阅读说明：本技术 发根农杆菌介导的植物转化系统及其方法 (Agrobacterium rhizogenes-mediated plant transformation system and method thereof ) 是由 文琴 于鲲 刘清利 许建平 于 2020-06-30 设计创作，主要内容包括：本申请涉及植物组织培养和转化领域,特别地涉及发根农杆菌(Agrobacterium rhizogenes)介导的植物(尤其是向日葵)转化技术。本申请建立了由发根农杆菌介导的向日葵转化,并获得复合植物的技术体系。(The present application relates to the field of plant tissue culture and transformation, in particular to Agrobacterium rhizogenes (Agrobacterium rhizogenes) mediated plant (especially sunflower) transformation techniques. The application establishes a technical system for transforming the sunflower mediated by agrobacterium rhizogenes and obtaining the composite plant.)

1. A method for agrobacterium rhizogenes-mediated genetic transformation of a plant, comprising the steps of:

1) seed disinfection and germination

Selecting filled plant seeds for sterilization;

sowing the disinfected seeds in a culture dish containing a culture medium, and placing the seeds in an environment suitable for seed germination for germination culture;

2) explant isolation

Selecting germinated seeds, cutting off a section of hypocotyl close to one end of the roots, and using the remaining seed parts as explants for infection;

3) infection and co-culture

Immersing the explant into the agrobacterium tumefaciens suspension for dip dyeing, and then transferring the explant to a culture dish for co-culture;

4) screening culture and root induction culture

After co-cultivation, the explants are transferred to a screening medium for cultivation and induced to root until composite plants are obtained.

2. The method of claim 1, wherein the sterilizing is performed by: sterilizing the surface with 75% alcohol, soaking in sodium hypochlorite solution for 15 min, and rinsing with ultrapure water for several times.

3. The method of claim 1, wherein the environment suitable for seed germination is an environment at room temperature with a light/dark replacement period of 16/8 hours.

4. The method of claim 1, wherein the co-culturing is performed for 2 to 5 days, filter paper is spread in the culture dish, and the filter paper contains the liquid co-culture medium thereon.

5. The method of claim 1, wherein in co-culturing, filter paper is plated in a petri dish, liquid medium is added, and then explants are placed on the filter paper.

6. The method according to claim 1, wherein the explant is from a seed that germinates from 1 to 7 days, preferably from a seed that germinates from 3 to 7 days.

7. The method of claim 1, wherein the seed used for explant isolation is either free of seed coat or has a portion of seed coat.

8. The method of claim 1, wherein the explant isolation is performed by: selecting germinated seeds, excising hypocotyls at the cotyledonary node part close to the growing point, and making a wound at the incision, and using the remaining seed part as an explant for infection.

9. The method of claim 1, wherein the explant isolation is performed by: selecting germinated seeds, cutting off hypocotyls at the cotyledon node part close to the growing point, making a wound at the cut, simultaneously removing partial cotyledons, and using the remaining seed part as an explant for infection.

10. The method according to claim 1, wherein the screening agent used is glufosinate-ammonium and its concentration is in the range of 4-8mg/L during the screening culture phase.

11. The method according to claim 1, wherein the screening agent is reduced or removed after one week of the screening culture in the screening culture stage, thereby proliferating the hairy root in a short period.

12. The method of claim 1, wherein the gene transformation into cells is enhanced by centrifugation or shaking while the explants are immersed in the Agrobacterium suspension for staining.

13. The method as claimed in claim 11, wherein the centrifugation is performed at a speed of 600 and 1000 rpm.

14. The method according to any one of claims 1 to 13, wherein the transformation product obtained by the method is a composite plant comprising not only hairy roots but also whole aerial parts.

15. The method according to any one of claims 1 to 13, wherein the plant is a dicotyledonous plant, in particular sunflower or soybean.

Technical Field

The invention relates to the field of biotechnology, in particular to the field of plant transformation technology, and more particularly to transgenic technology of dicotyledonous plants. The invention establishes an Agrobacterium rhizogenes (Agrobacterium rhizogenes) mediated transformation system for sunflower and other dicotyledonous plants.

Background

Agrobacterium rhizogenes, a gram-negative bacterium, causes plants to develop adventitious hairy roots at the wound site. Hairy root obtained by Agrobacterium rhizogenes-mediated plant transformation, grown in the absence of any plant in vitroUnder the action of the regulator, a large amount of lateral roots can be rapidly derived^[1]. Meanwhile, the Agrobacterium rhizogenes can integrate the T-DNA containing the foreign gene into the plant genome from Ri plasmid, or in case of co-transformation, the T-DNA region of the binary vector can be integrated into the plant genome^[2][3]. The agrobacterium rhizogenes-mediated transformation method has the advantages of rapidness and simplicity, and provides a powerful tool for gene function research and root biological physiological research^[4]。

Specifically, the application of the hair root has the following aspects. Hairy root transformation is particularly suitable for application in gene function studies involving root biology, such as those genes that play a role in symbiotic and pathogenic interactions, nutrient absorption and hormone transport^[5]. On soybeans, K599 can effectively induce hairy roots on cotyledons of multiple varieties, the hairy roots cultured in vitro can be used for breeding soybean cyst nematodes, and a simple and effective tool is provided for function research of the soybean cyst nematodes^[6]. The mass culture of hairy roots is a unique method for the accumulation of high-value secondary metabolites. Furthermore, hairy roots are also commonly used for the study of secondary metabolite metabolic pathways.

Besides, the hairy roots can also be applied to the fields of plant bioaugmentation, heavy metal bioaccumulation, plant growth restoration, artificial seeds and the like^[7][8]。

The composite plant is a plant form obtained by agrobacterium rhizogenes-mediated plant transformation. It comprises two parts: aerial parts, shoots, stems and leaves of non-transgenic wild-type, etc.; underground part, transgenic hairy root. The greatest advantage of this plant form is that the combination of transgenic hairy roots with non-transgenic stem and leaf parts forms a complete plant, greatly prolonging the survival time of the hairy roots and expanding the application of the hairy roots^[1]。

The application of the composite plant generally comprises the following aspects: firstly, the application of the composite plant comprises the application fields of all hair roots; secondly, the composite plant can be used for researching plant repair, plant nutrition and development related to interaction with rhizomes; and the method can also be applied to researches in aspects of vaccine development, induction, functional genomics and the like based on plants.

In our study, we applied the composite plant system to the pathological study of the parasitic plant, Orobanchecumana Wallr, on host plant infestation of germinating parasitic tumors.

Agrobacterium rhizogenes transformation systems have long been studied and used in the field of plant transformation, especially in dicotyledonous plants. However, Agrobacterium rhizogenes-mediated transformation of sunflower is rare. To date, we have searched only one report on transformation of sunflower hairy roots, and Yakupova et al obtained independent hairy roots by transforming sunflower cotyledons with Agrobacterium rhizogenes strains a4 and 15834^[9]. The system reported in the article has the disadvantages that the hairy roots obtained by the system are independent and are not composite plants, the operation is complicated, and the transformation period is long.

In order to solve the technical problems, the invention provides an agrobacterium rhizogenes-mediated plant transformation system which is simple in experimental operation, short in transformation period and free of genotype dependence, wherein a transformation product is a composite plant, and the physiological property of the composite plant is superior to that of an independent transgenic hairy root.

Further, advantages of the present invention over existing plant transformation systems include:

1. agrobacterium tumefaciens-mediated plant transformation and biolistic transformation are the most prominent methods used in the field of sunflower transformation at this stage^[10]. Both of these transformation methods have significant limitations, including: the long-period transformation and tissue culture process has large labor amount, consumes more resources, has obvious gene restriction, is difficult to realize high-flux production, and limits the development of gene function research. The agrobacterium rhizogenes-mediated sunflower transformation method has the obvious advantages of simple operation, short period and large flux^[11]。

2. The transformants obtained by general Agrobacterium rhizogenes-mediated plant transformation are all independent hairy roots. The transformant obtained by the invention is a composite plant, and the survival time of the transgenic hairy roots after leaving the culture medium environment can be prolonged to more than three weeks from less than one week. In addition, the isolated culture of the composite plant does not need a culture medium, so that the cost investment of time and resources is reduced, and the scientific research and production prospect of hairy roots is greatly expanded.

3. The traditional method for obtaining the composite plant usually adopts a plant which sprouts for about one week, agrobacterium injection is carried out on the stem of the plant, and after transgenic hairy roots sprout to a certain length, natural roots are removed. The method has complicated requirements on plant culture conditions, and requires special high-humidity sealed incubator in the whole process^[11]. Compared with the existing agrobacterium rhizogenes-mediated plant transformation method, the method has the advantages that the transformation process and the plant culture conditions are greatly simplified. This is because the transformed explants of the invention are selected from the seed part where the hypocotyl is attached to the cotyledon, and are the key to the cotyledonary node part containing the apical growth point. Such explants ensure that after Agrobacterium transformation the transformation product is a complete complex plant comprising both underground and above ground parts, i.e.transgenic hairy roots and non-transgenic stems and leaves. This is a direct method for obtaining complex plants, improving transformation efficiency.

4. The invention is not only suitable for the transformation of a plurality of sunflower varieties, but also successfully realizes the transformation on a plurality of soybean varieties and obtains a compound plant.

Disclosure of Invention

In general, the present invention relates to a method for agrobacterium rhizogenes-mediated genetic transformation of plants, comprising the steps of:

1) seed disinfection and germination

Selecting filled plant seeds for sterilization;

sowing the disinfected seeds in a culture dish containing a culture medium, and placing the seeds in an environment suitable for seed germination for germination culture;

2) explant isolation

Selecting germinated seeds, cutting off a section of hypocotyl close to one end of the roots, and using the remaining seed parts as explants for infection;

3) infection and co-culture

Immersing the explant into the agrobacterium tumefaciens suspension for dip dyeing, and then transferring the explant to a culture dish for co-culture;

4) screening culture and root induction culture

After co-cultivation, the explants are transferred to a screening medium for cultivation and induced to root until composite plants are obtained.

In a further embodiment, the sterilization is performed by: sterilizing the surface with 75% alcohol, soaking in sodium hypochlorite solution for 15 min, and rinsing with ultrapure water for several times.

In a further embodiment, the environment suitable for seed germination is an environment at room temperature and a light/dark replacement period of 16/8 hours.

In a further embodiment, the co-cultivation is performed for 2 to 5 days, with filter paper being spread in the culture dish and the liquid co-cultivation medium being contained on the filter paper.

In a further embodiment, during co-cultivation, filter paper is laid down in a petri dish, liquid medium is added, and the explant is then placed on the filter paper.

In a further embodiment, the explant is from a seed that germinates from 1 to 7 days, preferably from a seed that germinates from 3 to 7 days.

In a further embodiment, the seed used for explant isolation is either seedless or has a partial seed coat.

In a further embodiment, the explant isolation is performed by: selecting germinated seeds, excising hypocotyls at the cotyledonary node part close to the growing point, and making a wound at the incision, and using the remaining seed part as an explant for infection.

In a further embodiment, the explant isolation is performed by: selecting germinated seeds, cutting off hypocotyls at the cotyledon node part close to the growing point, making a wound at the cut, simultaneously removing partial cotyledons, and using the remaining seed part as an explant for infection.

In a further embodiment, the selection agent used during the selection culture stage is glufosinate-ammonium and its concentration is in the range of 4-8mg/L, preferably 4 mg/L.

In a further embodiment, the selection agent is reduced or removed during the selection culture phase after one week of selection culture, thereby proliferating the hairy roots in a short period of time.

In a further embodiment, gene transformation into cells is enhanced by centrifugation or shaking while the explants are immersed in the Agrobacterium suspension for transfection.

In a further embodiment, the centrifugation is carried out at a speed of 600 and 1000 rpm.

In a further embodiment, the transformation product obtained by the method is a composite plant, which includes not only hairy roots but also whole aerial parts.

In a further embodiment, the plant is a dicot, in particular sunflower or soybean.

The transformation product obtained by the method of the present invention is a complex plant including not only hairy roots but also the whole aerial parts of the plant.

The transformation method of the invention is not only suitable for various sunflower varieties, but also suitable for various soybean varieties, thereby being generally suitable for dicotyledonous plants.

Drawings

FIG. 1 is a schematic diagram of a plant binary expression vector 15312.

FIG. 2 is a schematic diagram of seed germination.

FIG. 3 schematic representation of explants. A: explant isolation method 1; b: explant isolation method 2.

FIG. 4 is a schematic representation of co-cultivation.

FIG. 5 is a schematic diagram of a composite plant.

FIG. 6 is a graph showing the expression of GFP in roots.

FIG. 7 is a schematic diagram of PCR detection.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.