Method for obtaining transgenic root system by using Chinese herbaceous peony rhizome
Through the non-tissue culture transgenic method mediated by Agrobacterium rhizos, the transgenic root system was obtained using peony rhizos, which solved the problem of slow progress in peony transgenic breeding technology, and achieved the effect of simplifying the breeding process and expanding germplasm resources.
Patent Information
- Application Number
- CN202510122715.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-26
- Publication Date
- 2025-04-25
AI Technical Summary
The genetically modified breeding technology of peony is progressing slowly, and the lack of a complete genomic sequence and mature tissue culture system has led to a model plant dependent on genetic function verification, and the breeding cycle of traditional breeding methods has long hindered the cultivation of new varieties and the progress of molecular research.
A non-tissue culture transgenic method mediated by Agrobacterium rhizobium was used to use peony rhizobium rhizobium rhizobium rhizobium rhizobium to obtain a stable transformed peony transgenic root system through infection and co-culture of plasmids and Agrobacterium rhizobium rhizobium.
The possibility of peony genetically modified breeding has been realized, the gene function verification process has been simplified, the breeding cycle has been reduced, the genetic gene bank of germplasm resources has been expanded, and technical support has been provided for new varieties cultivation and molecular research.
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Abstract
Description
Technical Field
[0001] The invention relates to the technical field of peony molecular breeding and transgenic seedling propagation, and in particular to a method for obtaining a transgenic root system by utilizing peony rhizomes. Background Art
[0002] Peony is a perennial herbaceous flower of the genus Paeonia in the family Paeoniaceae. It is also one of the oldest traditional famous flowers in Chinese history. Since ancient times, it has been known as the "flower phase" and the "May flower god". Its elegant flower posture, elegant color and unique fragrance are loved by people all over the world and are also the darling of the cut flower market. At the same time, peony is also a medicinal material widely planted all over the world. It has a long history of medical uses, such as pain relief, treatment of gynecological diseases, anti-tumor, and anti-infection. However, peony grows slowly and the traditional breeding cycle is long, which limits the further development of the peony industry. In the field of peony transgenic technology research, due to the lack of a complete genome sequence and the lack of a mature tissue culture system, the transgenic breeding technology of peony has progressed slowly, which seriously restricts the in-depth exploration of its molecular genetic mechanism and the breeding process of new varieties.
[0003] Plant transgenic technology refers to the process of introducing exogenous genes into plants and enabling them to be stably expressed, thereby obtaining transgenic plants. Traditional technologies mainly include Agrobacterium-mediated method, gene gun method, electric shock method, pollen tube channel method, etc., and most of them need to rely on tissue culture technology. Although domestic and foreign scholars have made certain progress in the tissue culture of Paeonia plants, they still face a series of technical difficulties such as explant contamination, browning and difficulty in redifferentiation. Therefore, transgenic technology that relies on tissue culture system is difficult to carry out on peony. In recent years, studies have found that using rhizogenes Agrobacterium to infect the rhizomes of plants can bypass tissue culture, produce transgenic roots in a non-sterile environment, and use the ability of these plant roots to produce transgenic plants. The root system of peony is a fleshy root with the ability to bud. In this context, the application prospects of Agrobacterium-mediated non-tissue culture transgenic method have attracted much attention.
[0004] Agrobacterium is a Gram-negative bacterium commonly found in natural soil. Currently, there are two main types of Agrobacterium used for genetic modification: Agrobacterium tumefaciens and Agrobacterium rhizogenes. Agrobacterium rhizogenes contains a root-inducing plasmid (Ri plasmid), which can be used to infect plants to produce adventitious roots. This type of adventitious root grows fast, has many hairy branches, and is not pathogenic to the host plant. It only causes the plant to produce adventitious roots. Many studies have shown that after the Ri plasmid of Agrobacterium rhizogenes induces plants to produce hairy roots and obtain regenerated plants, it can cause changes in the phenotype of the plant, such as extended flowering period, early flowering, male sterility, etc. These characteristics have important application value in plant variety improvement. Agrobacterium rhizogenes-mediated genetic modification technology is currently used in many plants such as sweet potatoes, mulberry trees, tea trees, dandelions, and sweet oranges.
[0005] For peony, the application of traditional genetic transformation technology is relatively difficult and the progress is relatively slow. First of all, the tissue culture technology of peony still faces many problems such as high browning rate and high contamination rate. Therefore, it is necessary to use tissue culture to complete the genetic transformation method, which is difficult to maturely apply on peony. Secondly, peony also has complex dormancy characteristics, which leads to a long breeding cycle and a low reproduction coefficient. Traditional breeding methods are also difficult to carry out rapid genetic improvement, which not only increases the years required for the cultivation of new varieties, but also hinders the establishment of a transgenic system. At present, the verification of peony gene function still needs to be carried out in model plants tobacco and Arabidopsis, but heterologous expression sometimes cannot accurately reflect the function of the gene, which also restricts the research progress at the molecular level of peony.
[0006] Therefore, it is very necessary to explore the transgenic technology and system of peony for transgenic breeding of peony and promote research at the molecular level. Summary of the invention
[0007] The technical purpose of the present invention is to provide a method for obtaining a transgenic root system using peony rhizomes. The method uses the rhizomes of peony as infection explants, utilizes rhizogenes, and innovative infection and co-cultivation steps to bypass tissue culture, thereby obtaining a stably transformed peony transgenic root system, laying a foundation for the subsequent breeding of new peony varieties and research on functional genomics.
[0008] To achieve the above object, the present invention adopts the following technical scheme: a method for obtaining a transgenic root system using peony rhizomes, comprising the following steps:
[0009] 1) Obtaining peony rhizomes as explants: Select 3-4 year old mature peony plants, remove the lower fleshy roots and fibrous roots, retain the buds and rhizomes, and cut the rhizomes, retaining 2-4 buds on each rhizome;
[0010] 2) Preparation of plasmid and Agrobacterium rhizogenes infection: The plasmid used was Super1300 containing the EGFP reporter gene, and the Agrobacterium type was Agrobacterium rhizogenes K599;
[0011] 3) Infecting peony explants: including three methods: soaking in original bacterial solution, resuspension soaking, and smearing;
[0012] 4) Root induction and cultivation: Perlite and peat are used as culture medium for planting;
[0013] 5) Detection of transgenic positive roots: Use a handheld laser transmitter to identify transgenic roots;
[0014] 6) Obtaining peony composite transgenic plants: For peony rhizomes that are successfully infected and produce positive roots, remove non-positive roots and plant them, and then complete winter vernalization to allow seedlings to emerge and grow.
[0015] Furthermore, the time for obtaining peony rhizomes as explants in step 1) is from September to November in autumn each year, which is the traditional time for peony division, and it is easier to obtain more new root systems after planting.
[0016] Further, in step 2), the plasmid and Agrobacterium rhizogenes bacterial solution are prepared, and the strain into which the exogenous plasmid is transferred is first cultured in TY liquid medium at 28° C. with shaking for 24 to 36 hours. After the culture, about 700 μL of the bacterial solution is spread on TY solid medium and cultured for 36 to 48 hours to obtain solid infection products.
[0017] Further, in step 2), the plasmid and Agrobacterium rhizogenes infection product were prepared, and the strain into which the exogenous plasmid was transferred was first cultured in liquid TY liquid medium at 28°C with shaking for 36-48h, and then the concentration reached OD 600 =1.0, and the original bacterial solution was obtained. The original bacterial solution was centrifuged and resuspended, and acetosyringone was added to the resuspended solution for induction, thereby obtaining a resuspended bacterial solution.
[0018] Furthermore, in step 3), the peony explants are infected by re-cutting and scratching the prepared peony rhizome incision to ensure that the incision in contact with the infection material is a fresh incision.
[0019] Further, step 3) infection methods mainly include three types, the first is to directly use the original bacterial solution to soak the peony rhizomes for infection for 1.5 hours. The second is to soak the peony rhizomes in the resuspended bacterial solution for infection for 1.5 hours. The third is to apply the solid infection material to the fresh cut of the peony rhizomes.
[0020] Further, in step 4), the substrate used for root induction and cultivation is a mixed substrate of peat and perlite in a ratio of 1:1. No fungicide or rooting hormone is used in this process, and the roots grow naturally from the infected rhizomes. After planting, the plants are placed in a greenhouse for cultivation at a temperature of 10-25°C, a relative air humidity of 60-80%, and a soil moisture content of 50-60%. After 30 to 60 days, new roots can sprout from the rhizomes.
[0021] Furthermore, in step 5), after the roots grow out, a handheld laser emitter is used to irradiate the roots. The roots that can emit green fluorescence are transgenic positive roots, and the roots that fail to emit green fluorescence are ordinary roots.
[0022] Furthermore, in step 5), after the root system is observed, the non-transgenic roots are removed, and only the positive roots with successful transgenic are retained. After planting, they are vernalized at low temperature in the same year, and can sprout normally in the second year. At this time, the plant is a composite plant containing a transgenic root system.
[0023] Compared with the prior art, the present invention has the following advantages and innovations:
[0024] 1) The method of the present invention for obtaining transgenic root system by using peony rhizomes has overcome the technical bottleneck that has always made it difficult to carry out transgenic operation on peony, and has enabled the research and verification of peony gene function to turn to the main body, no longer relying on some model plants, and also provided a technical solution for the exploration of transgenic technology for other similar species.
[0025] 2) The method of the present invention for obtaining transgenic root system by using peony rhizomes makes transgenic breeding of peony possible. This technology can be used to effectively expand the genetic gene pool of germplasm resources, and provide a technical solution for transgenic breeding of new varieties and improving existing traits.
[0026] 3) The method of the present invention for obtaining transgenic root system using peony rhizomes can bypass tissue culture, does not require expensive and cumbersome tissue culture technology, and avoids the disadvantages of high browning rate and high contamination rate in traditional tissue culture technology. Especially for some rare and precious germplasm resources, it can reduce the consumption and waste of materials.
[0027] 4) The present invention provides a method for obtaining transgenic root system using peony rhizomes. This technology uses acetosyringone to induce resuspension without relying on tissue culture, which can effectively improve the efficiency and provide the best infection technology for using rhizogenes K599 to infect the root neck of peony.
[0028] 5) The present invention provides a method for obtaining a transgenic root system using peony rhizomes. The transgenic technology used can directly infect the rhizomes of peony. The operation is simple, convenient and efficient. A stable transgenic root system can be obtained. The transgenic green fluorescence signal can still be detected after one year of cultivation. BRIEF DESCRIPTION OF THE DRAWINGS
[0029] Figure 1 The rhizome of peony root after cutting;
[0030] Figure 2 Preparation for infection;
[0031] Figure 3 To use the resuspended bacterial solution to soak and infect the peony rhizomes;
[0032] Figure 4 To infect the peony rhizomes with solid infectants;
[0033] Figure 5 The left side shows the root development after 60 days of infection. The right side shows the root under natural light and the left side shows the root under ultraviolet laser irradiation. The arrows indicate transgenic positive roots, which can be excited to emit green fluorescence.
[0034] Figure 6To remove non-positive roots, rhizomes must be allowed to emerge after natural vernalization;
[0035] Figure 7 This is the root system of the genetically modified composite rhizome after one year of growth. The left side is under natural light, and the right side is under ultraviolet laser irradiation, which can be stimulated to emit green fluorescence. DETAILED DESCRIPTION
[0036] The specific implementation of the present invention is further described in detail below in conjunction with the accompanying drawings and examples. The following examples are used to illustrate the present invention, but are not intended to limit the scope of protection of the present invention, which is subject to the claims.
[0037] The materials for the experiment were from the Xiaotangshan Base of the National Flower Engineering Center (Changping District, Beijing). Robust peony plants with strong growth and no diseases or insect pests were selected.
[0038] Example 1
[0039] The present embodiment provides a method for obtaining a transgenic root system using peony rhizomes, which specifically comprises the following steps:
[0040] Step 1. Obtain peony rhizomes as explants: Select 3-4 year old mature peony plants, remove the lower fleshy roots and fibrous roots, retain the buds and rhizomes, and cut the rhizomes, retaining 2-4 buds on each rhizome; this step is performed from September to November each autumn, which is the traditional division time for peony, and it is easier to obtain more new roots after planting.
[0041] Step 2: Preparation of plasmid and Agrobacterium rhizogenes infection: The plasmid used was Super1300 containing the EGFP reporter gene, and the Agrobacterium type was Agrobacterium rhizogenes K599; the strain into which the exogenous plasmid was transferred was first cultured in liquid TY liquid medium at 28°C for 36-48h with shaking until the concentration reached OD 600 =1.0, and obtain the original bacterial solution.
[0042] Step 3: Infect the peony explants. In this embodiment, the peony rhizomes are directly immersed in the original bacterial solution for 1.5 hours. During the infection, the prepared peony rhizomes should be recut and scratched to ensure that the incision in contact with the infected object is a fresh incision.
[0043] Step 4: Root induction and cultivation: Use perlite and peat as the culture medium for planting. The matrix used for root induction and cultivation is a 1:1 mixed matrix of peat and perlite. No fungicide or rooting hormone is used in this process. The roots grow naturally from the rhizomes after infection. After planting, place it in a greenhouse for cultivation at a temperature of 10-25℃, a relative air humidity of 60-80%, and a soil moisture content of 50-60%. After 30 to 60 days, new roots can sprout from the rhizomes.
[0044] Step 5: Detection of transgenic positive roots: Use a handheld laser transmitter to identify transgenic roots. After the roots grow out, use a handheld laser transmitter to illuminate the roots. Roots that can emit green fluorescence are transgenic positive roots, and roots that fail to emit green fluorescence are ordinary roots.
[0045] Step 6. Obtaining peony composite transgenic plants: For peony rhizomes that are successfully infected and produce positive roots, remove the non-transgenic roots and only retain the positive roots that have been successfully genetically modified. After planting, they are subjected to low-temperature vernalization in the same year. After completing winter vernalization, they can sprout normally in the second year. At this time, the plants are composite plants containing transgenic roots.
[0046] Example 2
[0047] Compared with Example 1, the difference is that in the preparation of plasmid and Agrobacterium rhizogenes infection in step 2 of this embodiment, the strain into which the exogenous plasmid is transferred is first cultured in liquid TY liquid culture medium at 28°C with shaking for 36-48h, and then the concentration reaches OD 600 =1.0, the original bacterial solution was centrifuged and resuspended, and acetosyringone was added to the resuspended solution for induction, thereby obtaining a resuspended bacterial solution.
[0048] Compared with Example 1, the difference is that in step three of this embodiment, the peony explants are infected, and this embodiment is a resuspension and soaking treatment, and the peony rhizomes are immersed in the resuspension bacterial solution for infection for 1.5 hours.
[0049] Example 3
[0050] Compared with Example 1, the difference is that the plasmid and Agrobacterium rhizogenes infection material preparation in step 2 of this embodiment is solid infection material preparation, and the strain into which the exogenous plasmid is transferred is first cultured in TY liquid medium at 28° C. with shaking for 24 to 36 hours. After the culture, about 700 μL of the bacterial liquid is smeared on TY solid medium and cultured for 36 to 48 hours to obtain solid infection material.
[0051] Compared with Example 1, the difference is that in step three of this embodiment, the peony explants are infected, and this embodiment is a smearing treatment, in which the solid infection material is smeared on the fresh incision of the peony rhizome.
[0052] Comparative Example 1
[0053] Compared with Example 1, the difference is that after the rhizome is cut, no infection operation is performed on the explant, and the explant is directly planted and managed conventionally.
[0054] The results of Example 1-3, a method for obtaining a transgenic root system using peony rhizomes, are shown in Table 1 below.
[0055] Table 1
[0056]
[0057] The calculation method of the above theoretical survival rate is: Survival rate = number of surviving explants / total number of explants
[0058] The calculation method of the above theoretical positive rate is: Positive rate = number of explants producing positive root systems / total number of explants
[0059] As can be seen from Table 1, the use of the present invention to treat the root neck of peony with transgenic technology will affect the survival rate. The survival rate of the control group is 93%. In Example 2, the survival rate of infection with resuspended bacterial solution is the highest, which is 80%. From the perspective of positive rate, the highest is that the rhizome is treated with resuspended bacterial solution in Example 2, and the positive rate is 40%. In summary, the best method for obtaining transgenic root system using peony rhizomes is to soak them in resuspended bacterial solution, and both the survival rate and the positive rate have reached the highest.
[0060] In the foregoing description, the present invention has been comprehensively and meticulously introduced through general description, specific implementation methods and experimental verification. However, the above content only relates to the preferred embodiments of the present invention and is not a limitation of the present invention. Based on the core idea of the present invention, it can be appropriately adjusted. Therefore, any modification or improvement made without departing from the core purpose of the present invention should be regarded as the protection scope of the present invention.
Claims
1. A method for obtaining transgenic root system using peony rhizomes, characterized in that: The following steps are involved: 1) Obtaining peony rhizomes as explants: Select 3-4 year old mature peony plants, remove the lower fleshy roots and fibrous roots, retain the buds and rhizomes, and cut the rhizomes, retaining 2-4 buds on each rhizome; 2) Preparation of plasmid and Agrobacterium rhizogenes infection: The plasmid used was Super1300 containing the EGFP reporter gene, and the Agrobacterium type was Agrobacterium rhizogenes K599; 3) Infecting peony explants: including three methods: soaking in original bacterial solution, resuspension soaking, and smearing; 4) Root induction and cultivation: Perlite and peat are used as culture medium for planting; 5) Detection of transgenic positive roots: Use a handheld laser transmitter to identify transgenic roots; 6) Obtaining peony composite transgenic plants: For peony rhizomes that are successfully infected and produce positive roots, remove non-positive roots and plant them, and then complete winter vernalization to allow seedlings to emerge and grow.
2. The method for obtaining transgenic root system using peony rhizomes according to claim 1, characterized in that: The time for obtaining peony rhizomes as explants in step 1) is from September to November in autumn each year, which is the traditional time for peony division, and it is easier to obtain more new root systems after planting.
3. The method for obtaining transgenic root system using peony rhizomes according to claim 1, characterized in that: In step 2), the plasmid and Agrobacterium rhizogenes culture solution are prepared by first culturing the strain into which the exogenous plasmid has been transferred in a TY liquid medium at 28° C. with shaking for 24 to 36 hours. After the culture, about 700 μL of the culture solution is spread on a TY solid medium and cultured for 36 to 48 hours to obtain a solid infection product.
4. The method for obtaining transgenic root system using peony rhizomes according to claim 1, characterized in that: Preparation of plasmid and Agrobacterium rhizogenes infection in step 2), the strain into which the exogenous plasmid was transferred was first cultured in liquid TY medium at 28°C with shaking for 36-48h, and then the concentration reached OD 600 =1.0, and the original bacterial solution was obtained. The original bacterial solution was centrifuged and resuspended, and acetosyringone was added to the resuspended solution for induction, thereby obtaining a resuspended bacterial solution.
5. The method for obtaining transgenic root system using peony rhizomes according to claim 1, characterized in that: In step 3), the peony explants are infected by re-cutting and scratching the prepared peony rhizome incision to ensure that the incision in contact with the infection material is a fresh incision.
6. The method for obtaining transgenic root system using peony rhizomes according to claim 1, characterized in that: Step 3) There are three main infection methods. The first is to directly soak the peony rhizomes in the original bacterial solution for 1.5 hours. The second is to soak the peony rhizomes in the resuspended bacterial solution for 1.5 hours. The third is to apply the solid infection material to the fresh cut of the peony rhizomes.
7. The method for obtaining transgenic root system using peony rhizomes according to claim 1, characterized in that: In step 4), the substrate used for root induction and cultivation is a mixed substrate of peat and perlite in a ratio of 1:
1. No fungicide or rooting hormone is used in this process, and the roots grow naturally from the infected rhizomes. After planting, the plants are placed in a greenhouse for cultivation at a temperature of 10-25°C, a relative air humidity of 60-80%, and a soil moisture content of 50-60%. After 30 to 60 days, new roots can sprout from the rhizomes.
8. The method for obtaining transgenic root system using peony rhizomes according to claim 1, characterized in that: In step 5), after the roots grow out, a handheld laser emitter is used to irradiate the roots. The roots that can emit green fluorescence are transgenic positive roots, and the roots that fail to emit green fluorescence are ordinary roots.
9. The method for obtaining transgenic root system using peony rhizomes according to claim 1, characterized in that: After observing the root system, the non-transgenic roots are removed and only the positive roots with successful genetic modification are retained. After planting, they are vernalized at low temperature in the same year and can sprout normally in the second year. At this time, the plant is a composite plant containing a transgenic root system.