Application of 6-benzylaminopurine in promoting growth of rhizobium
By soaking alfalfa seeds with 6-benzyl aminopurine solution at a concentration of 15 to 17 mg·L-1, the growth and proliferation of endogenous rhizobia in alfalfa was promoted, and the problem of insufficient research on rhizobia growth in the prior art was solved, and the problem of improving the Plant nitrogen fixation efficiency.
Patent Information
- Application Number
- CN202411489168.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-24
- Publication Date
- 2025-02-11
AI Technical Summary
The prior art studies on the proliferation and proliferation effects of rhizobia, which is difficult to effectively promote its growth and affects the nitrogen fixation efficiency of plants.
Alfalfa seeds were soaked in 15-17 mg·L-1 concentration of 6-benzyl aminopurine solution and planted in nutrient soil to promote the growth and proliferation of rhizobia endogenous alfalfa.
Through this method, the proliferation and growth of endogenous rhizobia in alfalfa plants was significantly promoted, and new ideas for nitrogen fixation for legume plants were provided.
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Abstract
Description
Technical Field
[0001] The invention relates to the technical field of plant endophytes, and in particular to application of 6-benzylaminopurine in promoting the growth of rhizobia. Background Art
[0002] Viola yedoensis, also known as wild violet, light-petal violet, etc. It is a perennial herb belonging to the order Paramecia, Violaceae, without an above-ground stem, 4 to 14 cm high, and can reach more than 20 cm in fruiting period. The lower part of the leaf is triangular ovate or narrow ovate, and the upper part is longer, oblong, narrow ovate-lanceolate or oblong-ovate. The flower is medium-sized, purple or lavender, rarely white, and the throat color is lighter with purple stripes; the capsule is oblong, 5 to 12 mm long, and the seeds are ovoid, 1.8 mm long, and light yellow. The flowering and fruiting period is from mid-to-late April to September. The whole plant is used for medicinal purposes, which can clear away heat and detoxify, cool blood and reduce swelling; the young leaves can be used as wild vegetables. It can be used as an ornamental flower in early spring.
[0003] Biological nitrogen fixation refers to the process by which nitrogen-fixing microorganisms convert free nitrogen in the atmosphere into nitrogen-containing compounds, which then become nitrogen that can be absorbed by plants. There are three main types of biological nitrogen fixation in nature: symbiotic nitrogen fixation, autogenic nitrogen fixation, and combined nitrogen fixation. Among them, the symbiotic nitrogen fixation system formed by legumes and rhizobia is considered to be the most environmentally friendly, economically effective, and sustainable way for plants to obtain nitrogen. Rhizobia is a symbiotic nitrogen-fixing bacterium widely distributed in the soil. It can form nodules with plants and can fix inorganic nitrogen in the atmosphere into combined nitrogen that can be absorbed and utilized by plants.
[0004] Cytokinin is a general term for a class of plant growth substances that have physiological functions such as promoting cell division. Cytokinin can promote cell division and expansion, promote flowering in short-day plants, and promote flowering in long-day plants. Cytokinin can also promote lateral bud germination, break apical dominance, delay aging, and prolong flowering period. However, there are few studies on the proliferation and growth-promoting effects of cytokinin on rhizobia. Summary of the invention
[0005] The purpose of the present invention is to provide the application of 6-benzylaminopurine in promoting the growth of rhizobia.
[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:
[0007] The invention provides application of 6-benzylaminopurine in promoting the growth of rhizobia.
[0008] Preferably, the concentration of 6-benzylaminopurine is 15-17 mg·L -1 .
[0009] The present invention also provides the use of 6-benzylaminopurine in promoting the growth and / or proliferation of endophytic rhizobia of alfalfa, wherein the concentration of the 6-benzylaminopurine is 15-17 mg·L -1 .
[0010] The present invention also provides a method for promoting the growth and / or proliferation of endophytic rhizobia of alfalfa, comprising the following steps:
[0011] (1) soaking alfalfa seeds containing rhizobia in a 6-benzylaminopurine solution;
[0012] (2) Planting the alfalfa seeds from step (1) in nutrient soil.
[0013] Preferably, the concentration of the 6-benzylaminopurine solution is 15 to 17 mg / L -1 .
[0014] Preferably, the soaking time is 1.5 to 2.5 hours.
[0015] The invention also provides the use of 6-benzylaminopurine in promoting nitrogen fixation in plants.
[0016] Preferably, the plant comprises a plant from the family Leguminosae.
[0017] Preferably, the leguminous plant includes alfalfa.
[0018] By adopting the above technical solution, the present invention has the following beneficial effects: The present invention has found through experiments that 15-17 mg·L -1 The 6-benzylaminopurine solution can effectively promote the proliferation and growth of endophytic rhizobia in alfalfa plants. The present invention also provides a method for promoting the growth and / or proliferation of endophytic rhizobia in alfalfa, and the specific method is: soaking alfalfa seeds containing rhizobia in a 6-benzylaminopurine solution, and then planting the alfalfa seeds in nutrient soil. The method can effectively promote the proliferation and growth of endophytic rhizobia in alfalfa plants, and provides a new idea for nitrogen fixation in leguminous plants. DETAILED DESCRIPTION
[0019] The present invention provides the use of 6-benzylaminopurine in promoting the growth of rhizobia. Preferably, the concentration of the 6-benzylaminopurine is 15-17 mg·L -1 , more preferably 15.5 to 16.5 mg·L -1 , more preferably 16 mg·L -1 .
[0020] The present invention also provides the use of 6-benzylaminopurine in promoting the growth and / or proliferation of endophytic rhizobia of alfalfa, wherein the concentration of the 6-benzylaminopurine is 15-17 mg·L -1, more preferably 15.5 to 16.5 mg·L -1 , more preferably 16 mg·L -1 .
[0021] The present invention also provides a method for promoting the growth and / or proliferation of endophytic rhizobia of alfalfa, comprising the following steps:
[0022] (1) soaking alfalfa seeds containing rhizobia in a 6-benzylaminopurine solution;
[0023] (2) Planting the alfalfa seeds from step (1) in nutrient soil.
[0024] In the present invention, firstly, alfalfa seeds are soaked in iodine solution for disinfection, wherein the effective iodine content of the iodine solution is 0.45-0.55%, more preferably 0.48-0.53%, more preferably 0.50%, the soaking time is 2-4 minutes, more preferably 2.5-3.5 minutes, more preferably 3 minutes, and after soaking, the seeds are washed with water for 3-5 times, more preferably 4 times, the water is preferably sterile water, and the disinfection of the alfalfa seeds is preferably carried out in a sterile operating table.
[0025] The present invention soaks the sterilized seeds in a 6-benzylaminopurine solution, wherein the concentration of the 6-benzylaminopurine solution is 15-17 mg·L -1 , more preferably 15.5 to 16.5 mg·L -1 , more preferably 16 mg·L -1 ; The treatment time is 1.5 to 2.5 hours, more preferably 2 hours.
[0026] Then the soaked seeds are planted in a cultivation container filled with nutrient soil. Preferably, a double layer of non-woven fabric is placed in the cultivation container to prevent soil loss in the cultivation container and ensure permeability. When the alfalfa plants grow to the branching stage, the alfalfa plants are transplanted from indoors to outdoors for planting.
[0027] The present invention also provides the use of 6-benzylaminopurine in promoting nitrogen fixation in plants; preferably, the plants include leguminous plants; preferably, the leguminous plants include alfalfa.
[0028] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.
[0029] The 6-benzylaminopurine (6-BA) (cytokinin) used in the present invention was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.
[0030] TY solid medium: pancreatic peptone 5.0 g·L -1, yeast powder 3.0g·L -1 , CaCL2·H2O 1.3g·L -1 , agar 16.0 g·L -1 , distilled water 1000mL, pH=7.0.
[0031] TY liquid medium: pancreatic peptone 5.0 g·L -1 , yeast powder 3.0g·L -1 , CaCL2·6H2O 1.3g·L -1 , distilled water 1000mL, pH=7.0.
[0032] YMA solid medium: K2HPO4·3H2O 0.5 g·L -1 , MgSO4·7H2O 0.2g·L -1 , NaCl0.1g·L -1 , mannitol 10g·L -1 , yeast powder 1g·L -1 , concentration 2.5 g·L -1 Congo red solution 10mL·L -1 , agar 15 g·L -1 , distilled water 1000mL, pH=7.0.
[0033] Example 1 Preparation of Cyan Fluorescent Protein Labeled Sinorhizobium Strain
[0034] Cyan fluorescent protein (CFP) is a mutant of green fluorescent protein gfp. After the encoding gene is expressed, it does not damage the host cell and does not require a substrate. It is suitable for in vivo detection, has low energy consumption, and can produce brighter blue-green or cyan fluorescence. It is used to detect the nodule occupancy rate of rhizobia and trace rhizobia infection and nodulation. The process is intuitive, accurate and easy to operate. Therefore, the Chinese rhizobium strain Ensifermeliloti LZgn5f (R.gn5f) labeled with cyan fluorescent protein was used for the experiment. Its original strain is the endogenous rhizobium of alfalfa Ensifermeliloti LZgn5. By incubating the culture medium with 50 μg·mL of gentamicin (Gm), the CFP-labeled Rhizobium sinensis strain Ensifermeliloti LZgn5f was used to detect the nodule occupancy rate of rhizobia and trace the nodule infection of rhizobia. The process is intuitive, accurate and easy to operate. -1 and spectinomycin (Spe) 50 μg·mL -1 Activate the donor bacteria E. coli 4517 and the helper bacteria E. coli 2073 in LB solid medium and culture at 28℃ for 16-18h. Transfer the strains to LB solid medium containing 50μg mL -1 Gm and 50 μg mL -1 Spe in LB liquid medium, 37°C, 120 r·min -1 The culture was shaken until the bacterial growth logarithmic phase (OD600 =0.5). 1.5 mL of each of the bacterial liquids of the donor bacteria, the auxiliary bacteria, and the recipient bacteria (competent cells of Ensifer meliloti LZgn5) were mixed and incubated at 8000 r.min. -1 Centrifuge for 5 min to obtain a mixed bacterial precipitate. Discard the supernatant and add 1 mL of antibiotic-free TY liquid medium to the precipitate. Shake well and rotate at 8000 r / min. -1 Centrifuge for 5 minutes, discard the supernatant and keep the mixed bacterial precipitate, then take a 0.22μm sterile filter membrane and stick it on the surface of the TY solid plate, 3 pieces per dish; take the bacterial precipitate and add 1mL sterile water to break it into a concentrated bacterial solution, add 0.2mL of concentrated bacterial solution in the center of the filter membrane, place it upright at 28℃ for 2h, and then invert it for culture. After 2-3 days, take the filter membrane with the culture grown and put it into a sterile cillin bottle, add 5mL sterile water to wash the bacteria, dilute it 10 times with sterile water, take 0.2mL of bacterial solution and smear it on the SM+Gm solid plate, and culture it at 28℃ for 7 days, pick 50 single colonies on the plate, number them and spot-inoculate them on antibiotic-free TY medium and nitrogen-free medium respectively, and culture them at 28℃ until colonies appear. The colonies were detected by WFH-204 ultraviolet analyzer at 336nm, and the cultures of single colonies that could emit cyan fluorescence were selected and transferred to Winogradsky nitrogen-free medium. The strains that could grow normally on the nitrogen-free plates were rhizobia labeled with cyan fluorescent protein and are now preserved in the Key Laboratory of Grassland Ecosystems of the Ministry of Education at Gansu Agricultural University.
[0035] Example 2
[0036] The seeds used in the present invention are the third generation seeds containing fluorescently labeled endophytic rhizobium R.gn5f. The first generation seeds are Medicagosativa L.Gannong No.5 grown for 4 years at the forage training base of Gansu Agricultural University. During the pod-setting period, the main root is slightly damaged and the root is directly watered, and 600 mg mL -1 The three root inoculation methods were inoculated with fluorescently labeled rhizobia Ensifermeliloti LZgn5f (gn5f) by irrigating with matrine (concentration 1.3%), and the roots were irrigated with sterile distilled water at the same time. The primary seeds were sown and the secondary seeds were obtained. The secondary seeds were sown again and the third-generation alfalfa seeds containing fluorescently labeled endophytic rhizobia R.gn5f were harvested and named A.GN5+R.gn5f. It was determined that the number of fluorescently labeled endophytic rhizobia R.gn5f contained in the third-generation seeds was 10-20 cfu / seed. -1 .
[0037] Example 3 Growth-promoting effect of 6-BA on rhizobia
[0038] The fluorescently labeled rhizobium strain R.gn5f stored in glycerol at -80°C was taken out and inoculated into TY solid plate medium for activation culture. Then a single colony was picked and transferred into TY liquid medium and placed at a speed of 120 r·min. -1 , cultured in a shaking incubator at 28°C until OD 600nm =1×10 9 cfu·mL -1 .
[0039] 5 mg of 6-BA was placed in a sterile conical flask, dissolved with a small amount of alcohol and filtered three times with a sterile filter membrane (diameter 0.22 μm), and then the volume was adjusted to 10 mL to prepare a mother solution. The mother solution was then used to prepare 12, 14, 16, 18 and 20 mg·L -1 of 6-BA solution.
[0040] Add the prepared 6-BA solution to 40 mL of bacterial solution and set the speed to 120 r / min. -1 The culture was carried out in a shaker at 28°C. The number of viable bacteria in each treatment solution was measured on the first, third, fifth and seventh days, and the hormone concentration most suitable for the growth of endophytic rhizobium R.gn5f was selected. The solution of distilled water was used as the control. The results are shown in Table 1.
[0041] Table 1 Effects of different concentrations of hormone 6-BA on rhizobium R.gn5f
[0042]
[0043] Note: The data in the table are mean ± standard error. Different lowercase letters in the same column indicate significant differences (P<0.05).
[0044] From Table 1, it can be seen that the appropriate concentration of 6-benzylaminopurine (6-BA) has a promoting effect on the growth of fluorescently labeled rhizobium R.gn5f. -1 The 6-BA in the first day (OD 600nm =1.04) to 3d (OD 600nm =1.32) had the most significant effect on the growth of Rhizobium R.gn5f; on the 5th day, 12 mg·L -1 (OD 600nm =1.38) had the strongest promoting effect, followed by 16 mg·L -1 (OD 600nm =1.35) of 6-BA, its promoting effect was significantly higher than that of the control group (OD 600nm =1.01), which was 33.66% higher than that of the control group; at 7 days, 12 mg·L -1 (OD 600nm =1.37) of 6-BA and 16 mg·L-1 (OD 600nm =1.37) had the most significant promoting effect of 6-BA. 600nm =1.01) were significantly increased by 35.64%. -1 The 6-BA helps the growth of rhizobium gn5f.
[0045] Example 4 Effect of 6-BA on the Growth of Rhizobia in Alfalfa Plants
[0046] The experiment was conducted in the greenhouse of Gansu Agricultural University and the Lanzhou Forage Experiment Station of Gansu Agricultural University. Alfalfa seeds containing cyan fluorescently labeled endophytic rhizobia were placed in a sterilized 50mL conical flask, soaked in iodophor (purchased from Lanzhou Boyu Biotechnology Co., Ltd., with an effective iodine content of 0.45%-0.55%) for 3 minutes, and washed with sterile water 4 times, each time for 1 minute. All the above operations were carried out in a sterile operating table. The disinfected seeds were soaked in a concentration of 16mg·L -1 The cells were treated with 6-BA for 2 h, and distilled water (CK) was used as the control.
[0047] The nutrient soil was placed in a flower pot with a diameter of 18 cm, a height of 13 cm, and a volume of 2.6 L. A double layer of non-woven fabric was placed in the pot to prevent soil loss and ensure permeability. Twenty sterilized seeds were sown in each cup, and the flower pots were placed in the greenhouse of Gansu Agricultural University for growth. After the alfalfa seedlings grew true leaves, 15 seedlings were planted in each cup, and each treatment was repeated 6 times. Sterile distilled water was used to replenish water during the growth of the seedlings. After indoor planting until the branching stage, they were transplanted to outdoor planting.
[0048] Sampling was carried out at the budding stage, flowering stage, pod-setting stage and maturity stage of alfalfa. The sampling tissues were roots, stems, leaves, flowers and ovaries in the flowering stage; roots, stems, leaves and pods in the pod-setting stage, and roots, stems, leaves and mature seeds in the maturity stage. Three plants were randomly selected for each treatment. Separate each tissue and weigh 1g (25 mature seeds) respectively, place it in a sterile 50mL triangular bottle, immerse each part in medical iodine tincture for 2 minutes in the clean bench, and then rinse it with sterile water 4 times until the rinse liquid is clear and transparent without foam. Place each surface of the sterilized plant tissue on the solid culture medium for 30 minutes and then take it out. The culture medium was placed at 28℃ for 48 hours. No colonies grew, indicating that the surface of the plant tissue had been thoroughly disinfected. Put each sterilized tissue in a sterile mortar, add 2mL of sterile water, grind it thoroughly, pour the grinding liquid into a 2mL sterilized centrifuge tube, and centrifuge it at 8000r·min -1 Centrifuge for 20 minutes, take 0.2 mL of the supernatant and apply it to YMA solid medium containing Congo red (after taking the supernatant from the root, use sterile water to make 10 -1 , 10 -2 , 10-3 dilution) and cultured at 28°C for 48 hours. After the culture was completed, the number of cyan fluorescent rhizobium colonies was observed and recorded under dark conditions using a portable ultraviolet lamp (wavelength 336nm), and the number of fluorescent rhizobia per gram of plant tissue was calculated. The results are shown in Table 2.
[0049] Table 2 Number of labeled endophytic rhizobia in each tissue
[0050]
[0051]
[0052]
[0053] Note: The data in the table are mean ± standard error. Different lowercase letters in the same row indicate significant differences (P<0.05).
[0054] As shown in Table 2, at the bud stage of alfalfa, the 6-BA treatment group had a -1 ) and when the root grinding solution was diluted 1000 times (1666.67 cfu·g -1 ) was significantly higher than that of the control group (p<0.05). When the root grinding solution was diluted 100%, the 6-BA treatment group (833.33 cfu·g -1 ) was significantly higher than that of the control group (166.67 cfu·g -1 )(p<0.05), and significantly increased by 399.99% compared with the control group.
[0055] At the flowering stage, the 6-BA treatment group had a high concentration of 3.33 cfu·g -1 ) and flower tissue (25.00cfu·flower -1 ) was significantly higher than that in the control group (p < 0.05), and significantly increased by 99.40% and 400% compared with the control group; in the leaves (3.33 cfu·g -1 ) was significantly higher than that of the control group (p < 0.05). In the case of root dilution of 1000, the number of labeled endophytic rhizobia in the roots of the 6-BA treatment group (1666.67 cfu·g -1 ) were significantly higher than those in the control group.
[0056] At the pod-setting stage, the 6-BA treatment group had a significant increase in the number of leaves (5.00 cfu·g -1 ) and pods (680.00 cfu·g -1) tissues were significantly higher than those in the control group (p < 0.05), and in the pods they were significantly increased by 148.78% compared with the control group; in other tissues, the levels of labeled endophytic rhizobia in the 6-BA treatment group were not significant compared with those in the control group.
[0057] At maturity, the labeled endophytic rhizobia in the 6-BA treated group were not significantly different from those in the control group, but in the seeds, the 6-BA treated group (33.33 cfu·seed -1 ) was higher than that of the control group (25.00 cfu·seed -1 ), so the appropriate concentration of 6-BA has a promoting effect on the labeled endophytic rhizobia in seeds.
[0058] As can be seen from the above examples, the present invention provides the use of 6-benzylaminopurine in promoting the growth of rhizobia. 6-benzylaminopurine promotes the proliferation and growth of endophytic rhizobia in alfalfa plants.
[0059] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.
Claims
1. Application of 6-benzylaminopurine in promoting the growth of rhizobia.
2. The use according to claim 1, characterized in that: The concentration of 6-benzylaminopurine is 15-17 mg·L -1 .
3. Use of 6-benzylaminopurine in promoting the growth and / or proliferation of endophytic rhizobia of alfalfa, wherein the concentration of 6-benzylaminopurine is 15-17 mg·L -1 .
4. A method for promoting the growth and / or proliferation of endophytic rhizobia of alfalfa, characterized in that: The following steps are involved: (1) soaking alfalfa seeds containing rhizobia in a 6-benzylaminopurine solution; (2) Planting the alfalfa seeds from step (1) in nutrient soil.
5. The method according to claim 4, characterized in that The concentration of the 6-benzylaminopurine solution is 15-17 mg·L -1 .
6. The method according to claim 4, characterized in that The soaking time is 1.5 to 2.5 hours.
7. Application of 6-benzylaminopurine in promoting nitrogen fixation in plants.
8. The use according to claim 7, characterized in that: The plants include plants of the family Leguminosae.
9. The use according to claim 8, characterized in that: The legumes include alfalfa.