very good consistency, therefore, the resistance levels of different sunflower varieties can be quickly identified.

Description

A Rapid Indoor Method for Identification of Antiretin in Sunflower

technical field

The invention belongs to the field of biotechnology, and relates to a method for rapidly inducing germination of sunflower sangritang under indoor conditions, that is, the petri dish filter paper method, and its application in rapid identification of sunflower sangritangia level.

Background technique

Sunflower Liedang (Orobanche cumana Wallr), also known as poisonous root grass, is an annual herbaceous parasitic seed plant, belonging to dicotyledonous plants, Orobanche cumana Wallr, mainly damages sunflower, watermelon, melon, pea, broad bean, carrot, celery, tobacco, flax , tomato, etc. Sunflower Liedang is a major disease that seriously threatens the development of sunflower industry at present. It occurs in all sunflower producing areas in my country, but it occurs most seriously in Xinjiang and Inner Mongolia. After the sunflower is listed as parasitic, the sunflower plant is short and thin, unable to form a flower disc, and finally causes the whole plant to die. Screening and cultivating sunflower varieties resistant to Lietang is currently the most effective measure to control Lietang. The establishment of a rapid screening and identification system of resistance levels of different sunflower varieties under indoor conditions is the premise and basis of sunflower resistance breeding. The current existing nutrient bowl identification method has many shortcomings in the operation process, such as the method needs a large number of dangling seeds and soil to be mixed in a certain proportion; the washing work before the counting of the dangling quantity in the later stage requires a lot of manpower and time ; This system is not conducive to observing the various developmental stages of Liedang. A method for rapid identification of sunflower anti-retidin level under indoor conditions established by our laboratory-petri dish filter paper method can avoid the above-mentioned shortcomings of the nutrient bowl identification method, and the identification results of this method are very good with the field test results. Consistency, therefore, enables the rapid identification of the level of resistance to Lietang in different sunflower cultivars.

Contents of the invention

Aiming at the deficiencies of the prior art, the present invention provides a method for rapidly inducing the germination of sunflower sunflowers under indoor conditions, that is, the culture dish filter paper method, which aims to solve the problem of slow germination of sunflower seeds, difficulty in observing the germination and parasitic process, and long experimental period. The problem of poor reproducibility, and the establishment of a method for rapidly inducing sunflower rowang germination under indoor conditions can also be applied to the rapid identification of sunflower resistance to rowang.

The object of the present invention is to provide a kind of method for quickly inducing sunflower rowan germination under indoor conditions, i.e. the petri dish filter paper method, the method comprises the following steps:

Step 1: Select plump sunflower seeds and sterilize with 0.1% sodium hypochlorite for 3 minutes, then add 75% alcohol for 3 minutes, then rinse with sterile water for 3 times and soak in sterile water for 2 hours, then take it out, cover with wet gauze and spray water Moisturize, and the sunflowers will germinate in about 5 days. The pre-germinated sunflower seedlings were cultured in a 13×13 cm plastic square dish covered with filter paper.

Step 2: After the cleaning and disinfection of the sunflower seeds, the root exudates of the sunflower seedlings were used for pretreatment. Put the sunflower seeds collected from the field through a sieve and add distilled water to wash them. Add 1ml of 0.1% sodium hypochlorite to the air-dried seeds for disinfection for 3 minutes, then add 75% alcohol for disinfection for 3 minutes, and finally rinse them with sterile water for 3 times . The sunflower seedlings were cultivated hydroponically in jars, and after three weeks of cultivation, the exudates of the sunflower roots were collected by filter sterilization. Spread the sterilized sunflower seeds as evenly as possible on the filter paper and completely wet the filter paper with the sunflower root exudates collected above. After sealing the petri dish with a parafilm, place it in a dark condition at 23-28°C for 7-10 days, and then use it to inoculate sunflowers.

Step 3: Inoculate the sunflower row as seeds. After the sunflower seedlings are in the square petri dish for 3-5 days, when the new root system is strong, move the pretreated sunflower row-dang seeds together with the filter paper into the petri dish with sunflower seedlings, seal the petri dish with a sealing film and place it in the cultured in a light incubator. Add distilled water every two days to keep the filter paper moist.

Step 4: Observation of the germination of Liedang seeds and formation of parasitic tumors and counting of parasitic rates. 1 week after inoculation, observe the number of germination of sunflower Liedang seeds under a 20X dissecting microscope; 3 weeks after inoculation, observe the formation of parasitic tumor-like structures of sunflower Liedang seeds with the naked eye, and use the following formula to calculate the parasitic rate of Liedang seeds.

Parasitic rate (%)=[number of parasitic tumors (pieces)/inoculated seed quantity (grain)] x100

The method for quickly inducing the germination of sunflower radang under indoor conditions provided by the invention can co-cultivate sunflower seedlings and sunflower radang in a square petri dish, induce the germination of sunflower radang and form a parasitic relationship with sunflower without cleaning Observe the germination process and parasitic structures under a microscope or with the naked eye. The invention achieves the purpose of simple, easy, and rapid induction of sunflower dandelion germination, can be applied in the experiment of identifying sunflower variety resistance level to dandelion, has strong practicability, and has strong popularization and application value.

Description of drawings

Fig. 1: the flow chart of the method for rapidly inducing sunflower rowan germination under a kind of indoor condition provided by the present invention;

Figure 2: Sunflower seedlings cultivated in square petri dishes;

Figure 3: Observing the germination of sunflower Liedang seeds under a microscope;

Figure 4: Formation of parasitic tumors;

Figure 5: Two different methods to identify the level of resistance to Lidan in different sunflower cultivars.

detailed description

Example 1

Fig. 1 has shown the flow chart of the method for rapidly inducing sunflower rowan germination under a kind of indoor condition provided by the present invention, and the operation method of the concrete steps of this method is as follows:

1. Disinfection and pretreatment of sunflower Liedang seeds

1.1. Cleaning of sunflower rowan seeds

The sunflower seeds collected from the field were passed through a sieve (diameter 0.18mm) to remove impurities. Collect the sunflower columns from which impurities have been removed and put them into a 1.5ml centrifuge tube (1/3 volume), then add distilled water, and centrifuge at 12000r/min for 15 minutes. After discarding the supernatant, spread the precipitate on the filter paper, dry it naturally, sweep it down with a brush, and store it in a 1.5ml centrifuge tube for later use.

1.2. Disinfection treatment of sunflower Liedang seeds

Add 1ml of 0.1% sodium hypochlorite (1.5ml centrifuge tube) to the cleaned sunflower seeds above, and let stand for 3 minutes after fully shaking. After centrifugation at 12000r/min, the supernatant was discarded, and 1ml of 75% alcohol was added to the sediment, shaken sufficiently, and allowed to stand for 3 minutes. After the supernatant was discarded by centrifugation, it was washed 3 times with sterile water. Pretreatment was carried out after the seeds were left to dry naturally.

1.3 Germination of sunflower seeds

Pick plump sunflower seeds and put them in a 9cm petri dish, add 5ml of 0.1% sodium hypochlorite to fully shake, and let stand for 3min. Then pour out the disinfectant, then add 5ml of 75% alcohol to the petri dish, shake it fully, and let it stand for 3 minutes. Rinse 3 times with sterile water after pouring off 75% alcohol and soak the sterilized sunflower seeds in sterile water. After 2 hours, take out the sunflower seeds and put them in a petri dish, and cover the sunflower seeds with moist gauze, then put a moist towel on the gauze for moisturizing. Spray water onto a towel once a day while moisturizing. After the sunflower seeds germinate (about 5 days), they can be used for the inoculation treatment of Liedang seeds.

1.4 Collection of sunflower root exudates

Rinse the pebbles with clean water 3 times (to ensure that there are no impurities and sand on them), put them into an aluminum lunch box, and sterilize them together with a 250ml jar at high temperature. Then, put 2/3 volume of sterilized pebbles in the sterilized jar. Inoculate the sunflower seedlings that have germinated in a jar, fix the root system with absorbent cotton, and then add sterilized water to completely submerge the sunflower root system. After culturing for three weeks, the sunflower root exudates were collected by filter sterilization, aliquoted into 50ml centrifuge tubes and stored at -4°C.

1.5 Pretreatment of sunflower rowdang seeds

Spread filter paper moistened with sterile water in a petri dish with a diameter of 9 cm, then spread the sterilized sunflower seeds as evenly as possible on the filter paper and completely wet the filter paper with the sunflower root exudates collected and preserved above. After sealing the petri dish with a parafilm, place it in a dark condition at 23-28°C for 7-10 days, and then use it to inoculate sunflowers.

2. Inoculation of sunflower rowdang seeds

Evenly spread the absorbent cotton in a 13×13cm plastic square dish, and then cover a layer of filter paper on it, soak the absorbent cotton and filter paper in the culture dish with distilled water and set aside. Put the pre-germinated sunflower seedlings into a petri dish (3 plants/dish), cover the root system of the seedlings with a small amount of moistened absorbent cotton (for fixing the sunflower seedlings), and cover the absorbent cotton with a layer of moist filter paper (guarantee The root system grows downward) (Figure 2). Place the petri dish upright in a light incubator (light at 28°C during the day, light intensity 66%; 18°C at night, keep dark conditions, and the relative humidity of the incubator is 50%). After 3-5 days, move the above pretreated sunflower rowdang seeds together with the filter paper into a petri dish with sunflower seedlings, seal the petri dish with a parafilm and place it in a light incubator for cultivation. Add distilled water every two days to keep the filter paper moist. Observation can be done after 1 week.

3. Observation of Germination of Liedang Seeds and Formation of Parasitic Tumors and Counting of Parasitic Rates

After 1 week of inoculation, observe the number of germinations of sunflower rowdang seeds in a 20X dissecting microscope (Fig. 3); after 3 weeks of inoculation, observe with the naked eye the formation of sunflower rowdang parasitic tumor-like structures (Fig. 4), and use the following formula to calculate column When the parasitic rate of seeds.

Parasitic rate (%)=[number of parasitic tumors (pieces)/inoculated seed quantity (grain)] x100

4. Accuracy of identification of sunflower antiretinoid level by petri dish filter paper method

In order to further confirm the accuracy of the identification results of the petri dish filter paper method, we identified the resistance levels of four sunflower varieties, Longshikui No. 2, LD5009 Longkuiza No. 2 and Chi KY11-23, under field and indoor conditions. , and compared the accuracy of the identification results of the Petri dish filter paper method. The results are shown in Figure 5. Under field conditions, the parasitism rates of Longshikui 2 and LD5009 were 89.0% and 91.0%, respectively, which belonged to Gaogan Liedang sunflower varieties; while the parasitization rates of Longkuiza 2 were 56%, respectively, which belonged to Ganliedang sunflower varieties; And Chi KY11-23 has a parasitic rate of 26.67%, which is a high-resistance variety. The identification results of the resistance levels of the above four sunflower varieties using the culture dish filter paper method showed that the parasitism rates of Longshikui 2 and LD5009 were 94.33% and 97.67% respectively; while Longkuiza 2 and Chi KY11- The 23 parasitic rates were 53.33% and 27.67%. The identification results of Petri dish filter paper method were in good agreement with the results of field identification. Therefore, we believe that the Petri dish filter paper method can accurately and quickly identify the resistance level of different sunflower varieties to Lietan under indoor conditions.

Claims (4)

1. a kind of method that rapid induction Helianthi Herba Orobanchess are sprouted under indoor conditionss, i.e. culture dish filter paper method, it is characterised in that have Following committed step：

(1) Sunflower Seedlings of advance accelerating germination are placed in the plastic square ware of the 13 × 13cm for being covered with filter paper and are cultivated；

(2) thing pretreatment is carried out with the root system secretion of Sunflower Seedlings after the cleaning of Helianthi Herba Orobanchess seed and sterilization；

(3) pretreatment good Helianthi Herba Orobanchess seed has been moved into Helianthi in company with filter paper by Helianthi Herba Orobanchess seed inoculation in the lump In the square culture dish of seedling, cultivate in being placed on illumination box after sealed by culture dish, addition distilled water is protected State of the card filter paper in moistening；

(4) counting of the observation and parasitic rate of the sprouting of Herba Orobanchess seed and the formation of thylacium.

2. the method that rapid induction Helianthi Herba Orobanchess are sprouted under the indoor conditionss described in claim 1, it is characterised in that use 13 Sunflower Seedlings and Herba Orobanchess seed are co-cultured the sprouting to induce Herba Orobanchess by the square culture dish of plastics of × 13cm.

3. the method that rapid induction Helianthi Herba Orobanchess are sprouted under the indoor conditionss described in claim 1, it is characterised in that Helianthi The pretreatment of Herba Orobanchess seed, by field gather Herba Orobanchess seed sieve after clean, after drying again with 0.1% sodium hypochlorite with 75% alcohol disinfecting, will pass through the Helianthi Herba Orobanchess seed disinfected and is uniformly laid on filter paper as far as possible and with searching above The Helianthi root exudates that collection is preserved moistens filter paper completely, is sealed after culture dish with sealed membrane, in 23-28 DEG C of dark condition It is lower to place 7-10 days, it is subsequently used for being inoculated with Helianthi.

4. application of the method described in claim 1 in the anti-Herba Orobanchess level identification of quick Helianthi, using above-mentioned indoor induction Helianthi Herba Orobanchess sprout system, and different Sunflower Varieties are carried out with the indoor identification of anti-Herba Orobanchess level, count posting for different cultivars Raw rate determines its anti-Herba Orobanchess level：

Parasitic rate (%)=[parasitic neoplastic quantity (individual)/inoculation Herba Orobanchess seed amount (grain)] x100.