A method for stable transformation of root system of various chrysanthemum by tissue culture

Abstract

The application discloses a kind of root system stable transformation methods for a variety of wild chrysanthemum tissue culture, comprising the following steps: S1, wild chrysanthemum seed is sown in substrate and is cultured;S2, when the seedling has 4-5 leaf blades, cut off leaf blade together with petiole;S3, preparation of infection solution;S4, cut off leaf blade is soaked in immersion solution, and vacuum pump is assisted to permeate;After treatment is completed, immersion solution is absorbed, then is placed in substrate, and is cultured again;S5, after culture grows root, is identified by PCR experiment and selects the plant containing transgenic root;S6, selected plant is transferred to substrate and is cultured again.The application uses the vacuum permeation infection mode to soil culture seedling petiole, obtains the wild chrysanthemum composite plant of transgenic root+wild type bud;Method is simple, short cycle, high efficiency, and suitable for a variety of wild chrysanthemum, and lay the foundation for transgenic function verification and genetic engineering breeding in wild chrysanthemum.

Description

Technical Field

[0001] This invention belongs to the field of wild chrysanthemum breeding technology, specifically relating to a root system stabilization transformation method for various wild chrysanthemum tissue cultures. Background Technology

[0002] Plant transgenic technology aims to integrate specific exogenous genes into the plant genome, thereby cultivating plants carrying desired characteristics. This technology is widely used in various fields such as exploring the molecular mechanisms of plant traits, improving plant varieties, and breeding new varieties. To date, important crops such as cotton, soybeans, rice, and apples have been successfully improved through transgenic technology, resulting in new varieties with superior characteristics such as high yield, high stress resistance (including multiple resistances), and high quality, laying a solid foundation for the future progress of agriculture.

[0003] wild chrysanthemum ( Chrysanthemum indicum L. Chrysanthemum indicum (also known as wild chrysanthemum) is a perennial herbaceous plant belonging to the genus Chrysanthemum in the family Asteraceae. It originated in Shanxi and Shaanxi provinces of China and is widely distributed in Northeast, North, Central, South, and Southwest China. In 2022, wild chrysanthemum was recognized as one of the "Sixteen Excellent Authentic Medicinal Herbs of Hubei Province," one of the "Ten Famous Hubei Herbs." Due to significant limitations imposed by the species' genotype and germplasm, traditional chrysanthemum breeding methods are often unsuccessful. Existing technologies, such as patent document CN102090342A, provide a method for establishing a high-efficiency regeneration system for purple wild chrysanthemum. This method utilizes a randomized block design with two hormones to screen for the optimal induction and differentiation medium. Comparing the differentiation of mature leaves near the base and young leaves at the tip, it was found that callus tissue from young leaves at the tip was less prone to browning and exhibited a higher regeneration rate compared to mature leaves near the base. However, these methods still rely on traditional tissue culture methods. The emergence of transgenic technology has provided new ideas and approaches for chrysanthemum breeding. Agrobacterium tumefaciens-mediated transgenic systems are one of the common methods for studying the function of chrysanthemum genes and obtaining genetically modified organisms; however, transgenic materials obtained through tissue culture have problems such as long cycle, low efficiency, and unstable genetic transformation.

[0004] In view of the above problems, this invention is proposed. Summary of the Invention

[0005] To address the aforementioned technical problems, the present invention aims to provide a root system stabilization transformation method for various wild chrysanthemum tissue cultures. This method employs a vacuum infiltration infection of the petioles of soil-grown seedlings to obtain a wild chrysanthemum composite plant consisting of transgenic roots and wild-type buds. The method is simple to operate, has a short cycle, high efficiency, and is applicable to various wild chrysanthemums, laying the foundation for subsequent transgenic function verification and genetic engineering breeding in wild chrysanthemums.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a root system stabilization transformation method for various wild chrysanthemum tissue cultures, comprising the following steps: S1. Sow wild chrysanthemum seeds in a substrate for cultivation; S2. When the seedlings have 4-5 leaves after being cultivated in step S1, cut off the leaves along with the petioles. S3. Prepare the infiltration solution; S4. Soak the leaves cut off in step S2 in the dyeing solution obtained in step S3, and use a vacuum pump to assist in the permeation; after the treatment is completed, absorb the dyeing solution, and then place them in the substrate for cultivation again. S5. After roots (red roots) have grown in step S4, identify and screen plants containing transgenic roots through PCR experiments; that is, if the identification is positive, continue to cultivate in soil. S6. Transfer the plants selected in step S6 to the substrate for further cultivation.

[0007] The above technical solution employs a vacuum infiltration infection method on the petioles of soil-grown seedlings, eliminating the need for a lengthy tissue culture process. This allows for the rapid acquisition of a large number of transgenic roots, resulting in a composite plant of transgenic roots and wild-type buds in wild chrysanthemum. This method is simple to operate, has a short cycle time, is highly efficient, and is applicable to various types of wild chrysanthemums, laying the foundation for subsequent transgenic functional verification and genetic engineering breeding in wild chrysanthemums.

[0008] As a preferred technical solution of the present invention, the substrate used in steps S1 and S4 is the same, which is prepared by mixing nutrient soil and vermiculite in a volume ratio of 1:1.

[0009] As a preferred technical solution of the present invention, the cultivation conditions in steps S1 and S4 are the same, specifically as follows: humidity 60~70%, light intensity 800~1200 Lux, cultivation temperature 20~24℃, and photocycle 16h light / 8h dark.

[0010] As a preferred technical solution of the present invention, the preparation of the infiltration solution in step S3 includes the following steps: take MSU440 transgenic bacterial culture, streak it on LB solid medium containing spectinomycin, incubate it upside down in an incubator, pick single clones and put them into liquid LB medium with spectinomycin added, shake and incubate, collect the bacterial cells by centrifugation, and finally resuspend them to obtain the infiltration solution.

[0011] As a preferred embodiment of the present invention, the concentration of spectinomycin in LB solid medium is 40-60 mg / L; the concentration of spectinomycin in liquid LB medium is 40-60 mg / L; and the composition of the resuspension solution used for resuspension is: MS + 2% sucrose + 100 µM AS.

[0012] As a preferred technical solution of the present invention, the preparation of the MSU440 transgenic bacterial culture includes the following steps: Add 2 µL of 35S-Ruby plasmid to MSU440 Agrobacterium rhizogenes competent cells, mix quickly and vigorously by hand or by pipetting, and then place the culture in ice for 5 minutes, liquid nitrogen for 5 minutes, a 37°C water bath for 5 minutes, and an ice bath for 5 minutes. Remove from the ice bath and allow to room temperature, add 700 µL of antibiotic-free TY liquid medium, and incubate at 28°C with shaking for 2 hours; centrifuge at 6000 rpm for one minute to collect the bacteria, and resuspend approximately 100 µL of the supernatant by gentle pipetting and spreading it onto a TY plate containing the appropriate antibiotic. Invert the plate and incubate at 28°C for 2-3 days; after colonies have grown, select single colonies and send them to the company for sequencing. Store the correctly sequenced bacterial cultures for later use.

[0013] As a preferred technical solution of the present invention, in step S6, the composition of the substrate is: perlite: vermiculite: humus = 1:1:2; the cultivation conditions are: light intensity 800~1200 Lux, cultivation temperature 20~24℃, and photoperiod of 16h light / 8h darkness.

[0014] As a preferred technical solution of the present invention, the wild chrysanthemum seeds are selected from one or more of the varieties of "Hubei Dawu", "Hunan Shaoyang" or "Chongqing Qijiang".

[0015] Secondly, this invention also aims to provide applications of the above-mentioned methods in resistance breeding and gene screening of Chrysanthemum indicum. For example, it is understood that the above-mentioned methods can be used in scenarios including but not limited to: ① obtaining transgenic roots + wild-type Chrysanthemum indicum plants containing traits of important medicinal value; for example, based on important traits such as flavonoid content, metabolic pathways can be studied through the transgenic roots of the indicative plants; ② conducting research on the improvement of resistance-related traits in Chrysanthemum indicum; ③ conducting research on the drought and salinity stress resistance of Chrysanthemum indicum roots.

[0016] Compared with the prior art, the present invention has the following beneficial effects: (1) The method provided by the present invention simplifies the long process of tissue culture required to obtain transgenic materials of wild chrysanthemum. By using Agrobacterium rhizogenes to mediate stable transformation of wild chrysanthemum leaf explants, a transgenic root material + wild-type bud wild chrysanthemum composite line can be obtained in a short time. (2) The wild chrysanthemum root transformation system constructed by the method provided in this invention can be used for a variety of wild chrysanthemum varieties, has wide applicability, is not limited to a single genotype, and is beneficial to providing a favorable method for varieties with difficult genetic transformation; (3) The transformation system established by the method provided by the present invention is stable, has a high transformation rate and a short time consumption, which provides important technical support for future research on the resistance and tolerance of wild chrysanthemum. At the same time, it will promote the development of molecular genetic improvement research on important traits of wild chrysanthemum and lay the foundation for obtaining wild chrysanthemum varieties with excellent target traits. (4) The method provided by the present invention, compared with other crop transformation methods, obtains wild chrysanthemum composite plants whose transgenic part is only the underground root, and there is no transgenic safety risk, making it possible to utilize and promote transgenic plants with excellent traits in the future. Attached Figure Description

[0017] Figure 1 This is a diagram of wild chrysanthemum seedlings cultivated in soil in an embodiment of the present invention until they have 4-5 fully expanded leaves; Figure 2 This is a diagram of a leaf with a petiole used as the acceptor material for transformation in an embodiment of the present invention. Figure 3 is a diagram of Agrobacterium rhizogenes infecting and assisting vacuum penetration in an embodiment of the present invention; Figure 4 This is a diagram showing the induction of transgenic roots for 20 days in an embodiment of the present invention; Figure 5 This is a qPCR detection diagram of transgenic roots in an embodiment of the present invention; Figure 6 This is a diagram of a complete composite plant in an embodiment of the present invention. Detailed Implementation

[0018] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0019] Unless otherwise specified, all raw and auxiliary materials used in this invention are readily available.

[0020] For example, the nutrient soil used in this invention was purchased from Danish Pinsai peat moss, brand name CPAI; the LB solid medium was purchased from Solarbio; the liquid LB medium was purchased from Solarbio; the MS medium was purchased from Phytotech; and the AS medium was purchased from Solarbio.

[0021] This invention provides a method for root system stabilization transformation in the tissue culture of various wild chrysanthemums, comprising the following steps: S1. Seeds of three wild chrysanthemum varieties, namely “Hubei Dawu”, “Hunan Shaoyang”, and “Chongqing Qijiang”, which had undergone vernalization at 4℃ for about 2 months, were evenly sown in moist soil with a ratio of 1:1 of nutrient soil and vermiculite for cultivation. The cultivation conditions were: humidity 65%, light intensity of about 1000 Lux, light cycle of 16h light / 8h darkness, and temperature controlled at 22±1℃. S2, When the seedlings have 4-5 fully expanded leaves as described in step S1 (see...) Figure 1 Use a scalpel to cut off leaves of uniform size, with good growth and petioles (see...). Figure 2 The incision is made at an angle to increase the contact area with the bacterial solution and improve the conversion efficiency. S3. Preparation of Infection Solution: Remove the MSU440 transgenic bacterial culture carrying the Ruby reporter system from the -80℃ freezer. (Add 2 µL of 35S-Ruby plasmid to MSU440 Agrobacterium rhizogenes competent cells. Mix quickly and vigorously by hand or by pipetting. Place the culture in ice for 5 minutes, in liquid nitrogen for 5 minutes, in a 37℃ water bath for 5 minutes, and in an ice bath for 5 minutes. Remove from the ice bath and allow to room temperature. Add 700 µL of antibiotic-free TY liquid medium and incubate at 28℃ with shaking for 2 hours. Centrifuge at 6000 rpm for one minute to collect the bacteria. Retain approximately 100 µL of supernatant and gently resuspend the bacterial cells. Spread the resuspended cells onto TY plates containing the appropriate antibiotics and incubate upside down at 28℃ for 2-3 hours.) After colonies have grown, single colonies are picked and sent to the company for sequencing. Stranded cultures with correct sequencing results are stored for later use. (The following text appears to be unrelated and possibly a separate instruction: Streak the culture medium on LB solid medium containing 50 mg / L spectinomycin and incubate in an inverted incubator at 28°C for 2 days. Then, pick single colonies and add them to 100 ml of liquid LB medium supplemented with 50 mg / L spectinomycin. Incubate at 220 rpm and 28°C with shaking until OD reaches [missing information].) 600 =0.9, centrifuged at 5000 rpm for 5 min to collect the bacterial cells, and then resuspended in MS + 2% sucrose + 100 µM AS for 1.5 h before being used as the infection solution; S4. Immerse the petioles of the wild chrysanthemum leaves cut off in step S2 in the staining solution obtained in step S3, and use a vacuum pump to assist in the permeation (see...). Figure 3 After treatment, the dye solution was dried and then placed in moist soil with a ratio of nutrient soil to vermiculite of 1:1 for cultivation. The cultivation conditions were: humidity 65%, light intensity of about 1000 Lux, light cycle of 16h light / 8h dark, and temperature controlled at 22±1℃. S5. After roots have grown in step S4, observe and count the red hairy roots induced by MSU440 Agrobacterium rhizogenes with a Ruby reporter system to preliminarily identify the transgenic roots. Then, further identify them using experiments such as qPCR (see [link]). Figure 5); S6. Transfer the composite plants containing transgenic roots selected in step S6 to a substrate of perlite:vermiculite:humus in a ratio of 1:1:2, and culture them under conditions of 16 hours of light, 8 hours of darkness, and 22±1℃. (See [link to relevant documentation]) Figure 6 ).

[0022] Obviously, this invention utilizes Agrobacterium rhizogenes-mediated stable transformation of Chrysanthemum indicum without the need for tissue culture. It successfully transfers a vector carrying a betaine marker gene into Chrysanthemum indicum somatic cells, and obtains regenerated transgenic roots through induced culture, creating a transgenic root + wild-type bud composite Chrysanthemum line. It establishes a simple, rapid, and multi-variety Chrysanthemum root transformation system, providing technical support for research on molecular breeding of Chrysanthemum indicum.

[0023] The applicant declares that the present invention is illustrated by the above embodiments, but the present invention is not limited to the above embodiments, that is, it does not mean that the present invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of individual raw materials in the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for root system stabilization transformation in the tissue culture of various wild chrysanthemums, characterized in that, Includes the following steps: S1. Sow wild chrysanthemum seeds in a substrate for cultivation; S2. When the seedlings have 4-5 leaves after being cultivated in step S1, cut off the leaves along with the petioles. S3. Prepare the infiltration solution; S4. Soak the leaves cut off in step S2 in the dyeing solution obtained in step S3, and use a vacuum pump to assist in the permeation; after the treatment is completed, absorb the dyeing solution, and then place them in the substrate for cultivation again. S5. After the roots have grown in step S4, identify and screen plants containing transgenic roots through PCR experiments. S6. Transfer the plants selected in step S6 to the substrate for further cultivation.

2. The root system stabilization transformation method for various wild chrysanthemum tissue cultures according to claim 1, characterized in that, The same substrate is used in steps S1 and S4, which is prepared by mixing nutrient soil and vermiculite in a volume ratio of 1:

1.

3. The root system stabilization transformation method for various wild chrysanthemum tissue cultures according to claim 1, characterized in that, The cultivation conditions in steps S1 and S4 are the same, specifically as follows: humidity 60-70%, light intensity 800-1200 Lux, cultivation temperature 20-24℃, and photoperiod of 16h light / 8h darkness.

4. The root system stabilization transformation method for tissue culture of various wild chrysanthemums according to claim 1, characterized in that, The preparation of the infiltration solution in step S3 includes the following steps: take the MSU440 transgenic bacterial culture, streak it on LB solid medium containing spectinomycin, incubate it upside down in an incubator, pick a single clone and put it into liquid LB medium with spectinomycin added, shake and incubate, centrifuge to collect the bacterial cells, and finally resuspend it to obtain the infiltration solution.

5. The root system stabilization transformation method for various wild chrysanthemum tissue cultures according to claim 4, characterized in that, In LB solid medium, the concentration of spectinomycin is 40-60 mg / L; in liquid LB medium, the concentration of spectinomycin is 40-60 mg / L; the composition of the resuspension solution used is: MS + 2% sucrose + 100 µM AS.

6. The root system stabilization transformation method for various wild chrysanthemum tissue cultures according to claim 4, characterized in that, The preparation of the MSU440 transgenic bacterial suspension includes the following steps: 35S-Ruby plasmid is added to MSU440 Agrobacterium rhizogenes competent cells, quickly mixed, and then treated sequentially in ice, liquid nitrogen, and water bath, followed by room temperature; then, antibiotic-free TY liquid medium is added, shaken and cultured, centrifuged, and the bacteria are collected. The supernatant is collected, the resuspended bacterial blocks are spread on TY plates containing the corresponding antibiotics, and placed in an incubator for culture; after colonies grow, they are sequenced and screened.

7. The root system stabilization transformation method for tissue culture of various wild chrysanthemums according to claim 1, characterized in that, In step S6, the composition of the substrate is: perlite: vermiculite: humus = 1: 1: 2; the cultivation conditions are: light intensity 800~1200 Lux, cultivation temperature 20~24℃, and photoperiod of 16h light / 8h darkness.

8. The root system stabilization transformation method for tissue culture of various wild chrysanthemums according to claim 1, characterized in that, Wild chrysanthemum seeds are selected from one or more varieties from "Hubei Dawu", "Hunan Shaoyang", or "Chongqing Qijiang".

9. The application of the method according to any one of claims 1 to 8 in the breeding of wild chrysanthemum resistance and the screening of wild chrysanthemum genes.

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