Method for improving photosynthetic characteristics and biomass of equisetum arvense and equisetum arvense cultivation method

Abstract

The present application belongs to the technical field of plant cultivation, and particularly relates to a method for improving photosynthetic characteristics and biomass of Ilex asprella and a method for cultivating Ilex asprella. The present application uses any one of three methods of seed soaking, seed irrigation and seed germination for treating Ilex asprella seeds, and inoculates rhizobium, so as to improve the photosynthetic characteristics and biomass of Ilex asprella. The method for cultivating Ilex asprella with high photosynthetic characteristics and biomass comprises the following steps: 1) separating rhizobium; 2) preparing rhizobium liquid with sterile water; 3) treating Ilex asprella seeds by seed germination with the rhizobium liquid to obtain Ilex asprella germinated seeds treated by seed germination with the rhizobium liquid; and 4) culturing the Ilex asprella germinated seeds to obtain Ilex asprella with high photosynthetic characteristics and biomass. The Ilex asprella seedlings treated by seed germination with the rhizobium liquid have higher photosynthetic characteristics and biomass. The present application determines the influence of different inoculation methods on the photosynthetic characteristics and biomass of Ilex asprella seedlings, and provides technical support for cultivating high-quality Ilex asprella.

Description

[0001] Priority application

[0002] This application claims priority to Chinese invention patent application filed on December 29, 2023, with application number 202311845021.9 and entitled "Method for Improving Photosynthetic Characteristics and Biomass of *Hippophae rhamnoides* and Method for Cultivating *Hippophae rhamnoides*", the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention belongs to the field of plant cultivation technology, specifically relating to a method for improving the photosynthetic characteristics and biomass of *Hippophae rhamnoides*, as well as a method for cultivating *Hippophae rhamnoides*. Background Technology

[0004] *Indigofera ferseudotinctoria* Matsum., a semi-shrub belonging to the genus *Indigofera* in the legume family, is widely distributed in southwestern my country. *Indigofera ferseudotinctoria* possesses advantages such as drought tolerance, heat tolerance, tolerance to poor soil, a well-developed root system, strong growth, high biomass, and good palatability. Its plant contains a high level of crude protein and is rich in trace elements and amino acids, meeting the standards of typical high-quality protein feed. It has great potential for utilization in southern regions lacking high-protein forage. In recent years, due to its long flowering period and the fact that its pods do not easily fall off after leaf fall, *Indigofera ferseudotinctoria* has been widely used in urban greening, wetlands, parks, and slope protection.

[0005] Rhizobia can exist in plant tissues as endophytes, forming unique microenvironments. Current research confirms the presence of rhizobia in legume tissues. Rhizobia can form a symbiotic relationship with legumes, forming nodules in the roots to fix nitrogen and promote growth. Endophytic rhizobia can synthesize compounds that promote plant growth, improve water and nutrient absorption, jointly fix nitrogen, and enhance plant stress resistance. In existing research, Chi Feng et al. demonstrated that rhizobia can invade and colonize plant roots and migrate from the plant interior to the stems and leaves. Within the host legume, rhizobia can exist in a symbiotic state and as an endophytic ecotype, distributed in the root nodules and throughout the plant body. Zhang Shuqing et al. also demonstrated through experiments that rhizobia can enter alfalfa roots and migrate to various parts of the stem, increasing its biomass. Legume growth requires rhizobia; therefore, inoculation with rhizobia can promote legume growth.

[0006] However, most current research focuses on the germination of *Hippophae rhamnoides* seeds, and there are few reports on the effects of inoculation on the photosynthetic characteristics and biomass of *Hippophae rhamnoides* seedlings. Furthermore, the effects of different inoculation methods of *Hippophae rhamnoides* strains on the photosynthetic characteristics and biomass of *Hippophae rhamnoides* seedlings are still unclear. Summary of the Invention

[0007] In view of this, this invention studies the effects of different inoculation methods of *Hippophae rhamnoides* strains on the photosynthetic characteristics and biomass of *Hippophae rhamnoides*, clarifies the impact of inoculation methods on the biomass of *Hippophae rhamnoides*, and screens out effective inoculation methods that are beneficial to improving the photosynthetic characteristics and biomass of *Hippophae rhamnoides*, providing technical support for achieving high-quality and high-yield production of *Hippophae rhamnoides*.

[0008] One of the objectives of this invention is to provide a method for improving the photosynthetic properties and biomass of *Hippophae rhamnoides*.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] To improve the photosynthetic characteristics and biomass of *Hippophae rhamnoides*, the seeds were treated with one of three methods: seed soaking in bacterial solution, irrigation with bacterial solution, or bud soaking in bacterial solution, and then inoculated with rhizobia.

[0011] Preferably, the rhizobium has the accession number CCTCC NO:M20232454.

[0012] As a preferred method, the seeds of *Hippophae rhamnoides* are treated by soaking the buds in a fungal solution and then inoculated with rhizobia.

[0013] Furthermore, the bacterial solution soaking includes: germinating seeds using the petri dish filter paper method, soaking the germinated seeds in a rhizobium solution to obtain broomcorn germinated seeds treated with bacterial solution soaking.

[0014] Furthermore, the soaking time in the bacterial solution is 0.5-1 hour, preferably 0.5 hours.

[0015] Furthermore, the germinated seeds are inoculated with rhizobia when the radicle is 3-4 cm long.

[0016] Furthermore, the seed soaking in the bacterial solution includes: soaking the disinfected seeds in a rhizobium solution for 0.5-1 hour, then removing and planting them. The soaking time is preferably 0.5 hours.

[0017] Furthermore, the bacterial solution irrigation includes: after the *Hippophae rhamnoides* seedlings have grown true leaves, adding the rhizobium solution to the roots of the seedlings under sterile conditions.

[0018] Furthermore, during the bacterial solution irrigation, no bacterial solution is applied to any part of the seedlings other than the root system.

[0019] Furthermore, in the bacterial solution irrigation, the amount of Rhizobium bacterial solution added is 25-40 mL, more preferably 30 mL.

[0020] The second objective of this invention is to provide a method for cultivating horsehair with high photosynthetic characteristics and biomass.

[0021] To achieve the above objectives, the present invention adopts the following technical solution:

[0022] The cultivation method of *Hippophae rhamnoides* with high photosynthetic characteristics and biomass includes the following steps:

[0023] (1) Isolation of rhizobia;

[0024] (2) Use sterile water to prepare the rhizobium inoculum solution;

[0025] (3) The seeds of *Hippophae rhamnoides* were treated with a fungal solution to obtain germinated *Hippophae rhamnoides* seeds treated with a fungal solution.

[0026] (4) Cultivate the germinated seeds of horsehair obtained in step (3) to obtain horsehair with high photosynthetic characteristics and biomass.

[0027] Furthermore, in step (1), the rhizobium is shaped as a round protrusion and has an opaque white or watery, translucent milky white appearance; the colony diameter is 5-8 mm, preferably 6 mm.

[0028] Further, step (2) includes: activating, culturing, centrifuging, and washing the rhizobia obtained in step (1), and then preparing a rhizobia culture solution with sterile water.

[0029] Furthermore, the culture medium was TY liquid medium.

[0030] Furthermore, the culture conditions were 160 r / min and 28℃ in a shaker.

[0031] Preferably, step (2) includes the following steps: after activating the rhizobia, transfer them to TY liquid medium and culture them in a shaker at 160 r / min and 28°C until the optical density value (OD) reaches the specified value. 600 When the bacterial count (value) is ≥0.5, centrifuge at 10000 r / min for 10 min, discard the supernatant, wash off the bacterial cells with sterile water, shake well to disperse, and then prepare OD200 with sterile water. 600 Bacterial suspension with a value of 0.5.

[0032] Furthermore, in step (4), after the germinated seeds of *Hippophae rhamnoides* are sown, nitrogenous nutrient solution is used to supplement nutrients and sterile water is used to supplement moisture.

[0033] As a preferred option, step (4) includes the following steps: taking samples on the 45th day after inoculation; washing and sterilizing fine sand three times, then placing it into a plastic cup with mesh holes at the bottom and putting it into a hydroponic box; sowing 30 treated seeds (disinfected seeds, seed soaked in bacterial solution, and germinated seeds soaked in bacterial solution) in each cup, and covering the surface with about 2cm of dry sand; adding Hoagland nitrogen-containing nutrient solution to each flowerpot once, and watering with sterile water before germination; watering with Hoagland nitrogen-free nutrient solution once every 15 days after germination, and watering with sterile water during this period.

[0034] As a preferred method, the sterilization conditions are: high-temperature sterilization at 121℃ for 26 minutes.

[0035] The beneficial effects of this invention are as follows:

[0036] This invention found that different inoculation methods resulted in variations in the photosynthetic characteristics and biomass of *Hippophae rhamnoides* seedlings. Specifically, the aboveground fresh weight, underground fresh weight, aboveground dry weight, and underground dry weight of *Hippophae rhamnoides* seedlings treated with rhizobium inoculum solution increased by 202.37%, 207.07%, 218.07%, and 201.75% respectively compared to CK3; transpiration rate, stomatal conductance, photosynthetic rate, and intercellular CO2 concentration increased by 91.95%, 134.83%, 142.64%, and 10.37% respectively compared to CK3; and chlorophyll a content, chlorophyll b content, carotenoid content, and chlorophyll a+b content increased by 37.93%, 9.74%, 80.82%, and 74.29% respectively compared to CK3. These results were significantly better than those achieved by seed soaking and irrigation with rhizobium inoculum solution. Attached Figure Description

[0037] Figure 1 The figures show the results of the detection of the effect of Rhizobium inoculum sapling soaking on the biomass of *Hippophae rhamnoides*. In the figures, A is the aboveground fresh weight of *Hippophae rhamnoides* seedlings after Rhizobium inoculum sapling soaking, B is the underground fresh weight of *Hippophae rhamnoides* seedlings after Rhizobium inoculum sapling soaking, C is the aboveground dry weight of *Hippophae rhamnoides* seedlings after Rhizobium inoculum sapling soaking, and D is the underground dry weight of *Hippophae rhamnoides* seedlings after Rhizobium inoculum sapling soaking.

[0038] Figure 2 The figures show the results of the detection of the effect of Rhizobium inoculum sapling soaking on the photosynthetic characteristics of Hippophae rhamnoides seedlings. In the figures, A is the detection result of the transpiration rate of Hippophae rhamnoides seedlings after Rhizobium inoculum sapling soaking, B is the detection result of the stomatal conductance of Hippophae rhamnoides seedlings after Rhizobium inoculum sapling soaking, C is the detection result of the net photosynthetic rate of Hippophae rhamnoides seedlings after Rhizobium inoculum sapling soaking, and D is the detection result of the intercellular CO2 concentration of Hippophae rhamnoides seedlings after Rhizobium inoculum sapling soaking.

[0039] Figure 3 The figure shows the results of the detection of the effect of Rhizobium inoculum slurry soaking on the chlorophyll content of Hippophae rhamnoides. Detailed Implementation

[0040] The technical solution of the present invention will be described more clearly and completely below with reference to specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0041] In this embodiment of the invention, the preservation information of Rhizobium R3 is as follows:

[0042] Culture name: Rhizobimindigoferae 1-21; Accession number: CCTCC NO: M20232454; Depository institution: China Center for Type Culture Collection; Deposit address: Wuhan University, Luojia Mountain, Wuchang District, Wuhan City, Hubei Province; Deposit date: December 4, 2023; Applicant institution: Institute of Grassland Science, Chongqing Academy of Animal Sciences; Applicant: Zhu Ruifen.

[0043] Example 1

[0044] 1. Isolation of Rhizobia: In May 2022 (flowering period), root nodules were randomly selected from the roots of 5 plants in the *Hippophae rhamnoides* cultivar plot at the Shuanghe Forage Experimental Base in Rongchang District, Chongqing. The sterilized nodules were placed in a sterile mortar, 2 mL of sterile water was added, and the mixture was thoroughly ground. The resulting mortar was then poured into a 5 mL sterile centrifuge tube and centrifuged at 4000 rpm. -1 Centrifuge for 5 minutes, then prepare a solution of 10 μL with sterile water. -1 10 -2 10 -3 Dilute the solution, and spread 0.2 mL of the supernatant onto nitrogen-free solid medium. Incubate at 28°C for 6-7 days. Select single colonies, streak them onto YMA Congo Red solid medium, and incubate at 28°C for 48 hours. Select typical strains that do not absorb pigment and conform to the morphological characteristics of rhizobia (round colonies with smooth edges, raised center, and relatively flat shape; colonies are opaque white or watery translucent milky white) for isolation and purification. Streak normally growing single colonies onto YMA Congo Red solid medium and incubate at 28°C for 48 hours. Select normally growing single colonies on YMA solid medium for streak isolation, purification, and culture until single colonies are formed. Measure and record the colony diameter, morphology, appearance, and viscosity. Number the strains that conform to the morphological characteristics (R1-R5), as shown in Table 1. In YMA liquid medium at 180 rpm... -1 The culture was expanded and cultured on a shaker at 28°C for 18 hours, and then stored in an ultra-low temperature freezer at -80°C with glycerol.

[0045] Table 1. Morphological characteristics of strains

[0046]

[0047]

[0048] 2. Preparation of Rhizobium inoculum: A strain of Rhizobium R3, taken from an ultra-low temperature freezer, was activated and transferred to TY liquid medium, incubated at 160 rpm. -1 Incubate at 28°C in a shaker until the optical density (OD) value is reached. 600 When the value is ≥0.5, 10000 r·min -1Centrifuge for 10 minutes, discard the supernatant, wash off the bacterial cells with sterile water, shake well to disperse, and then prepare OD200 with sterile water. 600 Bacterial suspension with a value of 0.5.

[0049] 3. Select healthy, plump, and uniformly sized horsebane seeds. Disinfect them with povidone-iodine for 2 minutes in a sterile operating table, rinse them 4 times with sterile water, and then blot them dry with sterile filter paper before use.

[0050] 4. Treat the seeds of *Hippophae rhamnoides* using three methods: seed soaking in bacterial solution (P), irrigation with bacterial solution (G), and bud soaking in bacterial solution (Y). After treatment, cultivate *Hippophae rhamnoides* seedlings.

[0051] Seed soaking in bacterial solution (P): 30 sterilized seeds were soaked in 30 mL of bacterial solution for 30 minutes and then planted. Soaking in sterile water was CK1.

[0052] Bacterial solution irrigation (G): After the broomcorn seedlings have grown true leaves, under sterile conditions, use a 50mL syringe to add 30mL of bacterial solution to the roots of the seedlings. Ensure that no part of the seedlings other than the roots are exposed to bacterial solution. Irrigation with sterile water is CK2.

[0053] Seed germination test (Y): Seed germination was conducted using the petri dish and filter paper method. Sterilized *Hippophae rhamnoides* seeds were placed in sterile petri dishes lined with a single layer of filter paper, 30 seeds per dish. After 5-6 days, germinated seeds (radicles 3-4 cm long) were selected and immersed in 30 mL of bacterial solution for 30 minutes. The germinated seeds were then transplanted into plastic cups and immersed in sterile water (CK3).

[0054] 5. Cultivation of *Hippophae rhamnoides* seedlings

[0055] Samples were taken on day 45 post-inoculation. After being washed and sterilized three times with fine sand (121℃, high-temperature sterilization for 26 min), the sand was placed into plastic cups (8.5cm in diameter, 12cm in height, 500g / cup) with mesh holes at the bottom. 1 Place the seeds in a hydroponic container (31cm long, 19cm wide, 10.5cm high). Sow 30 treated seeds per container (disinfected seeds, soaked in bacterial solution, and germinated seeds soaked in bacterial solution), and cover the surface with about 2cm of dry sand. Add Hoagland nitrogen-containing nutrient solution to each pot once, and water with sterile water before germination. After germination, water with Hoagland nitrogen-free nutrient solution once every 15 days, and water with sterile water during this period.

[0056] 6. Testing of various indicators

[0057] (1) Photosynthetic parameters were measured 45 days after inoculation: From 9:00 AM to 11:30 AM on February 21-23, 2022, photosynthetic parameters of the tested *Hippophae rhamnoides* plants were measured using a TPS-2 portable photosynthesis meter. Normally growing plants were randomly selected from each cup, and photosynthetic parameters were measured on the fourth fully unfolded, healthy true leaf from the top downwards. Each treatment was repeated three times, and three leaves were selected from each replicate for measurement. Transpiration rate (T...) r ), porosity (G) s ), photosynthetic rate (P) n ), intercellular CO2 concentration (C i The parameters were directly measured by the TPS-2 portable photosynthesis instrument.

[0058] Result: As Figure 2 As shown, the transpiration rate, stomatal conductance, net photosynthetic rate, and intercellular CO2 concentration of *Hippophae rhamnoides* seedlings treated with *Rhizobium* inoculum solution increased by 91.95%, 134.83%, 142.64%, and 10.37%, respectively, compared to CK3. The transpiration rate of *Hippophae rhamnoides* seedlings treated with *Rhizobium* inoculum solution increased by 29.46% and 11.33%, respectively, compared to seed soaking and irrigation treatments. The stomatal conductance of *Hippophae rhamnoides* seedlings treated with *Rhizobium* inoculum solution increased by 74.38% and 10.60%, respectively, compared to seed soaking and irrigation treatments. The net photosynthetic rate of *Hippophae rhamnoides* seedlings treated with *Rhizobium* inoculum solution increased by 101.47% and 94.35%, respectively, compared to seed soaking and irrigation treatments. The intercellular CO2 concentration of *Hippophae rhamnoides* seedlings treated with *Rhizobium* inoculum solution increased by 7.54% and 8.26%, respectively, compared to seed soaking and irrigation treatments.

[0059] (2) Chlorophyll content: Chlorophyll was extracted with 95% alcohol and the absorbance was measured with a UV-Vis spectrophotometer at wavelengths of 665 nm and 649 nm. The contents of chlorophyll a, chlorophyll b, carotenoids and chlorophyll a+b were calculated.

[0060] Result: As Figure 3 As shown, the contents of chlorophyll a, chlorophyll b, carotenoids, and chlorophyll a+b increased by 37.93%, 9.74%, 80.82%, and 74.29% respectively compared with CK3. The chlorophyll a content of *Hippophae rhamnoides* seedlings treated with rhizobium inoculum solution increased by 21.26% and 16.03% respectively compared with seed soaking and irrigation treatments. The chlorophyll b content of *Hippophae rhamnoides* seedlings treated with rhizobium inoculum solution increased by 6.68% compared with irrigation treatment. The carotenoid content of *Hippophae rhamnoides* seedlings treated with rhizobium inoculum solution increased by 378.13% and 48.54% respectively compared with seed soaking and irrigation treatments. The chlorophyll a+b content of *Hippophae rhamnoides* seedlings treated with rhizobium inoculum solution increased by 41.20% and 33.80% respectively compared with seed soaking and irrigation treatments.

[0061] (3) Aboveground and underground biomass: Weigh the fresh weight of the aboveground biomass using a balance; then place it in an oven at 105℃ for 20 minutes, and then dry it at 80℃ until constant weight, and weigh its dry weight.

[0062] Result: As Figure 1 As shown, the aboveground fresh weight, underground fresh weight, aboveground dry weight, and underground dry weight of *Hippophae rhamnoides* seedlings treated with *Rhizobium* inoculum solution increased by 202.37%, 207.07%, 218.07%, and 201.75% respectively compared with CK3; the aboveground fresh weight of *Hippophae rhamnoides* seedlings treated with *Rhizobium* inoculum solution increased by 38.17% and 79.23% respectively compared with seed soaking and irrigation treatments; the underground fresh weight of *Hippophae rhamnoides* seedlings treated with *Rhizobium* inoculum solution increased by 12.82% and 63.47% respectively compared with seed soaking and irrigation treatments; the aboveground dry weight of *Hippophae rhamnoides* seedlings treated with *Rhizobium* inoculum solution increased by 25.18% and 97.08% respectively compared with seed soaking and irrigation treatments; and the underground dry weight of *Hippophae rhamnoides* seedlings treated with *Rhizobium* inoculum solution increased by 23.36% and 124.03% respectively compared with seed soaking and irrigation treatments.

Claims

1. A method for improving the photosynthetic characteristics and biomass of *Hippophae rhamnoides*, characterized in that, The seeds of *Hippophae rhamnoides* were inoculated with rhizobia using a fungal germination method; the rhizobia were... Rhizobim indigoferae 1-21, deposited on December 4, 2023 at the China Center for Type Culture Collection, accession number CCTCC NO:M20232454; the above-mentioned germination by bacterial solution includes: seed germination using the petri dish filter paper method, soaking the germinating seeds in a solution of rhizobium to obtain germinating seeds of *Hippophae rhamnoides* treated with bacterial solution.

2. The method according to claim 1, characterized in that, The soaking time in the bacterial solution is 0.5-1 hour.

3. The method according to claim 1, characterized in that, The germinated seeds are inoculated with rhizobia when the radicle is 3-4 cm long.

4. A method for cultivating *Hippophae rhamnoides* with high photosynthetic characteristics and biomass, characterized in that... Includes the following steps: (1) Isolation of rhizobia Rhizobim indigoferae 1-21, its accession number is CCTCC NO:M20232454; (2) Use sterile water to prepare the rhizobium inoculum solution; (3) The seeds of *Hippophae rhamnoides* were treated with a fungal solution to obtain germinated *Hippophae rhamnoides* seeds treated with a fungal solution. (4) Cultivate the germinated seeds of horsehair obtained in step (3) to obtain horsehair with high photosynthetic characteristics and biomass.

5. The method according to claim 4, characterized in that, In step (1), the rhizobium is morphologically round protrusions and has an opaque white or watery, translucent milky white appearance; the colony diameter is 5-8 mm.

6. The method according to claim 4, characterized in that, Step (2) includes: activating, culturing, centrifuging, and washing the rhizobia obtained in step (1), and then preparing a rhizobia culture solution with sterile water.

7. The method according to claim 6, characterized in that, The culture medium is TY liquid medium.

8. The method according to claim 6, characterized in that, The culture conditions were 160 r / min and 28℃ in a shaker.

9. The method according to claim 4, characterized in that, In step (4), after the germinated seeds of horsebane are sown, nitrogenous nutrient solution is used to supplement nutrients and sterile water is used to supplement moisture.

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