Application of agrobacterium tumefaciens C53 in promoting green keeping of green plants

Abstract

The invention relates to application of agrobacterium tumefaciens C53 in promoting plant green keeping, and belongs to the technical field of microorganisms and application thereof, the classification name of the agrobacterium tumefaciens C53 is Agrobacterium tumefaciens strain C53, the agrobacterium tumefaciens C53 is preserved in China Center for Type Culture Collection on November 5, 2024, the preservation number is CCTCC NO: M 20242432, and the preservation number is CCTCC NO: M 20242432. The agrobacterium tumefaciens C53 obtained through separation promotes plant green keeping through the dual effects of generating cytokinin and promoting soil nitrification to promote plants to secrete cytokinin, and has the advantages of being low in production cost, convenient to use and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms and their applications, and relates to Agrobacterium tumefaciens for promoting green plants to remain green and an application thereof. Background Art

[0002] For green plants, staying green is not only beneficial to improving the nutritional value of plants such as green vegetables and fruits, but the time that plants such as flowers stay green is also an important factor affecting whether consumers are willing to buy them. How to keep green plants green for longer has become a key issue for researchers, and studying their post-harvest storage and preservation technology is of great significance. Therefore, it is crucial to focus on research on using soil bacteria to promote the greening of green plants.

[0003] Agrobacterium tumefaciens is widely present in natural environments such as soil and river sediments. It has multiple functions. Currently, there are many reports on the research of Agrobacterium tumefaciens in the fields of biological control and environmental protection, but there are no reports on its role in promoting the greening of green plants.

[0004] Invention Technology The technical objectives of the present invention are: 1. To provide an application of Agrobacterium tumefaciens C5_3 in promoting the greening of plants; 2. To provide a method for promoting the greening of green plants; the present invention explores the function of Agrobacterium tumefaciens C5_3 screened from the soil and finds that it has the effect of promoting the greening of green plants, thus providing a new microbial resource for keeping plants green and fresh.

[0005] In order to achieve the above object, the specific scheme adopted by the present invention is: application of Agrobacterium tumefaciens C5_3 in promoting plant greening, wherein the Agrobacterium tumefaciens C5_3 is classified and named Agrobacterium tumefaciens strain C5_3 was deposited in the China Center for Type Culture Collection on November 5, 2024, with the deposit number CCTCC NO: M20242432.

[0006] Furthermore, the green plant is wheat or peony.

[0007] A method for promoting green plants to stay green, inoculating soil with Agrobacterium tumefaciens C5_3, which is classified and named Agrobacterium tumefaciens strain C5_3 was deposited in the China Center for Type Culture Collection on November 5, 2024, with the deposit number CCTCC NO: M 20242432.

[0008] Furthermore, the green plant is wheat or peony.

[0009] Furthermore, the peony is a potted plant and is inoculated during the budding period of the peony seedlings.

[0010] Beneficial effects: The Agrobacterium tumefaciens C5_3 screened from the soil in the present invention can effectively promote green plants to remain green. The bacterial agent prepared with Agrobacterium tumefaciens C5_3 as raw material has the advantages of low production cost and easy use. It is suitable for use in areas where green plants such as vegetables, fruits, and flowers are planted, providing a new microbial resource for keeping plants green and fresh.

[0011] After Agrobacterium tumefaciens C5_3 is inoculated into the soil, the plant produces root cytokinins through nitrification. This process begins in the plant's root apical meristem, which is the key site for cytokinin production. −− When stimulated, this area increases the synthesis of cytokinins, which are then transported to the stems and leaves of the plant through the xylem sap, delaying leaf aging and enhancing photosynthesis, increasing leaf chlorophyll content, and keeping the plant green.

[0012] Bacterium deposit information: The Agrobacterium tumefaciens C5_3 described in the present invention is classified and named Agrobacterium tumefaciens strain C5_3 has been deposited in the China Center for Type Culture Collection. The deposit date is November 5, 2024. The deposit address is Wuhan University, Wuhan, China. The deposit number is CCTCC NO: M 20242432. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is a colony morphology of Agrobacterium tumefaciens C5_3, which has the function of promoting plant greening, in the isolation medium; Figure 2 This is a colony morphology diagram of Agrobacterium tumefaciens C5_3, which has the function of promoting plant greening, in heterotrophic LB medium; Figure 3 This is an optical microscope image of the colony morphology of Agrobacterium tumefaciens C5_3, which has the effect of promoting plant greening. DETAILED DESCRIPTION

[0014] The technical solution of the present invention is described more clearly and completely below in conjunction with the accompanying drawings and embodiments.

[0015] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0016] The inoculation amounts described in the following examples are all inoculated according to the corresponding volume ratios.

[0017] Example 1: Isolation and screening of Agrobacterium tumefaciens C5_3 that promotes plant greening 20g of soil from the experimental farm of Henan University of Science and Technology was taken and added to the enrichment medium, the formula of which was: (NH4)2SO40.5g, KH2PO40.75g, NaH2PO40.25g, MnSO4∙4H2O 0.01g, MgSO4∙7H2O 0.03 g, CaCO35.0 g, 2.5g agar, deionized water was added to 1000ml, pH value was 7.0-7.2, sterilized at 121℃ for 20 min, and cultured at 28℃ in the dark for 15-25 days. Then, Griess reagent was used to detect the presence of ammonia oxidizing bacterial strains, and the enrichment culture solution showing the growth of ammonia oxidizing bacterial strains was transferred to a new enrichment medium for continued culture. The process was repeated twice to obtain a pure bacterial enrichment solution.

[0018] Take 0.1ml of the enriched culture fluid obtained above, spread it on the agar plate separation medium, and culture it at 28℃ and in the dark for 7 days. The formula of the agar plate separation medium is: (NH4)2SO40.5g, KH2PO40.75g, NaH2PO40.25g, MnSO4∙4H2O 0.01g, MgSO4∙7H2O 0.03 g, CaCO35.0 g, 2.5g agar, deionized water to 1000ml, pH 7.0~7.2, sterilize at 121℃ for 20 minutes. After 7 days, select a single colony from the above separation medium and spread it on the agar plate separation medium. Repeat this process 5~10 times to obtain a plate culture medium containing pure bacterial colonies.

[0019] On the plate medium containing pure bacterial colonies obtained above, 5 to 10 pure colonies were transplanted into an enrichment medium (formula as above), and cultured at 28°C and in the dark for 15 days to obtain the microbial agent of Agrobacterium tumefaciens C5_3 described in this study. The above Agrobacterium tumefaciens with the function of promoting greening of green plants is named C5_3, and its classification is named Agrobacterium tumefaciens strain C5_3 was deposited in the China Center for Type Culture Collection on November 5, 2024, with the deposit number being CCTCC NO: M 20242432.

[0020] Example 2: Determination of the effect of Agrobacterium tumefaciens C5_3 producing cytokinins to promote wheat greening Select wheat seeds with full grains and uniform size, disinfect them with NaClO solution, rinse them with sterile water until there is no odor, absorb the surface moisture and sow them into culture dishes, cover them, and culture them in an incubator at 23°C for 24 to 36 hours. Discard the seeds that have not germinated or have germinated unevenly, and keep the seeds that have germinated evenly. Continue to culture. When the malt reaches the lid of the dish, remove the lid and continue to culture under light conditions.

[0021] Prepare bacterial fermentation broth, the formula of which is: 3 g beef extract, 10 g peptone, 5 g NaCl, and deionized water to make up to 1000 ml, with a pH value of 7.0-7.2.

[0022] Take 6 test tubes, add 5 ml of the prepared bacterial fermentation liquid to each tube, sterilize at 121°C for 30 minutes, and randomly divide the 6 test tubes into two groups, namely test group a and group b, with 3 replicates in each group. Inoculate one loop of the above strain into each tube of bacterial fermentation liquid in test group b, place in a shaker, and culture at 37°C, 180 rpm for 24 hours.

[0023] When the wheat grows to a height of 10 cm, cut off the first true leaf of the wheat and put it in sterile water for later use. Add 5 ml of sterile water to each tube of bacterial fermentation liquid in experimental group b and dilute it. Shake well for later use. Take another test tube of experimental group a and add 5 ml of sterile water to dilute it and shake well as a control.

[0024] Take out the wheat leaves in sterile water, cut them into small pieces of about 1 cm, and put them into the test tubes of test groups a and b, 5 pieces in each tube. After placing them in a dark room at 25℃ for 4 days, visually inspect the greenness of the leaves in 4 levels: dark green, light green, light yellow and yellow. Obtain the greenness of the leaves of test groups a and b, Ga0 and Gb0, and set them aside.

[0025] Comparing the sizes of Ga0 and Gb0 mentioned above, if Gb0 is greener than Ga0, it means that the Agrobacterium tumefaciens C5_3 strain itself can promote the greenness of green plants.

[0026] The results showed that Ga0 and Gb0 were yellow and light green respectively, and Gb0 was greener than Ga0, indicating that inoculation of A6 could help green plants stay green and fresh.

[0027] Example 3: Determination of the effect of Agrobacterium tumefaciens C5_3 on promoting the greening of peony leaves and its growth Six pots of peony seedlings in the budding stage with the same variety and growth state were selected, each pot had one peony, and the pots were filled with soil, and each kilogram of soil contained 45.8g of organic matter. The 10 pots of peonies were randomly divided into 2 groups, each with 3 pots, namely test groups a and b, with three replicates in each group.

[0028] 200 ml of the above-mentioned microbial agent was added to the soil of each pot in the experimental group b, and 200 ml of the above-mentioned pure enrichment solution was added to the soil of each pot in the experimental group a as a control. The peonies of the experimental groups a and b were placed in a greenhouse to promote flowering.

[0029] Sixty days later, the peony seedlings entered the flowering stage after the bud stage, and the chlorophyll contents of the peony flowers of the test groups a and b were measured, and the chlorophyll contents Ca0 and Cb0 of the test groups a and b were obtained for later use.

[0030] Compare the magnitudes of Ca0 and Cb0 above. If Ca0 < Cb0, it indicates that inoculating with strain C5_3 can promote the green retention of peony leaves by increasing the chlorophyll content.

[0031] The results show that Ca0 and Cb0 are 24.51 and 26.83 respectively, Ca0 < Cb0, and Cb0 is 9.47% higher than Ca0. It indicates that inoculating with strain C5_3 can promote the green retention of peony leaves by increasing the chlorophyll content.

[0032] It should be noted that the above-described embodiments should be understood as illustrative and not limiting the scope of protection of the present invention. The scope of protection of the present invention is subject to the claims. For those skilled in the art, without departing from the essence and scope of the present invention, some non-essential improvements and adjustments made to the present invention still fall within the scope of protection of the present invention.

Claims

1. Application of Agrobacterium tumefaciens C5_3 in promoting plant greening, characterized in that: The Agrobacterium tumefaciens C5_3 is classified and named Agrobacterium tumefaciens strain C5_3 was deposited in the China Center for Type Culture Collection on November 5, 2024, with the deposit number being CCTCC NO: M 20242432.

2. The use of Agrobacterium tumefaciens C5_3 in promoting plant greening according to claim 1, characterized in that: The green plants are wheat or peony.

3. A method for promoting green plants to remain green, characterized in that: Inoculate soil with Agrobacterium tumefaciens C5_3, which is classified as Agrobacterium tumefaciens strain C5_3 was deposited in the China Center for Type Culture Collection on November 5, 2024, with the deposit number CCTCC NO: M 20242432.

4. A method for promoting green plants to remain green according to claim 3, characterized in that: The green plants are wheat or peony.

5. A method for promoting green plants to remain green according to claim 4, characterized in that: The peony is potted and inoculated during the budding period of the peony seedlings.

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