Bacterial liquid preparation for promoting healing of grafting unions of muskmelon and pumpkin and application of bacterial liquid preparation

Abstract

The invention relates to a plant grafting technology in agricultural horticulture, in particular to a bacterial liquid preparation for promoting healing of grafting unions of muskmelons and pumpkins and application of the bacterial liquid preparation, and provides a bacterial liquid preparation which is simple in operation method, outstanding in application effect and capable of promoting healing of grafting unions of the muskmelons and the pumpkins according to physiological and molecular mechanisms of CmGH9B3 infection liquid in grafting healing of the muskmelons and the pumpkins. The bacterial liquid preparation can significantly improve the grafting healing efficiency of muskmelon and pumpkin and the quality of grafted seedlings. In the attaching grafting process of the muskmelon and the pumpkin, the muskmelon scion hypocotyl is soaked in a bacterial liquid preparation to be infected for 30 min, the muskmelon scion hypocotyl and the pumpkin rootstock are fixed through a grafting clamp, callus formation at a grafting opening and connection between the scion and a vascular bundle bridge of the rootstock can be promoted, and then healing of the grafting opening of the muskmelon and the pumpkin is accelerated; the grafting healing rate and survival rate are improved, and the grafted seedling quality is guaranteed.

Description

Technical Field

[0001] This invention relates to plant grafting technology in agriculture and horticulture, specifically to a bacterial liquid preparation for promoting the healing of the graft union between melon and pumpkin and its application. Background Technology

[0002] Plant grafting, an ancient horticultural technique, is used to improve the yield, quality, and stress resistance of single varieties and is widely used in the cultivation and variety improvement of horticultural crops. However, in actual production, differences in rootstock-scion combinations, environmental control, and management methods often lead to low survival rates of melon and pumpkin grafted seedlings, resulting in high cultivation costs and inconsistent quality, which seriously affects the economic benefits of the greenhouse melon industry. GH9B3 (Glycosyl hydrolase 9B3) is a key gene of β-1,4-glucanase, involved in cell wall remodeling and intercellular adhesion, playing a crucial role in plant wound healing.

[0003] Currently, there is a lack of effective means and methods to improve the grafting healing efficiency of melon and pumpkin grafted seedlings during cultivation. The main approach is to control environmental conditions such as temperature, light, and humidity in the facility to improve the healing efficiency and ensure the quality of the grafted seedlings. However, due to significant environmental differences between different regions and facilities, management methods vary and there is no unified standard. Therefore, this invention aims to develop a bacterial liquid preparation that can significantly improve the grafting healing efficiency of melon and pumpkin, thereby effectively shortening the grafting healing cycle, controlling graft union healing efficiency, improving seedling quality, and reducing seedling cultivation costs. This will provide technical and product support for ensuring the profitability of greenhouse melon production. Summary of the Invention

[0004] The purpose of this invention is to provide a bacterial liquid preparation for promoting the healing of the graft union between melon and pumpkin and its application. It can promote the formation of callus tissue at the graft union and the connection of vascular bundles between the scion and the rootstock, thereby accelerating the healing of the graft union between melon and pumpkin, improving the graft healing rate and survival rate, and ensuring the quality of grafted seedlings.

[0005] The objective of this invention is achieved through the following technical solution: This invention provides a recombinant bacterium that promotes the healing of the graft union between melon and pumpkin. The recombinant bacterium includes a vector containing the CmGH9B3 gene, the sequence of which is shown in SEQ ID NO.5.

[0006] Furthermore, the recombinant bacteria were constructed based on Agrobacterium K599; the primer sequences for amplifying the CmGH9B3 gene are shown in SEQ ID NO.1 and SEQ ID NO.2.

[0007] The present invention also provides a method for preparing the recombinant bacteria, comprising the following steps: (1) Extract RNA from melon and reverse transcribe it into cDNA. Use the cDNA as a template to amplify the target fragment CmGH9B3 gene. (2) The target fragment CmGH9B3 gene was ligated to the 1300 vector and transformed into Escherichia coli DH5α competent cells; (3) Select a single colony for bacterial PCR identification, sequence and compare to obtain the correct positive clone, and transfer the obtained positive clone into K599 Agrobacterium tumefaciens competent cells to obtain recombinant Agrobacterium.

[0008] Furthermore, in step (1), the amplification reaction system consists of 25 μL of high-fidelity DNA polymerase, 2 μL of CmGH9B3-1300-F, 2 μL of CmGH9B3-1300-R, 1 μL of cDNA, and 20 μL of RNase-Free ddH2O.

[0009] Furthermore, in step (1), the amplification reaction program is 95℃ for 5 min, 1 cycle; 95℃ for 30 s, 55℃ for 30 s, 72℃ for 100 s, 35 cycles; 72℃ for 10 min.

[0010] Furthermore, in step (2), the specific steps of the ligation are as follows: 2 μL of the target fragment CmGH9B3 gene, 3 μL of 1300 vector plasmid, 5 μL of 2xInfluence, ligation in a metal bath at 50°C for 30 min.

[0011] The present invention also provides a bacterial liquid preparation, wherein the bacterial liquid preparation comprises the recombinant bacteria described above.

[0012] The present invention also provides the application of the recombinant bacteria or the bacterial liquid preparation described above in promoting the healing of the graft union between melon and pumpkin.

[0013] Furthermore, the application includes immersing the hypocotyl of a melon scion in the bacterial solution preparation.

[0014] Furthermore, the soaking time is 30 minutes.

[0015] The beneficial effects of this invention are as follows: Based on the physiological and molecular mechanisms of CmGH9B3 infection in the grafting healing of melon and pumpkin, this invention has developed a simple and effective bacterial solution that significantly improves the grafting healing efficiency and seedling quality of melon and pumpkin. During the grafting process, the hypocotyl of the melon scion is first soaked in the bacterial solution for 30 minutes. Then, it is fixed to the pumpkin rootstock using a grafting clip. This promotes callus formation at the graft union and the connection of vascular bundles between the scion and rootstock, thereby accelerating the grafting healing process, increasing the grafting healing rate and survival rate, and ensuring the quality of the grafted seedlings.

[0016] The microbial agent in this invention can be stored at 4℃ in the dark for 10 days, which can significantly improve the grafting healing efficiency of melon and pumpkin, increase the grafting survival rate by 8.0%, enhance the root vitality of melon and pumpkin grafted seedlings, promote root growth, and have a significant root strengthening effect. It significantly improves the root-to-shoot ratio, seedling strength index and growth rate of melon and pumpkin grafted seedlings, and significantly improves the quality of grafted seedlings. Moreover, it is simple to apply and easy to operate, and has no significant impact on the grafting efficiency of grafting workers. Attached Figure Description

[0017] 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.

[0018] Figure 1 This is a quantitative graph illustrating the success rate of infection of self-rooted melon seedlings according to the present invention. Figure 2 This invention relates to a method for infecting grafted seedlings of melon and pumpkin. Figure 3 This is a quantitative diagram of CmGH9B3 expression during the grafting healing process of melon and pumpkin grafted seedlings according to the present invention; Figure 4 This is a quantitative graph showing the impact of the present invention on the healing efficiency and graft survival rate of melon and pumpkin grafted seedlings during the grafting healing process; Figure 5 This is a quantitative diagram showing the impact of the present invention on the root growth characteristics of grafted melon and pumpkin seedlings; Figure 6 This is a quantitative graph showing the effects of the present invention on the root-to-shoot ratio, seedling vigor index, and growth rate of grafted melon and pumpkin seedlings. Detailed Implementation

[0019] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0020] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0021] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0022] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0023] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0024] Based on the inventors' previous research on the physiological and molecular mechanisms of CmGH9B3 infection solution in the grafting healing of melon and pumpkin, a bacterial liquid preparation was developed to improve the grafting healing efficiency and seedling quality of melon and pumpkin. The preparation is simple to use and has significant effects. The influence of the bacterial liquid preparation on the grafting healing efficiency of melon and pumpkin was analyzed through tests such as acid fuchsin absorption. The influence of the bacterial liquid preparation on the quality of grafted seedlings was evaluated by measuring growth indicators (plant height, stem diameter, dry weight, fresh weight, root-to-shoot ratio, seedling vigor index, and growth rate). The aim is to improve the grafting healing efficiency of melon and pumpkin and ensure the quality of grafted seedlings through this invention. In the following examples, the melon used is the "Leopard" variety provided by Yangling Qianpu Agricultural Development Co., Ltd.

[0025] Example 1

[0026] Preparation and application method of bacterial suspension preparation for promoting the healing of graft union between melon and pumpkin The preparation method of the bacterial solution that promotes the healing of the graft union between melon and pumpkin in this embodiment is as follows: 1) Extract RNA from melon and reverse transcribe it into cDNA. Use the cDNA as a template to amplify the target fragment CmGH9B3 gene (the specific sequence is shown in SEQ ID NO.5). The size of the target fragment is 1488bp.

[0027] The primer sequence is as follows: CmGH9B3-1300-F (SEQ ID NO.1): 5'-ATGGCTTCTCCCATCTCAAAT-3'; CmGH9B3-1300-R (SEQ ID NO. 2): 5'-ATGTTTGCCCGAGAAGAATG-3'.

[0028] The amplification reaction system is as follows: 25 μL of high-fidelity DNA polymerase, 2 μL of CmGH9B3-1300-F, 2 μL of CmGH9B3-1300-R, 1 μL of cDNA, and 20 μL of RNase-Free ddH2O.

[0029] The amplification reaction procedure is as follows: 95℃ for 5 min, 1 cycle; 95℃ for 30 s, 55℃ for 30 s, 72℃ for 100 s, 35 cycles; 72℃ for 10 min.

[0030] 2) Take 2 μL of the target fragment amplification product, 3 μL of the 1300 vector plasmid, and 5 μL of 2xInfluence, and ligate in a metal bath at 50℃ for 30 min. Ligate the target fragment with the 1300 vector and transform it into *E. coli* DH5α competent cells. Pick single colonies for colony PCR identification, and sequence alignment to obtain correct positive clones. Transform the obtained positive clones into K599 *Agrobacterium rhizogenes* competent cells to obtain recombinant *Agrobacterium*, which will be used for subsequent hypocotyl infection experiments in melon.

[0031] 3) such as Figure 1 As shown, the timeliness and concentration of the bacterial culture preparation were screened based on the expression level of CmGH9B3. Infection success rate (%) = (Number of seedlings with significantly upregulated CmGH9B3 expression / Total number of seedlings) × 100%. After shaking, the prepared OD... 600 Agrobacterium tumefaciens bacterial solution with a concentration of 1.0 was left to stand at room temperature in the dark for 3 hours, and then left to stand at 4°C in the dark for 10 days to obtain a bacterial solution preparation that promotes the healing of the graft union between melon and pumpkin.

[0032] The method of using the bacterial liquid preparation that promotes the healing of the graft union between melon and pumpkin in this embodiment is as follows: The optimal time for grafting is when the melon scion has two leaves and a bud, and the cotyledons of the pumpkin rootstock are fully expanded. When grafting, first use a grafting knife to make a slanted cut 0.8-1.0 cm below the cotyledons of the melon scion, extending to 2 / 3 of the hypocotyl length, with a cut length of 0.5 cm. Soak the wedge-shaped surface in a bacterial solution for 30 minutes (just enough to cover the wedge-shaped surface). Figure 2 (As shown in the image), then remove one cotyledon and the growing point of the pumpkin rootstock, and use a grafting clip to fix the melon scion to the pumpkin rootstock.

[0033] Example 2 Effect of CmGH9B3 expression level on graft healing process using bacterial culture preparation To clarify the effect of the bacterial culture preparation prepared in this invention on the expression levels of melon and pumpkin grafted seedlings, this embodiment uses melon stem tissue from identified positive grafted seedlings, with normally injected empty grafts as a control. RNA was extracted and reverse transcribed into cDNA. qRT-PCR analysis was performed using CmGH9B3 real-time quantitative primers, with the 18S gene as an internal reference.

[0034] The real-time quantitative primer sequences are as follows: CmGH9B3-F (SEQ ID NO.3): 5'-GAACCCAGGATCGGATGTTG-3' CmGH9B3-R (SEQ ID NO.4): 5'-GCAGAGGAAAGCGAATCACTAT-3' The results are as follows Figure 3 As shown, by Figure 3 It was found that during the grafting healing process, the expression level of CmGH9B3 in plants treated with the bacterial solution was significantly higher than that in the control group. This indicates that the bacterial solution of the present invention can significantly promote the expression of CmGH9B3 in melon scions.

[0035] Example 3 Effects of bacterial culture preparations on graft healing efficiency and graft survival rate of melon and pumpkin grafts On days 5, 6, 7, 8, and 9 post-grafting, 1 cm stem segments were horizontally cut above and below the graft union. The pumpkin rootstock was then placed vertically, face down, in a 1% acidic fuchsin solution for 40 minutes. A stem segment 2.5 mm above the graft union was then manually cut horizontally, and the absorption of acidic fuchsin was observed under a microscope. This process was repeated three times, with three plants per replicate. Grafting survival rate (%) = (Number of surviving seedlings / Number of grafted seedlings) × 100%.

[0036] The final result is as follows Figure 4 As shown, by Figure 4It can be seen that the bacterial liquid preparation of the present invention can significantly improve the grafting healing speed of melon and pumpkin, and the grafting survival rate is significantly higher than that of the control by 8.0%. Therefore, the bacterial liquid preparation of the present invention has a significant effect on improving the grafting healing efficiency of melon and pumpkin.

[0037] Example 4 The effect of bacterial liquid preparation on the quality of grafted melon and pumpkin seedlings (1) Use Win RHIZO root scanner to scan the root system and analyze the total root length, root surface area, average root diameter, root projected area and number of root branches.

[0038] (2) Determination of root activity using the TTC-triphenyltetrazolium chloride method: Remove impurities and soil from the roots as much as possible and wash them clean with water. Weigh 0.5g of root tip sample and place it in a 15ml centrifuge tube (the root tip must be completely immersed in the solution). Add 10ml of an equal volume mixture of 0.4% TTC solution and phosphate buffer, and fully immerse the root in the solution. The ratio of TTC:A solution:B solution is 5:2:3. Incubate in the dark at 37℃ for 2 hours, and then add 2ml of sulfuric acid to stop the reaction. Blank test: Sulfate buffer (2ml) + root tip (0.5g) + TTC (5ml) + A solution (2ml) + B solution (3ml). Take out the root, absorb the surface moisture, and place it in a mortar. Add 4ml of ethyl acetate and a small amount of quartz sand, and grind (until the root is white and the liquid is red). Transfer the red extract to a test tube and wash the mortar 2-3 times with a small amount of ethyl acetate. Transfer the ethyl acetate used for washing to a test tube, and finally adjust the volume of ethyl acetate to 10ml. The absorbance at 485 nm was measured using a spectrophotometer.

[0039] from Figure 5 As can be seen, the root vigor of the grafted melon and pumpkin seedlings treated by this invention was significantly increased by 10.61%, the total root length significantly increased by 23.52%, and the total root surface area and total root tip number significantly increased by 26.57% and 24.19%, respectively. Figure 6 As can be seen, this invention can significantly improve the root-to-shoot ratio, seedling vigor index, and growth rate of grafted seedlings. It is evident that this invention has a significant effect on strengthening the roots of melon and pumpkin grafted seedlings, and also significantly improves the quality of the grafted seedlings.

[0040] Among them, root-to-shoot ratio = root fresh weight / aboveground fresh weight; seedling vigor index = (stem diameter at graft union / scion height) * total plant dry weight; growth rate (G value) = total plant dry weight / seedling time.

[0041] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A recombinant bacterium that promotes the healing of the graft union between melon and pumpkin, characterized in that, The recombinant bacteria include a vector containing the CmGH9B3 gene, the sequence of which is shown in SEQ ID NO.

5.

2. The recombinant bacteria according to claim 1, characterized in that, The recombinant bacteria were constructed based on Agrobacterium K599; the primer sequences for amplifying the CmGH9B3 gene are shown in SEQ ID NO.1 and SEQ ID NO.

2.

3. The method for preparing recombinant bacteria according to any one of claims 1-2, characterized in that, Includes the following steps: (1) Extract RNA from melon and reverse transcribe it into cDNA. Use the cDNA as a template to amplify the target fragment CmGH9B3 gene. (2) The target fragment CmGH9B3 gene was ligated to the 1300 vector and transformed into Escherichia coli DH5α competent cells; (3) Select a single colony for bacterial PCR identification, sequence and compare to obtain the correct positive clone, and transform the obtained positive clone into K599 Agrobacterium tumefaciens competent cells to obtain recombinant Agrobacterium.

4. The preparation method according to claim 3, characterized in that, In step (1), the amplification reaction system consists of 25 μL of high-fidelity DNA polymerase, 2 μL of CmGH9B3-1300-F, 2 μL of CmGH9B3-1300-R, 1 μL of cDNA, and 20 μL of RNase-FreeddH2O.

5. The preparation method according to claim 3, characterized in that, In step (1), the amplification reaction program is 95℃ for 5 min, 1 cycle; 95℃ for 30 s, 55℃ for 30 s, 72℃ for 100 s, 35 cycles; 72℃ for 10 min.

6. The preparation method according to claim 3, characterized in that, In step (2), the specific steps of the ligation are as follows: 2 μL of the target fragment CmGH9B3 gene, 3 μL of 1300 vector plasmid, 5 μL of 2xInfluence, ligation in a metal bath at 50°C for 30 min.

7. A bacterial liquid preparation, characterized in that, The bacterial preparation includes the recombinant bacteria as described in any one of claims 1-2.

8. The application of the recombinant bacteria as described in any one of claims 1-2 or the bacterial liquid preparation as described in claim 7 in promoting the healing of the graft union between melon and pumpkin.

9. The application as described in claim 8, characterized in that, The application involves immersing the hypocotyl of a melon scion in the bacterial solution.

10. The application as described in claim 9, wherein the soaking time is 30 minutes.

Images