Genetic transformation method of Ningxia wolfberry based on adventitious bud induction

Abstract

The invention discloses a Ningxia wolfberry genetic transformation method based on adventitious bud induction. The agrobacterium rhizogenes are adopted for genetic transformation, and the problem that buds of traditional Ningxia wolfberry leaves are difficult to regenerate is effectively solved. The agrobacterium rhizogenes can effectively induce formation of hairy roots of the Ningxia wolfberry, so that the technical bottleneck in the regeneration process of the Ningxia wolfberry is broken through, and the transformation efficiency is remarkably improved. In the transformation process, although the Ningxia wolfberry hairy roots induced by agrobacterium rhizogenes successfully obtain transgenic markers, the original adventitious bud regeneration capacity is inhibited. By means of the dedifferentiation and repeated induction culture technology, the redifferentiation capacity of the hairy root induced callus is effectively recovered, the regeneration bud forming efficiency of the hairy root induced callus is remarkably improved, and a transgenic Ningxia wolfberry plant is obtained.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of plant biotechnology, and particularly relates to a genetic transformation method of Lycium barbarum based on adventitious bud induction. BACKGROUND

[0002] Lycium is a genus of Solanaceae, and has strong stress resistance and is a pioneer cultivation tree species in saline-alkali areas. Lycium barbarum is the only native medicinal Lycium plant recorded in Chinese Pharmacopoeia, and has a history of more than 500 years of artificial cultivation. The fruit of Lycium barbarum is rich in functional active substances such as Lycium polysaccharide, flavonoids, betaine and carotenoids, and has health functions such as antioxidant, anti-radiation, anti-tumor, anti-aging, liver cell protection and immune enhancement. With the in-depth research on the pharmacological components of Lycium and the increasing demand for modern health care, the planting area and consumption of Lycium have been increasing year by year. By 2018, the artificial cultivation area of Lycium in China reached 2.15 million mu, with a production value of more than 10 billion yuan, becoming a sunrise industry for the development of western agriculture in China.

[0003] Establishing an efficient genetic transformation system is the key to in-depth study of gene function identification and precision breeding of Lycium. At present, Lycium ruthenicum is generally used as the receptor for genetic transformation of Lycium. However, the somatic differentiation of Lycium barbarum, the only medicinal Lycium in China, to generate adventitious buds is low, which makes it difficult to establish a genetic transformation system and limits the application of this species in the field of gene function research and new variety creation. Agrobacterium rhizogenes-mediated genetic transformation system has the advantages of simple operation and high transformation efficiency, and is a new method for introducing exogenous genes into plant cells in recent years. However, the transgenic hairy roots induced by this method have difficulty in regenerating adventitious buds, which makes it difficult to obtain complete transgenic plants, and restricts the further development and application of this technology. Therefore, it is of great significance to establish a hairy root genetic transformation system of Lycium barbarum based on Agrobacterium rhizogenes and to obtain complete transgenic plants by inducing hairy root cells to undergo dedifferentiation and redifferentiation according to the principle of plant cell totipotency, which can improve the medicinal quality of Lycium barbarum and create new stress-resistant varieties. SUMMARY

[0004] To solve the above problems, the present application provides a genetic transformation method of Lycium barbarum, which comprises the establishment of a sterile culture system of Lycium barbarum, Agrobacterium rhizogenes-mediated genetic transformation, the acquisition of transgenic hairy roots, callus induction, adventitious bud and adventitious root induction, and the transplantation and identification of regenerated plants. Finally, transgenic Lycium barbarum is obtained.

[0005] To this end, the application provides a genetic transformation method of Lycium barbarum L., which comprises the following steps:

[0006] (1) performing surface sterilization and aseptic culture on mature seeds of Lycium barbarum L. to obtain aseptic tissue culture seedlings, and taking stem segments of the aseptic tissue culture seedlings as genetic transformation receptors;

[0007] (2) using Agrobacterium rhizogenes carrying a RUBY reporter gene to infect the stem segments, performing co-culture, and inducing the occurrence of hairy roots;

[0008] (3) cutting the hairy roots, inoculating them on a callus induction medium to generate calli, and inducing the formation of adventitious buds through periodic alternate culture;

[0009] (4) cutting the adventitious buds, inoculating them on a rooting medium to induce adventitious roots, and obtaining regenerated plants;

[0010] (5) performing PCR detection of the RUBY gene on the regenerated plants to screen positive transgenic plants;

[0011] (6) transplanting and domesticating the positive plants to obtain Lycium barbarum L. transgenic plants capable of natural growth.

[0012] According to the application, the surface sterilizing agent is a sodium trichloroisocyanurate solution with an effective chlorine concentration of 0.1% and containing 0.2% Tween-80.

[0013] According to the application, the Agrobacterium rhizogenes is a K599 strain carrying a pCAMBIA3302 / Pro35S::RUBY expression vector.

[0014] According to the application, the preparation of the infection solution comprises: culturing the bacterial solution to OD 600 0.4, collecting it by centrifugation, resuspending it in a MS medium containing 0.2 mmol / L acetyl-syringone (AS), and standing for 1-3 hours.

[0015] According to the application, the stem segments are infected by injection, the injection position is about 1-2 cm from the lower end of the stem segment, and after infection, the stem segments are stood for 5 minutes and then inoculated in a co-culture medium for dark culture for 3 days.

[0016] According to the application, the formulations of the co-culture medium, the sterilization medium, the callus induction medium, the adventitious bud induction medium, and the adventitious root induction medium are respectively:

[0017] · Co-culture medium: MS+AS 0.2 mmol / L+sucrose 30 g / L+plant gel 2.5 g / L, pH 5.8;

[0018] • Bacterial-free medium: MS + Cef 150 mg / L + Tim 150 mg / L + sucrose 30 g / L

[0019] + phytagel 2.5 g / L, pH 5.8;

[0020] • Callus induction medium: MS + 6-BA 0.6 mg / L + NAA 0.5 mg / L + Cef 150 mg / L + Tim 150 mg / L

[0021] + PPT 5 mg / L, pH 5.8;

[0022] • Adventitious shoot induction medium: MS + 6-BA 0.6 mg / L + NAA 0.1 mg / L + Cef 150 mg / L + Tim 150

[0023] mg / L + PPT 5 mg / L, pH 5.8;

[0024] • Adventitious root induction medium: ½ MS + IBA 0.25 mg / L + Cef 150 mg / L + Tim 150 mg / L + sucrose 15

[0025] g / L + phytagel 2.5 g / L, pH 5.8.

[0026] According to the present application, the above-mentioned RUBY reporter gene renders the hairy roots, callus, adventitious shoots and regenerated plants purple red by synthesizing betalains, and is combined with PCR detection for positive screening.

[0027] According to the present application, the above-mentioned transplanting substrate is a mixture of perlite and TS-1 nutrient soil at a volume ratio of 1:1, which is sterilized at 121°C, 0.11 MPa for 20 min and then used.

[0028] According to the present application, the above-mentioned transgenic regenerated plants are domesticated for 25 days under conventional maintenance conditions in a greenhouse, and the survival rate of the transgenic plants after transplanting is more than 90%.

[0029] The application discloses a high-efficiency genetic transformation method of Lycium barbarum L., aims to solve the technical bottleneck problem that genetic transformation of the Lycium barbarum L. is difficult to be stably carried out, constructs a high-efficiency, repeatable and convenient-to-screen transformation system, and improves the obtaining efficiency of the Lycium barbarum L. transgenic plant. The method takes a sterile tissue culture seedling of the Lycium barbarum L. as a genetic transformation receptor, adopts a plant expression vector carrying a RUBY report gene to construct an Agrobacterium rhizogenes competent cell, realizes genetic transformation on the Lycium barbarum L. cell through an agrobacterium-mediated method, and induces the occurrence of a hairy root. Then, through activation of the dedifferentiation and redifferentiation processes of the hairy root cell, and in combination with a PCR molecular detection technology, the transgenic regenerated plant is successfully obtained.

[0030] The application adopts the RUBY gene for regulating biosynthesis of betalain as a visual screening marker. The transgenic Lycium barbarum L. plant shows obvious purple red characteristics due to expression of the RUBY gene, can realize initial screening of the transformed plant without relying on an antibiotic screening agent, thereby improving the screening efficiency, reducing screening errors, cooperating with further verification through PCR, and improving the accuracy and stability of the transformation system.

[0031] The application discloses a Lycium barbarum L. genetic transformation method based on adventitious bud induction. The application effectively overcomes the problem that it is difficult to regenerate a bud from a traditional Lycium barbarum L. leaf through genetic transformation by using Agrobacterium rhizogenes. The Agrobacterium rhizogenes can effectively induce the formation of a hairy root of the Lycium barbarum L., thereby breaking through the technical bottleneck in the regeneration process, and significantly improving the transformation efficiency. In the transformation process, although the hairy root of the Lycium barbarum L. induced by the Agrobacterium rhizogenes has successfully obtained a transgenic marker, the original adventitious bud regeneration ability is inhibited. Through dedifferentiation and repeated induction culture technology, the application effectively restores the redifferentiation ability of the hairy root-induced callus, significantly improves the formation efficiency of the regenerated bud, and obtains the transgenic Lycium barbarum L. plant. BRIEF DESCRIPTION OF DRAWINGS

[0032] The drawings described herein are used to provide further understanding of the present application, constitute a part of the present application, and the illustrative embodiments of the present application and the description thereof are used to explain the present application, and do not constitute improper limitations on the present application. In the drawings:

[0033] Figure 1 It is a Lycium barbarum L. tissue culture seedling of the present application.

[0034] Figure 2 It is a hairy root generated from a Lycium barbarum L. explant of the present application.

[0035] Figure 3 It is a callus induced by a red hairy root of the present application.

[0036] Figure 4 It is an adventitious bud induced from a callus of the present application.

[0037] Figure 5 Adventitious roots are induced from the adventitious shoots of the application.

[0038] Figure 6 An electrophoretogram of PCR amplification products of regenerated plants of the application.

[0039] Figure 7 Ningxia wolfberry regenerated plants of the application are transplanted and acclimatized to survive.

[0040] Figure 8 A flow chart of the Ningxia wolfberry genetic transformation method based on adventitious shoot induction provided by the application. DETAILED DESCRIPTION

[0041] The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0042] The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0043] In order to make the purpose, technical solutions and advantages of the application clearer and more apparent, the application will be further described in detail below in conjunction with the drawings and specific examples. It should be understood that the specific examples described herein are only used to explain the application and do not limit the application.

[0044] Example 1

[0045] Example 1 of the application is used to illustrate the establishment of a Ningxia wolfberry aseptic culture system, which is as follows:

[0046] 1. Seed source and pretreatment:

[0047] Ningxia wolfberry seeds were provided by the Ningxia Institute of Wolfberry Research, Ningxia Academy of Forestry Sciences. To ensure sterility, the seeds were first placed in a 10 mL sterile centrifuge tube and 5 mL of 70% ethanol solution (v / v) was added. On the clean bench, the ethanol solution was removed by pipette after horizontal shaking for 30 seconds. Then, the seeds were soaked with 5 mL of sterile water for 3 minutes, and then the sterile water was removed.

[0048] 2. Disinfection treatment:

[0049] 5 mL of 0.1% sodium chloroisocyanurate solution (containing 0.2% Tween-80, v / v) was added to the same centrifuge tube, and shaken at 100 r / min for 10 minutes on a shaker. Then, the centrifuge tube was transferred to the clean bench, and the disinfectant was removed by pipette, and the seeds were soaked with 5 mL of sterile water for 3 minutes each time to completely remove the disinfectant. After removing the sterile water, the seeds were inoculated on the germination medium (MS medium containing 1 / 2 macroelements).

[0050] 3. Inoculation and culture:

[0051] The treated seeds were inoculated on MS medium containing 1 / 2 macroelements. The medium was sterilized by high temperature and high pressure (121℃, 0.11MPa, 20min) to ensure sterility. The inoculated seeds were placed in a plant tissue culture room with the following culture conditions: 16 hours light (light intensity 36μmol / m 2 / s), 8 hours dark, and temperature maintained at 24-26℃. The culture was checked and contaminated cultures were removed every day to ensure a clean culture environment.

[0052] 4. Seed germination and subculture:

[0053] After the seeds started to germinate under suitable culture conditions, subculture was performed every 28 days. During the subculture, the leaves of the wolfberry tissue culture seedlings were removed, and the stems (containing 2-3 axillary buds) were cut into small pieces and vertically inoculated on fresh MS medium. Finally, the well-grown tissue culture seedlings were selected as the genetic transformation material (see Figure 1 ).

[0054] The medium and inoculation tools were sterilized by high temperature and high pressure (121℃, 0.11MPa, 20min) and used after cooling.

[0055] Example 2

[0056] Example 2 of the present application is used to illustrate Agrobacterium rhizogenes-mediated genetic transformation of Ningxia wolfberry, as follows:

[0057] 1. Culture of Agrobacterium rhizogenes:

[0058] Agrobacterium rhizogenes K599 containing pCAMBIA3302 / Pro 35S ::RUBY was preserved in the laboratory. On a clean bench, the glycerol bacteria stored at -80℃ were dipped with an inoculation ring, and streaked on solid YEB medium containing kanamycin (Kan, 50mg / L) and streptomycin (Str, 50mg / L), and cultured at 28℃ in the dark until single colonies appeared. Single colonies were picked and inoculated in 5mL liquid YEB medium (containing Kan 50mg / L and Str 50mg / L), and cultured at 28℃, 180r / min in the dark overnight; 1mL of the cultured bacteria was transferred to 100mL liquid YEB medium (containing Kan 50mg / L and Str 50mg / L), and cultured at 28℃, 180r / min in the dark until the OD 600 was 0.4-0.6. The cultured bacteria were centrifuged at 5000r / min for 10min, resuspended in 100mL liquid MS medium (containing acetosyringone 0.2mmol / L), and incubated at room temperature for 2h to obtain the infection solution.

[0059] 2. Infection of Lycium barbarum stem segments:

[0060] The Lycium barbarum tissue culture seedlings were cut into stem segments of 3-4 cm in length with a sterile scalpel on a clean bench. The infection solution was taken up with a 1 mL sterile medical syringe, and the needle was inserted into the lower end of the stem segment 1 cm from the end and the infection solution was injected. After 8 min, the surface contamination of the bacterial solution was absorbed with sterile filter paper, and the stem segments were vertically inoculated on the co-culture medium (MS medium containing 0.2 mmol / L acetosyringone), and cultured in the dark at 25°C for 3 days.

[0061] 3. Bacteria removal and hairy root induction:

[0062] The Lycium barbarum stem segments were transferred to the bacteria removal medium (MS medium containing 150 mg / L Cef and 150 mg / L Tim) on a clean bench, and cultured under the conditions of 16 h light (light intensity 36 μmol / m 2 / s) and 8 h dark, and a temperature of 24-26°C. After 7 days, the stem segments were transferred to the freshly prepared bacteria removal medium to wait for the growth of hairy roots, and the hairy root occurrence rate and red hairy root occurrence rate were counted after 28 days. Figure 2 The results showed (Table 1) that the hairy root occurrence rate (the number of stem segments with hairy roots / the total number of stem segments x 100%) was 72.93 ± 4.54%, the red hairy root occurrence rate (the number of stem segments with red hairy roots / the total number of stem segments x 100%) was 64.59 ± 1.00%, and the average number of red hairy roots per stem segment (the total number of red hairy roots / the number of stem segments with red hairy roots) was 3.08 ± 0.12.

[0063] Table 1. Results of Agrobacterium rhizogenes infection of Lycium barbarum

[0064]

[0065] 4. Callus induction:

[0066] The effects of the concentrations and proportions of 6-benzyladenine (6-BA) and 1-naphthaleneacetic acid (NAA) on the callus induction rate of Lycium barbarum hairy roots were determined by a concentration gradient test. The red hairy roots were cut into small pieces of about 1 cm in length, and horizontally inoculated on the callus induction medium (MS medium containing 6-BA 0.2-1.0 mg / L, NAA 0.5 mg / L, Cef 150 mg / L, Tim 150 mg / L and PPT 5 mg / L, Figure 3 left), under the conditions of 16 h light (light intensity 36 μmol / m 2The red hairy roots were cultured in the dark for 8 h at 24-26 °C, and the callus induction rate was counted after 28 days. The results (Table 2) showed that the 6-BA concentration had a significant effect on the callus induction rate of the red hairy roots. The highest callus induction rate (96.88%) was obtained when the 6-BA concentration was 0.6 mg / L, and the second highest callus induction rate (87.10%) was obtained when the 6-BA concentration was 0.8 mg / L. The callus induction rate was lower (6.45-68.75%) when the 6-BA concentration was <0.6 mg / L or >0.8 mg / L. The purple-red callus was selected and transferred to fresh medium every 28 days (right), and the obtained callus proliferation material was used for adventitious shoot induction. Figure 3 The red hairy roots were cultured in the dark for 8 h at 24-26 °C, and the callus induction rate was counted after 28 days. The results (Table 2) showed that the 6-BA concentration had a significant effect on the callus induction rate of the red hairy roots. The highest callus induction rate (96.88%) was obtained when the 6-BA concentration was 0.6 mg / L, and the second highest callus induction rate (87.10%) was obtained when the 6-BA concentration was 0.8 mg / L. The callus induction rate was lower (6.45-68.75%) when the 6-BA concentration was <0.6 mg / L or >0.8 mg / L. The purple-red callus was selected and transferred to fresh medium every 28 days (right), and the obtained callus proliferation material was used for adventitious shoot induction.

[0067] Table 2 Callus induction results of the red hairy roots

[0068]

[0069] 5. Adventitious shoot induction:

[0070] The ability of the callus to regenerate adventitious shoots was gradually restored by alternating the concentrations and ratios of 6-BA and NAA. The obtained red callus was cut into small pieces of about 0.3 cm x 0.3 cm and was alternately transferred to the adventitious shoot induction medium (MS medium containing 6-BA 0.3-1.5 mg / L, NAA 0.1 mg / L, Cef 150 mg / L, Tim 150 mg / L, and PPT 5 mg / L) and the callus induction medium (MS medium containing 6-BA 0.6 mg / L, NAA 0.5 mg / L, Cef 150 mg / L, Tim 150 mg / L, and PPT 5 mg / L), and each culture lasted for 28 days. The culture conditions were 16 h light (light intensity 36 μmol / m 2 The adventitious shoot induction rate was counted once every cycle under the culture conditions of 16 h light (light intensity 36 μmol / m2 / s) and 8 h dark at 24-26 °C. The results (Table 3) showed that the culture times and 6-BA concentration had a significant effect on the adventitious shoot induction rate. The callus cultured in the medium with a 6-BA concentration of 0.6-1.5 mg / L began to produce adventitious shoots by the third cycle. The highest number of adventitious shoots was obtained on the medium with a 6-BA concentration of 0.6 mg / L by the fourth cycle, and each callus could obtain 2.34 adventitious shoots. The second highest number of adventitious shoots was obtained on the medium with a 6-BA concentration of 0.9 mg / L. The adventitious shoot induction rate was lower when the 6-BA concentration was <0.6 mg / L or >0.9 mg / L.

[0071] Table 3 Adventitious shoot induction results of the callus

[0072]

[0073] 6. Rooting of adventitious shoots:

[0074] To determine the effect of IBA concentration on the rooting of adventitious buds of wolfberry, red adventitious buds were surgically removed ( Figure 4 Cut off the roots and transfer them to adventitious root induction medium (MS medium containing IBA 0.00–1.00 mg / L, Cef 150 mg / L, and Tim 150 mg / L), and incubate under light for 16 h (light intensity 36 μmol / m²). 2 The plants were cultured for 21 days in darkness (8 hours) at 24–26°C, and the adventitious root induction rate and root number were statistically analyzed. The results (Table 4) showed that IBA concentration had no significant effect on the rooting rate of adventitious shoots, but it had a significant effect on the number of roots. The highest number of roots (25.14 shoots / shoot) was observed when the IBA concentration was 0.25 mg / L. Figure 5 However, without the addition of IBA, the number of adventitious shoots with roots was lower (13.20 shoots / shoot).

[0075] Table 4 Results of adventitious root induction from adventitious buds

[0076]

[0077] 7. Molecular detection:

[0078] DNA was extracted from the leaves of regenerated wolfberry plants using pCAMBIA3302 / Pro. 35S The ::RUBY vector served as a positive control, and wild-type wolfberry leaf DNA served as a negative control. The exogenous gene RUBY was detected by PCR. The primer sequences used were: F: 5'-CAACTTCCGCAAGATTACCG-3', R: 5'-CGAGATGTGGCTGAACTGAGAG-3'; the amplification program was: 94℃, 3 min; 95℃, 20 s; 53℃, 20 s; 72℃, 40 s, 40 cycles; 72℃, 10 min. Electrophoresis results showed that all regenerated plants obtained in this protocol were PCR positive, with a positive rate of 100%. Figure 6 ).

[0079] Example 3

[0080] Example 3 of the present invention illustrates the transplantation and domestication of transgenic Ningxia wolfberry, as detailed below:

[0081] 1. Preparation of transplanting substrate:

[0082] Mix perlite and TS-1 potting soil (Klasmann-Deilmann) at a volume ratio of 1:1 until homogeneous; add a small amount of distilled water to moisten, then sterilize by high-temperature moist heat (121℃, 0.11MPa, 20min) and cool to room temperature. Distribute the mixture into square seedling pots (7cm×7.5cm, diameter×height), and thoroughly moisten the transplanting substrate with sterile water.

[0083] 2. Plantlet transplanting

[0084] The rooted Ningxia wolfberry plants were taken out from the culture medium, washed with warm water at 40℃ to remove residual culture medium; and were transplanted into nutrient pots using a gun-shaped forceps and placed in a moistened seedling raising box (55 cm x 30 cm x 23 cm, length x width x height), and cultured under the conditions of 16 h light (light intensity 36 μmol / m 2 / s) and 8 h darkness, and a temperature of 24-26℃.

[0085] 3. Ventilation and domestication management:

[0086] During the culture, the ventilation holes on the transparent protective cover were gradually opened, and when the transplanted wolfberry plant top grew new leaves, the ventilation holes were completely opened, and after 7 days, the transparent protective cover was completely removed, and the seedling raising box was transferred to a greenhouse for routine maintenance (in summer, appropriate shading should be provided), and Hogeland solution was sprayed once every 14 days.

[0087] 4. Domestication effect and transgenic stability:

[0088] After the wolfberry plantlets were transplanted and domesticated for 25 days Figure 7 ), the transplanting survival rate reached 93.55% (29 / 31), and the obtained transgenic plants still showed obvious purple red, indicating that they could stably express the RUBY gene.

[0089] The process of the Ningxia wolfberry genetic transformation method based on adventitious bud induction provided by the present application is shown in Figure 8 As there is no report on successful genetic transformation of Ningxia wolfberry to obtain regenerated transgenic plants, the present application scheme cannot be compared with the prior art. However, the present application uses Agrobacterium rhizogenes instead of Agrobacterium tumefaciens, realizes the introduction and expression of exogenous genes in wolfberry somatic cells, and successfully induces the formation of regenerated plants, establishes a Ningxia wolfberry genetic transformation system, and has obvious novelty and practicality. In addition, based on the characteristic that the synthesis product (betacyanin) regulated by RUBY protein is purple red, the hairy roots, callus, adventitious buds and regenerated wolfberry plants with obvious purple red characteristics are sequentially screened by naked eye, and transgenic Ningxia wolfberry is obtained, which significantly improves the positive rate of transgenic plants, has outstanding substantial features, and can produce positive effects in application.

[0090] It should be noted that the above-described embodiments are preferred embodiments of the present application, and improvements and refinements made by those skilled in the art without departing from the principles of the present application should also be considered as the protection scope of the present application.

Claims

1. A method for genetic transformation of Lycium barbarum, characterized in that, The method comprises the following steps: (1) surface sterilization and aseptic culture of mature seeds of Lycium ruthenicum Murr, to obtain aseptic tissue culture seedlings, and stems of the aseptic tissue culture seedlings are used as genetic transformation receptors; (2) infection of the stems by Agrobacterium rhizogenes carrying a RUBY reporter gene, co-culture, and induction of hairy roots; (3) cutting of the hairy roots, inoculation on a callus induction medium to generate callus, and induction of adventitious buds by periodic alternate culture; (4) cutting of the adventitious buds, inoculation on a rooting medium to induce adventitious roots, and obtaining of regenerated plants; (5) RUBY gene PCR detection of the regenerated plants, and screening of positive transgenic plants; (6) transplantation and domestication of the positive plants, and obtaining of Lycium ruthenicum Murr transgenic plants capable of natural growth.

2. The method of claim 1, wherein, The surface sterilizing agent is a sodium trichloroisocyanurate solution with an effective chlorine concentration of 0.1% and containing 0.2% Tween-80.

3. The method of claim 1, wherein, The Agrobacterium rhizogenes is carrying pCAMBIA3302 / Pro 35S ::K599 strain of RUBY expression vector.

4. The method of claim 1, wherein, The preparation of the infection solution includes: culturing the bacterial solution to OD 600 0.4, and resuspended in liquid MS medium containing 0.2 mmol / L acetosyringone after centrifugal collection, and standing for 1-3 hours.

5. The method of claim 1, wherein, The stems are infected by injection, and the injection position is about 1-2 cm from the lower end of the stems; after infection, the stems are left to stand for 5 minutes, and then inoculated on a co-culture medium for dark culture for 3 days.

6. The method of claim 1, wherein, The formulations of the co-culture medium, the sterilization medium, the callus induction medium, the adventitious bud induction medium, and the adventitious root induction medium are as follows: the co-culture medium: MS+acetosyringone 0.2 mmol / L+sucrose 30 g / L+plant gel 2.5 g / L, pH 5.8; the sterilization medium: MS+Cef 150 mg / L+Tim 150 mg / L+sucrose 30 g / L+plant gel 2.5 g / L, pH 5.8; the callus induction medium: MS+6-BA 0.6 mg / L+NAA 0.5 mg / L+Cef 150 mg / L+Tim 150 mg / L+PPT 5 mg / L, pH 5.8; the adventitious bud induction medium: MS+6-BA 0.6 mg / L+NAA 0.1 mg / L+Cef 150 mg / L+Tim 150 mg / L+PPT 5 mg / L, pH 5.8; the adventitious root induction medium: 1 / 2MS+IBA 0.25 mg / L+Cef 150 mg / L+Tim 150 mg / L+sucrose 15 g / L+plant gel 2.5 g / L, pH 5.

8. The RUBY reporter gene makes the hairy roots, callus, adventitious buds, and regenerated plants present purple red by regulating betalain synthesis, and positive screening is performed by PCR detection. The transplanting substrate is a mixture of perlite and TS-1 nutrient soil at a volume ratio of 1:1, which is sterilized at 121°C and 0.11 MPa for 20 min before use.

7. The method of claim 1, wherein, The transgenic regenerated plants are domesticated in a greenhouse under conventional maintenance conditions for 25 days, and the survival rate of the transgenic plants after transplantation is more than 90%.

8. The method of claim 1, wherein, ​ 9. The method of claim 1, wherein, ​

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