Application and method of metformin in improving rooting of tissue culture seedlings of Pyrus x bretschneideri and culture medium

Abstract

The application discloses a kind of metformin in promoting pear tissue culture seedling rooting application, method and culture medium, belong to fruit tree tissue culture seedling rooting culture field.Method includes: selecting the healthy pear tissue culture seedling of 28 days, plant height>3.5 cm, roughness>2 mm, leaf number>10 pieces after subculture, retain 3-4 leaves;Inoculation is to rooting culture medium, is cultured 30 days under the condition of 22±2 ℃, illumination 2200 Lx, photoperiod 16h / 8h.Root culture medium is added with 5-20 mg / L metformin, and contains 25 g / L sucrose, 6 g / L agar, 2 g / L PVA, 1 mg / L IAA and 0.5 mg / L IBA with 1 / 2MS as base medium.Experiments show that adding metformin can make pear tissue culture seedling early rooting 2-3 days, and rooting rate is increased to 83.67%-89.04%, wherein 10 mg / L treatment comprehensive effect is optimal, and rooting rate reaches 89.04%, and the number of adventitious roots, primary root length and total root number are significantly better than control.The application is simple in operation, low in cost, can effectively improve the rooting quality and transplanting survival rate of pear tissue culture seedling, and is suitable for factory seedling raising.

Description

Technical Field

[0001] This invention belongs to the field of fruit tree tissue culture seedling rooting technology, and specifically relates to the application, method and culture medium of metformin in improving the rooting of pear tissue culture seedlings. Background Technology

[0002] Duli ( Pyrus betulifolia Bunge is a commonly used rootstock for pear cultivation in northern regions, possessing numerous excellent characteristics such as drought resistance, disease resistance, and salt tolerance. Conventional propagation of wild pear primarily relies on seed, but seedlings exhibit significant genetic variability, making it difficult to retain the superior traits of the parent plants. Tissue culture technology, while preserving the superior traits of the parent plants, also offers advantages such as being unrestricted by season, having a high propagation coefficient, and producing uniform seedlings, making it an effective method for achieving industrialized production of fruit tree seedlings.

[0003] Pyrus pyrifolia tissue culture generally includes four stages: establishment of sterile explants, subculture, proliferation culture, and rooting acclimatization and transplanting. Among these, rooting acclimatization and transplanting of test-tube seedlings is a key technical step in tissue culture propagation, and the quality of rooting directly affects the survival rate after transplanting. The in vitro rooting ability of Pyrus pyrifolia tissue culture seedlings is affected by the rooting induction method and the composition of the culture medium. Currently, rooting induction of pear tissue culture seedlings mainly relies on auxins, commonly including indoleacetic acid (IAA), indolebutyric acid (IBA), and naphthaleneacetic acid (NAA). In existing techniques, rooting culture of Pyrus pyrifolia tissue culture seedlings often uses 1 / 2 MS medium supplemented with an appropriate concentration of IBA or IAA. Rooting generally begins in 14 days, with a rooting rate of about 60%, indicating significant room for improvement in rooting efficiency.

[0004] Metformin (MET) is a biguanide hypoglycemic drug widely used in the clinical treatment of type 2 diabetes. Research on the application of metformin in the rooting of fruit tree tissue culture seedlings remains lacking. Therefore, exploring the application of metformin in the rooting culture of pear tissue culture seedlings has significant theoretical and practical value for enriching the types of plant growth regulators induced by rooting in pear rootstock tissue culture and improving rooting efficiency. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the purpose of this invention is to provide an application, method, and culture medium for metformin in improving the rooting of *Pyrus pyrifolia* tissue culture seedlings, thereby solving the technical problems of low rooting rate and long rooting cycle in existing technologies.

[0006] The technical solution adopted in this invention is: a method for improving the rooting effect of pear tissue culture seedlings using metformin, the key technical points of which include the following steps: (1) Select robust pear tissue culture seedlings that have undergone subculture, have a plant height of at least 3.5 cm, a diameter of at least 2 mm, and at least 10 leaves, and retain 3 to 4 leaves; (2) The tissue culture seedlings of the pear were inoculated into the rooting medium and cultured for 30 days at 22±2℃, light intensity of 2200 Lx and photoperiod of 16h / 8h. The rooting medium is based on 1 / 2 MS medium with the addition of 5-20 mg / L metformin, which is added after being filtered and sterilized.

[0007] In the above scheme, the rooting medium also includes 25 g / L sucrose, 6 g / L agar, 2 g / L polyvinyl alcohol (PVA), 1 mg / L indoleacetic acid (IAA) and 0.5 mg / L indolebutyric acid (IBA).

[0008] In the above scheme, the amount of metformin added is 10 mg / L.

[0009] In the above scheme, the subculture medium in step (1) consists of: MS medium 4.4 g / L, sucrose 30 g / L, agar 6 g / L, polyvinyl alcohol (PVA) 2 g / L, 6-benzylaminopurine (6-BA) 1 mg / L, and indolebutyric acid (IBA) 0.1 mg / L.

[0010] A culture medium for rooting tissue culture seedlings of Pyrus pyrifolia, the key technical feature of which is that the culture medium is based on 1 / 2MS medium and supplemented with 5-20 mg / L metformin, which is added after being filtered and sterilized.

[0011] In the above-mentioned scheme, the culture medium also includes 25 g / L sucrose, 6 g / L agar, 2 g / L polyvinyl alcohol (PVA), 1 mg / L indoleacetic acid (IAA) and 0.5 mg / L indolebutyric acid (IBA).

[0012] In the above scheme, the amount of metformin added is 10 mg / L.

[0013] The application of metformin in improving the rooting effect of pear tissue culture seedlings is characterized by adding metformin as a rooting promoter to the rooting culture medium of pear tissue culture seedlings. The amount of metformin added is 5-20 mg / L, and the metformin is added after being filtered and sterilized.

[0014] In the above scheme, compared with the rooting medium without metformin, the addition of metformin increased the rooting rate of pear tissue culture seedlings to 83.67% to 89.04%, and the rooting time was advanced by 2 to 3 days.

[0015] Compared with the prior art, the beneficial effects of the present invention are: (1) Tissue culture seedlings in MET medium rooted 3 days earlier than those in traditional rooting medium; (2) Improved rooting rate of Pyrus pyrifolia tissue culture seedlings: the rooting rate of traditional rooting medium was about 60.00%, while the rooting rate of Pyrus pyrifolia tissue culture seedlings after adding MET reached 83.67%~89.04%; (3) Tissue culture seedlings in MET medium had more roots, longer roots, and more primary and secondary roots than those in traditional rooting medium; (4) 10 mg / L MET medium was the optimal concentration, which increased the average number of roots by 1.3 compared with traditional medium, with the average length of primary roots of rooted Pyrus pyrifolia tissue culture seedlings increasing by 3.81 mm, the number of secondary roots increasing by 1.7, and the total number of roots increasing by 3.6. The rooting rate reached 89.04%, which is nearly 30% higher than that of traditional culture medium; (5) Simple operation and low cost: MET is a commonly used drug, which is low in cost and easy to obtain, and can be directly added to the rooting medium. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 Daily rooting rate of *Pyracantha fortuneana* tissue culture seedlings in medium with different concentrations of metformin; Figure 2 Rooting of *Pyrus pyrifolia* tissue culture seedlings on day 30 in medium with different concentrations of metformin. Detailed Implementation

[0018] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the following description is provided in conjunction with the accompanying drawings. Figures 1-2 The present invention will be further described in detail below with reference to specific embodiments.

[0019] The metformin (MET) used in the following examples was a commercially available analytical grade reagent. Before use, it was prepared as a stock solution with sterile water, filtered for sterilization, and then used. In each example, the pH of the culture medium was adjusted to 5.8 (adjusted with 1 mol / L NaOH or HCl before sterilization). Agar, sucrose, and other components were sterilized together with the culture medium at high temperature (121°C, 20 min). Metformin and auxin-like substances (IAA, IBA) were sterilized by filtration through a 0.22 μm microporous membrane and added after the culture medium cooled to approximately 50–55°C and thoroughly mixed. Example 1:

[0020] A method for improving the rooting effect of pear tissue culture seedlings using metformin includes: Step 1: Prepare the subculture medium: MS medium 4.4 g / L, sucrose 30 g / L, agar 6 g / L, polyvinyl alcohol (PVA) 2 g / L, 6-benzylaminopurine (6-BA) 1 mg / L, indolebutyric acid (IBA) 0.1 mg / L. Adjust the pH to 5.8 before sterilization. Inoculate the pear tissue culture seedlings into the subculture medium. Culture conditions are: temperature 22±2℃, light intensity 2200 Lx, photoperiod (day / night) 16 h / 8 h, culture for 28 days. Select robust tissue culture seedlings with uniform growth (plant height > 3.5 cm, diameter > 2 mm, number of leaves > 10) for later use.

[0021] Step 2, prepare rooting medium: use 1 / 2 MS medium (2.2 g / L) as the basal medium, add 25 g / L sucrose, 6 g / L agar, 2 g / L PVA, 1 mg / L IAA, 0.5 mg / L IBA, and 5 mg / L metformin (added after filtration sterilization). Adjust the pH to 5.8 before sterilization.

[0022] Step 3: Take the pear tissue culture seedlings selected in Step 1, retain 3-4 leaves at the top, remove the excess callus tissue at the base with a sterile scalpel, inoculate them into the above-mentioned rooting medium, and culture them for 30 days at 22±2℃, 2200 Lx light, and a photoperiod of 16h / 8h.

[0023] Results: Adventitious roots began to appear on the 12th day after inoculation. After 30 days of culture, the rooting rate reached 85.29%, with 2.90 adventitious roots per plant, 3.67 primary roots, an average length of 13.62 mm for primary roots, 2.33 secondary roots, and a total of 6.00 roots. Example 2:

[0024] The difference between this embodiment and Example 1 is that the amount of metformin added to the rooting medium is 10 mg / L, while the rest of the operations are exactly the same.

[0025] Results: Adventitious roots began to appear on the 11th day after inoculation. After 30 days of culture, the rooting rate reached 89.04%, with 3.07 adventitious roots per plant, 5.50 primary roots, an average length of 16.57 mm for primary roots, 2.50 secondary roots, and a total of 8.00 roots. Example 3:

[0026] The difference between this embodiment and Example 1 is that the amount of metformin added to the rooting medium is 15 mg / L, while the rest of the operations are exactly the same.

[0027] Results: Adventitious roots began to appear on the 11th day after inoculation. After 30 days of culture, the rooting rate reached 88.89%, with 2.90 adventitious roots per plant, 4.00 primary roots, an average length of 13.15 mm for primary roots, 1.00 secondary roots, and a total of 5.00 roots. Example 4:

[0028] The difference between this embodiment and Example 1 is that the amount of metformin added to the rooting medium is 20 mg / L, while the rest of the operations are exactly the same.

[0029] Results: Adventitious roots began to appear on the 12th day after inoculation. After 30 days of culture, the rooting rate reached 83.67%, with 2.26 adventitious roots per plant, 6.00 primary roots, an average length of 16.12 mm for primary roots, 1.20 secondary roots, and a total of 7.20 roots.

[0030] Comparative Example 1: This control group serves as the control (CK), and metformin is not added to the rooting medium.

[0031] The difference between this comparative example and Example 1 is that metformin is not added to the rooting medium, but the rest of the operations are exactly the same.

[0032] Results: Adventitious roots began to appear on the 14th day after inoculation. After 30 days of culture, the rooting rate was only 60.00%, with 1.77 adventitious roots per plant, 3.60 primary roots, an average length of 12.76 mm for primary roots, 0.80 secondary roots, and a total of 4.40 roots.

[0033] Experimental example: The experiment was conducted in March 2025. Rooting culture of *Pyrus pyrifolia* tissue culture seedlings after 28 days of subculture was performed according to the methods described in Examples 1-4 and Comparative Examples 1-4. Each group contained 30 seedlings, with three replicates. After 30 days of culture, the rooting status of the *Pyrus pyrifolia* tissue culture seedlings in each group was recorded, and the results are shown in Tables 1 and 2.

[0034] Table 1. Effects of different concentrations of metformin on rooting rate and number of adventitious roots per plant of *Pyrus pyrifolia* tissue culture seedlings.

[0035] Table 2. Root system of rooted *Pyrus pyrifolia* tissue culture seedlings in different concentrations of metformin culture medium.

[0036] As shown in Tables 1 and 2, metformin concentration significantly affected the rooting effect of pear tissue culture seedlings. In the control group (CK, Comparative Example 1) without metformin, the average number of adventitious roots was only 1.77 per plant, the rooting rate was 60.00%, the average length of primary roots was 12.76 mm, and the total number of roots was 4.40, indicating poor root development. In treatment 1 (Example 1) with 5 mg / L metformin, the average number of adventitious roots increased to 2.90 per plant, the rooting rate increased to 85.29%, the average length of primary roots was 13.62 mm, and the total number of roots increased to 6.00, indicating that metformin has a significant root-promoting effect even at low concentrations. In Treatment 2 (Example 2, 10 mg / L), all indicators reached their optimal values, with a peak rooting rate of 89.04%, an average of 3.07 adventitious roots per plant, 5.50 primary roots, an average primary root length of 16.57 mm, 2.50 secondary roots, and a total of 8.00 roots. In Treatment 3 (Example 3, 15 mg / L), the rooting rate slightly decreased to 88.89%, the total number of roots decreased to 5.00, and the average length of primary roots decreased to 13.15 mm, indicating that some indicators began to decline after the metformin concentration exceeded 10 mg / L. In Treatment 4 (Example 4, 20 mg / L), the rooting rate further decreased to 83.67%, and the number of adventitious roots decreased to 2.26 per plant. Although the number of primary roots was still relatively high (6.00), the number of secondary roots decreased significantly (1.20). The above results indicate that metformin concentration and rooting effect exhibit a non-monotonic relationship of "low concentration promotes, high concentration inhibits", with the optimal concentration being 10 mg / L.

[0037] In summary, this invention, by adding 5–20 mg / L metformin (preferably 10 mg / L, added after filtration sterilization) to 1 / 2 MS basal medium, along with 25 g / L sucrose, 6 g / L agar, 2 g / L PVA, 1 mg / L IAA, and 0.5 mg / L IBA, and using robust *Pyrus pyrifolia* tissue culture seedlings subcultured for 28 days (subculture medium containing 30 g / L sucrose, 6 g / L agar, and 2 g / L PVA) as material, and culturing for 30 days under conditions of pH 5.8, 22±2℃, 2200 Lx light, and a photoperiod of 16h / 8h, can significantly advance the rooting time by approximately 2–3 days, increase the rooting rate to 83.67%–89.04%, and greatly improve both the quantity and quality of roots. This method is simple to operate, low in cost, and suitable for the industrial-scale rooting and seedling production of *Pyrus pyrifolia* tissue culture seedlings.

[0038] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for improving the rooting effect of pear tissue culture seedlings using metformin, characterized in that, Includes the following steps: (1) Select robust pear tissue culture seedlings that have undergone subculture, have a plant height of at least 3.5 cm, a diameter of at least 2 mm, and at least 10 leaves, and retain 3 to 4 leaves; (2) The tissue culture seedlings of Pyrus pyrifolia were inoculated into rooting medium and cultured for 30 days at 22±2℃, light intensity of 2200 Lx and photoperiod of 16h / 8h. The rooting medium is based on 1 / 2 MS medium with the addition of 5-20 mg / L metformin, which is added after being filtered and sterilized.

2. The method for enhancing the rooting effect of *Pyrus pyrifolia* tissue culture seedlings using metformin according to claim 1, characterized in that, The rooting medium also includes 25 g / L sucrose, 6 g / L agar, 2 g / L polyvinyl alcohol (PVA), 1 mg / L indoleacetic acid (IAA), and 0.5 mg / L indolebutyric acid (IBA).

3. The method according to claim 1, characterized in that, The amount of metformin added was 10 mg / L.

4. The method according to claim 1, characterized in that, The subculture medium in step (1) consists of: MS medium 4.4 g / L, sucrose 30 g / L, agar 6 g / L, polyvinyl alcohol (PVA) 2 g / L, 6-benzylaminopurine (6-BA) 1 mg / L, and indolebutyric acid (IBA) 0.1 mg / L.

5. A culture medium for rooting tissue culture seedlings of Pyrus pyrifolia, characterized in that, The culture medium is based on 1 / 2MS medium, with 5-20 mg / L metformin added. The metformin is added after being filtered and sterilized.

6. The culture medium for rooting tissue culture seedlings of *Pyrus pyrifolia* according to claim 5, characterized in that, The culture medium also includes 25 g / L sucrose, 6 g / L agar, 2 g / L polyvinyl alcohol (PVA), 1 mg / L indoleacetic acid (IAA), and 0.5 mg / L indolebutyric acid (IBA).

7. The culture medium according to claim 5, characterized in that, The amount of metformin added was 10 mg / L.

8. An application of metformin in improving the rooting effect of pear tissue culture seedlings, characterized in that, Metformin was added as a rooting promoter to the rooting medium of Pyrus pyrifolia tissue culture seedlings. The amount of metformin added was 5-20 mg / L, and the metformin was added after being filtered and sterilized.

9. The application according to claim 8, characterized in that, Compared with rooting medium without metformin, the addition of metformin increased the rooting rate of pear tissue culture seedlings to 83.67%–89.04% and advanced the rooting time by 2–3 days.

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