A method for genetic transformation of Agrobacterium tumefaciens mediated by

Abstract

The present invention discloses a method for genetic transformation of Scutellaria baicalensis mediated by Agrobacterium, which belongs to the field of plant genetic engineering. The method effectively inhibits the browning of tissue culture by combining low-temperature dark treatment and antioxidants; the callus of Scutellaria baicalensis is treated with lanthanum chloride solution, which significantly improves its tolerance level to Agrobacterium infection, and finally constructs an Agrobacterium-mediated genetic transformation system suitable for Scutellaria baicalensis. The highest transformation efficiency of the genetic transformation system is 2.7%, which reaches the general level of genetic transformation efficiency of sugarcane cultivars mediated by Agrobacterium. The present invention achieves a breakthrough from 0 to 1 in the genetic transformation of Scutellaria baicalensis, lays a necessary foundation for the construction of a more efficient technical system in the future, and plays an important role in promoting the mining of excellent genes of Scutellaria baicalensis and biotechnology breeding of sugarcane.

Description

Technical Field

[0001] The invention relates to the field of plant genetic engineering, and in particular to a method for genetic transformation of scutellaria mediated by Agrobacterium. Background Art

[0002] Sugarcane (Saccharum spp.) is my country's primary sugar crop, contributing more than 90% of the country's sugar production. The selection and breeding of sugarcane varieties is the key and foundation for the healthy development of my country's sugarcane industry and the full guarantee of sugar safety. Modern sugarcane breeding relies heavily on the infiltration of wild germplasm. Saccharum spontaneum is the most successful wild germplasm resource for breeding among sugarcane's closely related plants. At present, almost all sugarcane cultivars contain Saccharum spontaneum bloodlines. Saccharum spontaneum has excellent characteristics such as strong tillering, strong stress resistance, wide adaptability, and resistance to barrenness, making it the main source of genes for stress resistance and adaptability in modern sugarcane breeding. The mining of excellent genes in Saccharum spontaneum has become an important topic in sugarcane genetic improvement.

[0003] Genetic transformation is the premise and key to plant gene identification. At present, there have been several reports on the establishment and optimization of sugarcane genetic transformation technology systems. However, these studies are limited to sugarcane cultivars, and research on wild sugarcane resources is relatively rare. There is no report on the research of genetic transformation technology systems for Saccharum officinale. In recent years, with the completion of whole genome sequencing, Saccharum officinale has become the first species in the genus Saccharum with a reference genome sequence, providing a large number of candidate genes for sugarcane breeding. In order to identify the functions and breeding value of these candidate genes, it is urgent to establish a genetic transformation system for Saccharum officinale.

[0004] Agrobacterium-mediated transformation is a preferred method for genetic transformation of sugarcane and its closely related plants due to its low cost, low copy number and good repeatability. The present invention attempts to establish an Agrobacterium-mediated genetic transformation technology system in S. sutchuenensis. During the research process, it was found that unlike commonly used sugarcane cultivars such as ROC22, S. sutchuenensis genetic transformation faces a serious browning problem, and this browning runs through almost the entire process of the experiment; in particular, the severe browning after Agrobacterium infection is very likely to cause Agrobacterium contamination, which has become a key issue in determining the success of genetic transformation (see Figure 1 and Figure 2 ). This problem makes it impossible for the current Agrobacterium-mediated sugarcane genetic transformation technology system based on cultivated species to obtain positive transgenic seedlings in S. scabra (see Comparative Example 1 of the present invention). Reducing the intensity of Agrobacterium infection can alleviate browning to a certain extent, but it will also cause a significant reduction in genetic transformation efficiency (see Comparative Example 2 of the present invention). How to balance the contradiction between the Agrobacterium infection level and the browning of S. scabra callus has become a key issue in the efficient genetic transformation of S. scabra. Summary of the invention

[0005] The purpose of the present invention is to provide a method for genetic transformation of Scutellaria baicalensis mediated by Agrobacterium tumefaciens to solve the problems existing in the above-mentioned prior art. The method overcomes the problem that the genetic transformation of Scutellaria baicalensis is prone to browning, successfully constructs an Agrobacterium tumefaciens mediated genetic transformation system of Scutellaria baicalensis callus, and achieves a breakthrough from 0 to 1 in genetic transformation of Scutellaria baicalensis.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a method for genetic transformation of scutellaria mediated by Agrobacterium, comprising the following steps:

[0008] (1) Using the top growth point of the cut palm as an explant, induction culture was performed to obtain embryonic callus tissue;

[0009] (2) heat shocking the embryonic callus in a LaCl3 solution, then ultrasonically treating the embryonic callus, and then transferring the embryonic callus to an Agrobacterium infection solution for infection; transferring the infected embryonic callus to a co-culture medium for dark culture, and then performing a sterilization treatment using a LaCl3 solution, an antioxidant solution, and a Timentin solution;

[0010] (3) transferring the embryonic callus after sterilization treatment to a recovery medium for dark culture;

[0011] (4) the embryonic callus after recovery culture is transferred to a callus screening medium and a differentiation screening medium in sequence for differentiation screening to obtain differentiated seedlings;

[0012] (5) transferring the differentiated seedlings to a root induction medium to induce rooting, thereby obtaining resistant seedlings;

[0013] (6) The resistant seedlings are hardened, transplanted, and identified for transgenic properties using conventional methods (such as herbicide spraying detection, molecular detection, etc.) to obtain positive transgenic cut-thick plants.

[0014] Preferably, in step (1), the method for obtaining the explant is: taking the shoot of a 6-8 month old plant of the Scutellaria baicalensis, peeling off the outer old leaves and leaf sheaths, sterilizing, and standing in the dark at 18-22°C for 14-36 hours, then aseptically obtaining a 2-7 cm section above the top growth point of the shoot, peeling off the outer old leaves layer by layer, and retaining the innermost 2-3 heart leaves, which are the explant.

[0015] Preferably, in step (1), during the induction culture, the induction medium comprises the following components: 1 / 2MS + 2,4-D 2.0 mg / L + citric acid 150 mg / L + cysteine ​​100 mg / L + PVP 500 mgL + sucrose 30 g / L + agar powder 8.0 g / L; the culture conditions are: dark culture at 28°C, subculture once every 3 weeks.

[0016] Preferably, in step (2), the embryonic callus is heat-shocked in a LaCl3 solution at 45°C for 3-5 min, and the concentration of the LaCl3 solution is 10-20 mM;

[0017] The ultrasonic treatment conditions are: 40kHz ultrasonic oscillation for 1-3min.

[0018] Preferably, in step (2), the dyeing solution comprises the following components: 1 / 2MS + 2,4-D 0.5 mg / L + sucrose 10-20 g / L + glucose 10-20 g / L + acetosyringone 20-30 mg / L;

[0019] The conditions of the impregnation treatment are: vacuum treatment at room temperature 28-44 kPa for 5-15 minutes, and then standing in the dark for 15-20 minutes;

[0020] Preferably, in step (2), the co-cultivation medium comprises the following components: 1 / 2MS + 2,4-D 1.0 mg / L + citric acid 150 mg / L + cysteine ​​100 mg / L + sucrose 10-15 g / L + glucose 15-20 g / L + acetosyringone 20 mg / L; the co-cultivation conditions are: 22°C in the dark for 3 days.

[0021] Preferably, in step (2), the embryonic callus tissue after co-cultivation is first washed with sterile water until clear, and then soaked in LaCl3 solution, antioxidant solution, and timentin solution for 20 minutes respectively, wherein the concentration of the LaCl3 solution is 10-13.5mM; the antioxidant solution is an aqueous solution containing 150mg / L citric acid, 100mg / L cysteine, and 50mM 2-morpholineethanesulfonic acid, with a pH value of 5.8; and the concentration of the timentin solution is 300mg / L.

[0022] Preferably, in step (3), the recovery medium comprises the following components: 1 / 2MS + 2,4-D 2.0 mg / L + citric acid 150 mg / L + cysteine ​​100 mg / L + PVP 500 mg / L + sucrose 30 g / L + agar powder 8.0 g / L + timentin 200 mg / L; the culture conditions are: 28°C in the dark for 2 weeks.

[0023] Preferably, in step (4), the callus screening medium comprises the following components: 1 / 2MS + 2,4-D 2.0 mg / L + citric acid 150 mg / L + cysteine ​​100 mg / L + PVP 500 mg / L + sucrose 30 g / L + agar powder 8.0 g / L + timentin 200 mg / L + PPT 1.0 mg / L; the culture conditions are: dark culture at 28°C, subculture every 3 weeks;

[0024] The differentiation screening medium comprises the following components: 1 / 2MS+6-BA 1.0 mg / L+citric acid 150 mg / L+cysteine ​​100 mg / L+PVP 500 mg / L+sucrose 30 g / L+agar powder 8.0 g / L+timentin 200 mg / L+PPT 1.0 mg / L; the culture conditions are: temperature 28°C, light intensity 60-70 μmol / s / m 2 The photoperiod was 12 h light / 12 h dark, and subculture was performed every 3 weeks.

[0025] Preferably, in step (5), the root induction medium comprises the following components: 1 / 2MS + NAA 2.0 mg / L + activated carbon 1.5 g / L + sucrose 30 g / L + agar powder 8.0 g / L + timentin 200 mg / L + PPT 0.5 mg / L; the culture conditions are: temperature 28 ° C, light intensity 60-70 μmol / s / m 2 The photoperiod was 12 h light / 12 h dark, and subculture was performed every 3 weeks.

[0026] The present invention discloses the following technical effects:

[0027] Based on the existing technology for constructing sugarcane genetic transformation system, the present invention successfully established a genetic transformation technology system in Scutellaria scabra for the first time by optimizing the technical scheme, and the transformation efficiency was above 2.7%, reaching the general level (2-5%) of Agrobacterium-mediated sugarcane genetic transformation. The present invention achieved a breakthrough from 0 to 1 in the genetic transformation of Scutellaria scabra. More importantly, the present invention broke the limitation of Scutellaria scabra as an important wild germplasm resource of sugarcane and lack of genetic transformation system, which is of great significance for the biological research of Scutellaria scabra and the application of sugarcane breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The browning difference of the explants of the heart leaf of the hand-cut mulberry after 2 weeks of callus induction culture on different culture media; A is the control treatment, that is, no antioxidant is added to the callus induction culture medium in Comparative Example 1 of the present invention; B is the optimized callus induction culture medium, that is, the antioxidant combination is added in Example 3 of the present invention;

[0029] Figure 2 The effects of different genetic transformation methods on callus of cut-hand mites; A and B respectively used different immersion and sterilization treatment methods, wherein A is a conventional method, i.e., the genetic transformation method of comparative example 1 of the present invention; B is an optimized method of the present invention, i.e., the genetic transformation method of example 3 of the present invention;

[0030] Figure 3The results of transgenic detection of VN2 scissors-resistant differentiated seedlings by conventional herbicide spraying methods are as follows: 400 mg / L glufosinate was sprayed when the seedlings were 6 weeks old, and the results were observed after 1 week; A is the non-transgenic VN2 scissors-resistant seedlings constructed in Comparative Example 1 of the present invention; B is the positive transgenic VN2 scissors-resistant seedlings constructed in Example 3 of the present invention. DETAILED DESCRIPTION

[0031] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0032] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0033] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0034] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0035] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0036] The present invention is based on the fact that there is a serious browning problem in the process of genetic transformation of the hand-splitting secretion system using the current method of genetic transformation of the genus Saccharum, and the genetic transformation system of the hand-splitting secretion has not been effectively constructed so far. After long-term exploration, the applicant team effectively suppressed the browning phenomenon by placing the explant in the dark at low temperature and using a combination of antioxidants; more importantly, the principle that some rare earth metal ions can passivate plant hypersensitivity reactions was used to treat the hand-splitting secretion callus with lanthanum chloride (LaCl3) solution, which significantly improved its tolerance level to Agrobacterium infection, and finally constructed an Agrobacterium-mediated genetic transformation technology system suitable for hand-splitting secretion. The highest conversion efficiency of hand-splitting secretion in this system is 2.7%, which reaches the general level of genetic transformation efficiency of sugarcane cultivars mediated by Agrobacterium. In general, the present invention has achieved a breakthrough from 0 to 1 in the genetic transformation of hand-splitting secretion, laid a necessary foundation for the construction of a more efficient technical system in the future, and played an important role in promoting the mining of excellent genes of hand-splitting secretion and sugarcane breeding. In order to further explain the above technical scheme in detail, it is further described below in the form of specific embodiments.

[0037] Example 1

[0038] 1. Induction of dense embryonic callus in cut hands

[0039] Select 6-month-old VN2 plants of Scutellaria baicalensis, take their tips (15 cm below the top growth point to +1 cm leaf base), peel off the outer old leaves and leaf sheaths, wrap them with sterilized newspapers, and place them in the dark at 18°C ​​for 14 hours. After 75% alcohol surface disinfection, take the 2-7 cm section above the top growth point of Scutellaria baicalensis in the clean bench, peel off the outer old leaves layer by layer, keep the innermost 2-3 heart leaves, cut into 3×5 mm single-layer leaf blocks, inoculate the callus induction medium with the back of the leaves facing up, culture in the dark at 28°C, subculture once every 3 weeks, and obtain callus tissue; the callus induction medium is 1 / 2MS+2,4-D 2.0mg / L+citric acid 150mg / L+cysteine ​​100mg / L+PVP 500mg / L+sucrose 30g / L+agar powder 8.0g / L, pH 5.8.

[0040] 2. Agrobacterium infection

[0041] Pretreatment: The VN2 embryonic callus obtained by the previous step was selected for pretreatment before infection as follows: the callus was placed in a LaCl3 solution preheated to 45°C and with a concentration of 10mM for 3 minutes, and then transferred to an ultrasonic water bath at the same temperature and oscillated at 40kHz for 1 minute.

[0042] Infection: The pretreated VN2 embryonic callus was transferred to the Agrobacterium infection solution, vacuum treated at room temperature at 28kPa for 5 minutes, and then left in the dark for 20 minutes. The Agrobacterium infection solution was obtained by resuspending the EHA105 engineering bacteria culture with the plant binary expression vector in the infection medium (the method refers to the invention patent of Li Chunjia et al. "A method for efficient genetic transformation of sugarcane callus mediated by Agrobacterium" (ZL202210545664.0), the same below), and its concentration was adjusted to OD 600 =0.4; the infection medium contained the following components: 1 / 2MS+2,4-D 0.5 mg / L+sucrose 10 g / L+glucose 20 g / L+acetosyringone (AS) 20 mg / L, pH 5.2.

[0043] Co-cultivation: VN2 calli infected with Agrobacterium were placed on filter paper soaked in co-cultivation medium and cultured in the dark at 22°C for 3 days; the co-cultivation medium was 1 / 2MS + 2,4-D 1.0 mg / L + citric acid 150 mg / L + cysteine ​​100 mg / L + sucrose 10 g / L + glucose 20 g / L + AS 20 mg / L, pH 5.2.

[0044] Cleaning of bacteria: After co-culture, the VN2 callus was first washed with sterile water until clear, and then immersed in LaCl3 solution, antioxidant solution, and Timentin (Tim) solution for 20 min respectively; the concentration of LaCl3 solution was 10 mM; the antioxidant solution contained 150 mg / L citric acid, 100 mg / L cysteine, and 50 mM 2-Morpholinoethanesulfonic Acid (MES), pH = 5.8; the concentration of Tim solution was 300 mg / L.

[0045] 3. Screening and differentiation

[0046] Recovery culture: The VN2 callus after bacterial removal was inoculated into recovery medium and cultured in the dark at 28°C for 2 weeks; the recovery medium consisted of 1 / 2MS + 2,4-D 2.0 mg / L + citric acid 150 mg / L + cysteine ​​100 mg / L + PVP 500 mg / L + sucrose 30 g / L + agar powder 8.0 g / L + Tim 200 mg / L, pH 5.8.

[0047] Callus screening: The recovered VN2 calli were transferred to callus screening medium, cultured in the dark at 28°C, and subcultured every 3 weeks; the callus screening medium was 1 / 2MS + 2,4-D 2.0 mg / L + citric acid 150 mg / L + cysteine ​​100 mg / L + PVP 500 mg / L + sucrose 30 g / L + agar powder 8.0 g / L + Tim 200 mg / L + glufosinate (PPT) 1.0 mg / L, pH 5.8.

[0048] Differentiation screening: Resistant calli were transferred to differentiation screening medium at 28°C and light intensity of 60-70 μmol / s / m 2 Light culture was carried out under ambient conditions with a photoperiod of 12 h light / 12 h dark, and subculture was carried out every 3 weeks; the differentiation screening medium was 1 / 2MS + 6-BA 1.0 mg / L + citric acid 150 mg / L + cysteine ​​100 mg / L + PVP 500 mg / L + sucrose 30 g / L + agar powder 8.0 g / L + Tim 200 mg / L + PPT 1.0 mg / L, pH 5.8.

[0049] Rooting screening: The culture conditions of resistant differentiated seedlings on the rooting induction medium were consistent with the differentiation culture; the rooting screening medium was 1 / 2MS + NAA 2.0 mg / L + activated carbon 1.5 g / L + sucrose 30 g / L + agar powder 8.0 g / L + Tim 200 mg / L + PPT 0.5 mg / L, pH 5.8.

[0050] 4. Planting and identification

[0051] After hardening, transplanting and transgenic testing, the above-mentioned resistant differentiated seedlings are obtained to obtain positive transgenic cut-hand dense plants, which are then transplanted.

[0052] Example 2

[0053] 1. Induction of dense embryonic callus in cut hands

[0054] Select 8-month-old VN2 plants of the cutting hand, take their tips (15 cm below the top growth point to +1 cm leaf base), peel off the outer old leaves and leaf sheaths, wrap them with sterilized newspapers, and place them in the dark at 22°C for 36 hours. After 75% alcohol surface disinfection, take the 2-7 cm section above the top growth point of the cutting hand in the clean bench, peel off the outer old leaves layer by layer, keep the innermost 2-3 heart leaves, cut into 3×5 mm single-layer leaf blocks, inoculate the callus induction medium with the back of the leaves facing up, culture in the dark at 28°C, subculture once every 3 weeks, and obtain callus tissue; the callus induction medium is 1 / 2MS+2,4-D 2.0mg / L+citric acid 150mg / L+cysteine ​​100mg / L+PVP 500mg / L+sucrose 30g / L+agar powder 8.0g / L, pH 5.8.

[0055] 2. Agrobacterium infection

[0056] Pretreatment: The cut-hand dense embryonic callus obtained by induction in the previous step was selected for pretreatment before infection as follows: the callus was placed in a LaCl3 solution preheated to 45°C and with a concentration of 20mM for 5 minutes for heat shock, and then transferred to an ultrasonic water bath at the same temperature and oscillated at 40kHz for 3 minutes.

[0057] Infection: The pretreated cut embryogenic callus was placed in Agrobacterium infection solution, vacuum treated at room temperature at 44 kPa for 10 minutes, and then left in the dark for 15 minutes. The Agrobacterium infection solution was obtained by resuspending the EHA105 engineering bacteria culture with the plant binary expression vector in the infection medium, and its concentration was adjusted to OD 600 =0.5; the infection medium contained the following components: 1 / 2MS+2,4-D 0.5mg / L+sucrose 20g / L+glucose 10g / L+AS 30mg / L, pH 5.2.

[0058] Co-cultivation: The callus of P. tumefaciens infected with Agrobacterium was placed on filter paper soaked in co-cultivation medium and cultured in the dark at 22°C for 3 days; the co-cultivation medium was 1 / 2MS + 2,4-D 1.0 mg / L + citric acid 150 mg / L + cysteine ​​100 mg / L + sucrose 10 g / L + glucose 20 g / L + AS 20 mg / L, pH 5.2.

[0059] Cleaning of bacteria: After co-cultivation, the cut callus was first washed with sterile water until clear, and then immersed in LaCl3 solution, antioxidant solution, and Timentin solution for 20 min respectively; the concentration of the LaCl3 solution was 20 mM; the antioxidant solution contained 150 mg / L citric acid, 100 mg / L cysteine, and 50 mM 2-morpholineethanesulfonic acid, pH = 5.8; the concentration of the Tim solution was 300 mg / L.

[0060] 3. Screening and differentiation

[0061] Recovery culture: The cleaned callus was inoculated into recovery medium and cultured in the dark at 28°C for 1-2 weeks; the recovery medium was 1 / 2MS + 2,4-D 2.0 mg / L + citric acid 150 mg / L + cysteine ​​100 mg / L + PVP 500 mg / L + sucrose 30 g / L + agar powder 8.0 g / L + Tim 200 mg / L, pH 5.8.

[0062] Callus screening: The recovered calli of the cut palm were transferred to callus screening medium, cultured in the dark at 28°C, and subcultured every 3 weeks; the callus screening medium was 1 / 2MS+2,4-D 2.0mg / L+citric acid 150mg / L+cysteine ​​100mg / L+PVP 500mg / L+sucrose 30g / L+agar powder 8.0g / L+Tim 200mg / L+PPT 1.0mg / L, pH 5.8.

[0063] Differentiation screening: Resistant calli were transferred to differentiation screening medium at 28°C and light intensity of 60-70 μmol / s / m 2The cells were cultured under ambient conditions with a light cycle of 12 h light / 12 h dark, and subcultured every 3 weeks. The differentiation screening medium was 1 / 2MS+6-BA1.0 mg / L+citric acid 150 mg / L+cysteine ​​100 mg / L+PVP 500 mg / L+sucrose 30 g / L+agar powder 8.0 g / L+Tim 200 mg / L+PPT 1.0 mg / L, pH 5.8.

[0064] Rooting screening: The culture conditions of resistant differentiated seedlings on the rooting induction medium were consistent with the differentiation culture; the rooting screening medium was 1 / 2MS + NAA 2.0 mg / L + activated carbon 1.5 g / L + sucrose 30 g / L + agar powder 8.0 g / L + Tim 200 mg / L + PPT 0.5 mg / L, pH 5.8.

[0065] 4. Planting and identification

[0066] After hardening, transplanting and transgenic testing, the above-mentioned resistant differentiated seedlings are obtained to obtain positive transgenic cut-hand dense plants, which are then transplanted.

[0067] Example 3

[0068] 1. Induction of dense embryonic callus in cut hands

[0069] Select 7-month-old scissor-cut plants, take their tips (15 cm below the top growth point to +1 cm leaf base), peel off the outer old leaves and leaf sheaths, wrap them with sterilized newspapers, and place them in the dark at 22°C for 24 hours. After disinfecting the surface with 75% alcohol, take a 2-7 cm section above the top growth point of the scissor-cut plants in a clean bench, peel off the outer old leaves layer by layer, keep the innermost 2-3 heart leaves, cut into ~3×5 mm single-layer leaf blocks, inoculate the callus induction medium with the back of the leaves facing up, and culture them in the dark at 28°C. Subculture once every 3 weeks to obtain callus tissue (see Figure 1 Medium B and Figure 2 Callus induction medium was 1 / 2MS + 2,4-D 2.0 mg / L + citric acid 150 mg / L + cysteine ​​100 mg / L + PVP 500 mg / L + sucrose 30 g / L + agar powder 8.0 g / L, pH 5.8.

[0070] 2. Agrobacterium infection

[0071] Pretreatment: The cut-hand dense embryonic callus obtained by induction in the previous step was selected for pretreatment before inoculation. The method was as follows: the callus was placed in a LaCl3 solution preheated to 45°C and with a concentration of 13.5 mM for 5 minutes, and then transferred to an ultrasonic water bath at the same temperature and oscillated at 40 kHz for 1 minute;

[0072] Infection: The pretreated cut embryogenic callus was placed in Agrobacterium infection solution, vacuum treated at room temperature at 44 kPa for 10 minutes, and then left in the dark for 20 minutes. The Agrobacterium infection solution was obtained by resuspending the EHA105 engineering bacteria culture with the plant binary expression vector in the infection medium, and its concentration was adjusted to OD 600 =0.5; the infection medium contained the following components: 1 / 2MS+2,4-D 0.5mg / L+sucrose 15g / L+glucose 15g / L+AS20mg / L, pH 5.2.

[0073] Co-cultivation: The callus of P. tumefaciens infected with Agrobacterium was placed on filter paper soaked in co-cultivation medium and cultured in the dark at 22°C for 3 days; the co-cultivation medium was 1 / 2MS+2,4-D 1.0 mg / L+citric acid 150 mg / L+cysteine ​​100 mg / L+sucrose 15 g / L+glucose 15 g / L+AS 20 mg / L, pH 5.2.

[0074] Cleaning of bacteria: After co-cultivation, the cut callus was first washed with sterile water until clear, and then immersed in LaCl3 solution, antioxidant solution, and Timentin solution for 20 min respectively; the concentration of LaCl3 solution was 13.5 mM; the antioxidant solution contained 150 mg / L citric acid, 100 mg / L cysteine, and 50 mM 2-morpholineethanesulfonic acid, pH = 5.8; the concentration of Tim solution was 300 mg / L.

[0075] 3. Screening and differentiation

[0076] Recovery culture: The cleaned callus was inoculated into recovery medium and cultured in the dark at 28°C for 2 weeks; the recovery medium was 1 / 2MS + 2,4-D 2.0 mg / L + citric acid 150 mg / L + cysteine ​​100 mg / L + PVP 500 mg / L + sucrose 30 g / L + agar powder 8.0 g / L + Tim 200 mg / L, pH 5.8.

[0077] Callus screening: The recovered calli of the cut palm were transferred to callus screening medium, cultured in the dark at 28°C, and subcultured every 3 weeks; the callus screening medium was 1 / 2MS+2,4-D 2.0mg / L+citric acid 150mg / L+cysteine ​​100mg / L+PVP 500mg / L+sucrose 30g / L+agar powder 8.0g / L+Tim 200mg / L+PPT 1.0mg / L, pH 5.8.

[0078] Differentiation screening: Resistant calli were transferred to differentiation screening medium at 28°C and light intensity of 60-70 μmol / s / m 2Light culture was carried out under ambient conditions with a photoperiod of 12 h light / 12 h dark, and subculture was carried out every 3 weeks; the differentiation screening medium was 1 / 2MS + 6-BA 1.0 mg / L + citric acid 150 mg / L + cysteine ​​100 mg / L + PVP 500 mg / L + sucrose 30 g / L + agar powder 8.0 g / L + Tim 200 mg / L + PPT 1.0 mg / L, pH 5.8.

[0079] Rooting screening: The culture conditions of resistant differentiated seedlings on the rooting induction medium were consistent with the differentiation culture; the rooting screening medium was 1 / 2MS + NAA 2.0 mg / L + activated carbon 1.5 g / L + sucrose 30 g / L + agar powder 8.0 g / L + Tim 200 mg / L + PPT 0.5 mg / L, pH 5.8.

[0080] 4. Planting and identification

[0081] After hardening, transplanting and transgenic testing, the resistant differentiated seedlings are subjected to positive transgenic sugarcane seedlings, which are then transplanted.

[0082] Comparative Example 1

[0083] 1. Induction of dense embryonic callus in cut hands

[0084] Select 7-month-old VN2 plants of the cutting hand dense plant, take the top (15cm below the top growth point to +1cm leaf base), peel off the outer old leaves and leaf sheaths. After 75% alcohol surface disinfection, immediately take the 2-7cm section above the top growth point of the cutting hand dense plant in the clean bench, peel off the outer old leaves layer by layer, keep the innermost 2-3 heart leaves, cut into 3×5mm single-layer leaf blocks, inoculate the callus induction medium with the back of the leaf facing up, culture in the dark at 28℃, subculture once every 3 weeks, and obtain callus tissue (see Figure 1 Middle A and Figure 2 Callus induction medium was MS+2,4-D 3.0 mg / L+sucrose 30 g / L+agar powder 8.0 g / L, pH 5.8.

[0085] 2. Agrobacterium infection

[0086] The difference from "2. Agrobacterium infection" in Example 3 is:

[0087] (1) No preprocessing is performed.

[0088] (2) Infection: VN2 embryonic callus was placed in Agrobacterium infection solution, vacuum treated at room temperature at 44 kPa for 5 min, and then left in the dark for 25 min. Agrobacterium infection solution was obtained by resuspending the EHA105 engineering bacteria culture carrying the plant binary expression vector in the infection medium, and its concentration was adjusted to OD 600=0.5; the infection medium components are: MS+2,4-D 2.0mg / L+sucrose 10g / L+glucose 20g / L+AS20mg / L, pH 5.2.

[0089] (3) Co-culture: The co-culture medium was MS + 2,4-D 2.0 mg / L + sucrose 10 g / L + glucose 20 g / L + AS 20 mg / L, pH 5.2.

[0090] (4) Cleaning: After the above co-culture, the VN2 callus was first washed with sterile water until clear, and then immersed in 300 mg / L timentin solution for 20 min.

[0091] Other method steps are the same as in Example 3.

[0092] 3. Screening and differentiation

[0093] The difference from "3. Screening and differentiation" in Example 3 is that:

[0094] (1) Recovery culture: The recovery medium is MS + 2,4-D 3.0 mg / L + sucrose 30 g / L + agar powder 8.0 g / L + Tim 200 mg / L, pH 5.8.

[0095] (2) Callus screening: The callus screening medium was MS + 2,4-D 3.0 mg / L + sucrose 30 g / L + agar powder 8.0 g / L + Tim 200 mg / L + PPT 1.0 mg / L, pH 5.8.

[0096] (3) Differentiation screening: The differentiation screening medium is 1 / 2MS + 6-BA 2.0 mg / L + sucrose 30 g / L + agar powder 8.0 g / L + Tim 200 mg / L + PPT 1.0 mg / L, pH 5.8.

[0097] The other method steps are the same.

[0098] 4. Planting and identification

[0099] Same as "4. transplanting and identification" in Example 3.

[0100] Comparative Example 2

[0101] 1. Induction of dense embryonic callus in cut hands

[0102] Select 6-8 month old VN2 plants of the cutting hand, take their tips (15 cm below the top growth point to +1 cm leaf base), peel off the outer old leaves and leaf sheaths. After 75% alcohol surface disinfection, immediately take the 2-7 cm section above the top growth point of the cutting hand in the clean bench, peel off the outer old leaves layer by layer, keep the innermost 2-3 heart leaves, cut into 3×5 mm single-layer leaf blocks, inoculate the callus induction medium with the back of the leaves facing up, culture in the dark at 28℃, subculture once every 3 weeks, and obtain callus tissue; the callus induction medium is 1 / 2MS+2,4-D 2.0mg / L+sucrose 30g / L+agar powder 8.0g / L, pH 5.8.

[0103] 2. Agrobacterium infection

[0104] The difference from "2. Agrobacterium infection" in Example 3 is:

[0105] (1) No preprocessing is performed.

[0106] (2) Infection: Cut embryogenic callus was placed in Agrobacterium infection solution, vacuum treated at room temperature at 28 kPa for 5 min, and then left in the dark for 25 min. Agrobacterium infection solution was obtained by resuspending the EHA105 engineering bacteria culture with the plant binary expression vector in the infection medium, and its concentration was adjusted to OD 600 =0.2; the composition of the infection medium is: 1 / 2MS + 2,4-D 1.0mg / L + sucrose 10g / L + glucose 20g / L + AS 20mg / L, pH 5.2.

[0107] (3) Co-culture: The co-culture medium was 1 / 2MS + 2,4-D 1.0 mg / L + sucrose 10 g / L + glucose 20 g / L + AS 20 mg / L, pH 5.2.

[0108] (4) Cleaning: After co-cultivation, the VN2 calli were first washed with sterile water until clear, and then immersed in a 300 mg / L timentin solution for 20 min.

[0109] The other method steps are the same.

[0110] 3. Screening and differentiation

[0111] The difference from "3. Screening and differentiation" in Example 3 is that:

[0112] (1) Recovery culture: The recovery medium is 1 / 2MS + 2,4-D 2.0 mg / L + sucrose 30 g / L + agar powder 8.0 g / L + Tim 200 mg / L, pH 5.8.

[0113] (2) Callus screening: The callus screening medium was 1 / 2MS + 2,4-D 2.0 mg / L + sucrose 30 g / L + agar powder 8.0 g / L + Tim 200 mg / L + PPT 1.0 mg / L, pH 5.8.

[0114] (3) Differentiation screening: The differentiation screening medium is 1 / 2MS + 6-BA 1.0 mg / L + sucrose 30 g / L + agar powder 8.0 g / L + Tim 200 mg / L + PPT 1.0 mg / L, pH 5.8.

[0115] The other method steps are the same.

[0116] 4. Planting and identification

[0117] Same as "4. transplanting and identification" in Example 1.

[0118] Comparative Example 3

[0119] 1. Induction of dense embryonic callus in cut hands

[0120] The same as "1. Induction of dense embryonic callus from cut hands" in Example 3.

[0121] 2. Agrobacterium infection

[0122] The difference from "2. Agrobacterium infection" in Example 1 is:

[0123] (1) No preprocessing is performed.

[0124] (2) Infection: The cut embryogenic callus was placed in Agrobacterium infection solution, vacuum treated at room temperature at 28 kPa for 5 minutes, and then left in the dark for 25 minutes. The Agrobacterium infection solution was obtained by resuspending the EHA105 engineering bacteria culture containing the plant binary expression vector in the infection medium, and its concentration was adjusted to OD 600 =0.2-0.3; the infection medium contained the following components: 1 / 2MS+2,4-D1.0mg / L+sucrose 10g / L+glucose 20g / L+AS20mg / L, pH 5.2.

[0125] (3) Co-culture: The co-culture medium was 1 / 2MS + 2,4-D 1.0 mg / L + citric acid 150 mg / L + cysteine ​​100 mg / L + sucrose 10 g / L + glucose 20 g / L + AS 20 mg / L, pH 5.2.

[0126] (4) The concentration of LaCl3 solution is 10mM.

[0127] The other method steps are the same.

[0128] 3. Screening and differentiation

[0129] Same as “3. Screening and differentiation” in Example 3.

[0130] 4. Planting and identification

[0131] Same as "4. transplanting and identification" in Example 3.

[0132] Comparative Example 4

[0133] 1. Induction of dense embryonic callus in cut hands

[0134] The same as "1. Induction of dense embryonic callus from cut hands" in Example 3.

[0135] 2. Agrobacterium infection

[0136] The difference from "2. Agrobacterium infection" in Example 3 is:

[0137] (1) Pretreatment: The VN2 embryonic callus obtained by the previous step was selected for pretreatment before infection. The method was as follows: the callus was placed in a 10 mM LaCl3 solution preheated to 45°C for 5 min, and then transferred to an ultrasonic water bath at the same temperature and oscillated at 40 kHz for 1 min.

[0138] (2) Infection: Cut hand dense embryogenic callus was placed in Agrobacterium infection solution, vacuum treated at room temperature 28kPa for 5 minutes, and then left in the dark for 25 minutes. The Agrobacterium infection solution contained the following components: 1 / 2MS+2,4-D 1.0mg / L+sucrose 10g / L+glucose 20g / L+AS 20mg / L, pH 5.2; its bacterial solution OD 600 =0.2.

[0139] (3) Co-culture: The co-culture medium was 1 / 2MS + 2,4-D 1.0 mg / L + citric acid 150 mg / L + cysteine ​​100 mg / L + sucrose 10 g / L + glucose 20 g / L + AS 20 mg / L, pH 5.2.

[0140] (4) Bacterial sterilization: The concentration of LaCl3 solution is 10 mM.

[0141] Other method steps are the same as in Example 1.

[0142] 3. Screening and differentiation

[0143] Same as "3. Screening and differentiation" in Example 1.

[0144] 4. Planting and identification

[0145] Same as "implantation and identification" in Example 1.

[0146] The genetic transformation method disclosed in Examples 1-3 is a process of continuously optimizing the optimal parameter combination of the method using the VN2 of the cut hand. Genetic transformation was carried out using the methods of Examples 1-3, and transgenic seedlings were successfully obtained. The results are shown in Table 1.

[0147] Table 1 Agrobacterium-mediated genetic transformation of VN2

[0148]

[0149] Note: 1 Explant inoculation number, the number of explants inoculated on callus induction medium without contamination; 2 Browning contamination rate of infected callus, the rate of callus contaminated by excessive growth of Agrobacterium caused by browning due to Agrobacterium infection; 3 Transformation efficiency = number of positive seedlings / number of transformed calli × 100%. Only one positive seedling formed by the same callus was counted. The numbers in the table are the means of 3 replicates. ns, p>0.05.

[0150] As shown in Table 1, the number of explants and infected calli was highly consistent among the experimental groups of each embodiment, and no significant difference was observed. Under this background, there was no significant difference in the browning rate of explants, the browning contamination rate of infected calli, and the final transformation efficiency among different embodiments. This shows that the method of the present invention can effectively produce transgenic seedlings of Scutellaria baicalensis within the technical parameter range shown in the three embodiments; it also shows that the method of the present invention has strong robustness within a certain range. Among them, the transformation efficiency of Example 3 is slightly higher than that of the other two, which is a more optimized method.

[0151] Comparative Examples 1-4 partially reflect the invention process of the present method. Comparative Example 1 is an early attempt at genetic transformation of the cut-hand honey mediated by Agrobacterium. The transformation method is a relatively common method for genetic transformation of sugarcane at present, and antioxidants and LaCl3 are not used throughout the process. Comparative Example 2 is an attempt to solve the serious browning problem of Comparative Example 1. The concentration of various plant hormones is reduced throughout the process, and 1 / 2MS medium with half of the macroelements is used as the basic medium, and the concentration of the engineered bacteria impregnation liquid is reduced, but antioxidants and LaCl3 are still not used throughout the process. Comparative Example 3 is a group of fruitful attempts in a large number of exploratory experiments on the browning problem. The explants are pretreated with low temperature and darkness, and an antioxidant combination of citric acid, cysteine, and PVP is used, but LaCl3 is not used. Comparative Example 4 is a group of experimental results in a large number of exploratory experiments further carried out for the excessive growth of Agrobacterium caused by browning after impregnation. The cut-hand honey callus is treated with LaCl3 solution, which is close to the final optimization scheme, and the only difference is that the concentration of the impregnated bacterial liquid is low.

[0152] Table 2 Results of genetic transformation of VN2 mediated by different methods

[0153]

[0154] Note: 1 , 2 , 3 Definitions are the same as in Table 1. The numbers in the table are the means of more than 3 repeated experiments, and different letters indicate significant differences at the P < 0.05 level, Duncan test.

[0155] As can be seen from the results in Table 2, in Comparative Example 1, the browning of the callus induction and infection callus differentiation stages of the explants of the cut hand dense was very serious, and no transgenic plants were finally produced, indicating that the current general method of sugarcane transgenic is completely unsuitable for the cut hand dense. In Comparative Example 2, the browning of the callus induction stage of the explants of the cut hand dense was alleviated, but the browning rate was still unacceptable; the infection callus differentiation stage was not even improved, and no transgenic plants were finally produced, indicating that the conventional adjustment of the culture medium components could not effectively solve the browning problem of the cut hand dense. In Comparative Example 3, the browning of the cut hand dense was relatively fully alleviated, but the browning of the callus after Agrobacterium infection and the Agrobacterium outbreak caused by it were still relatively serious, and a small number of transgenic seedlings were finally obtained, indicating that the combined use of antioxidants is necessary to inhibit the browning of the cut hand dense at different stages. In Comparative Example 4, the browning of the callus induction and differentiation screening stages of the cut hand dense was effectively inhibited, and several transgenic seedlings were finally obtained, and the genetic transformation efficiency reached and exceeded 1%, indicating that the idea of ​​using LaCl3 to inhibit the hypersensitive reaction of the cut hand dense infection callus is correct. The inventors further assumed that the combined use of the antioxidant and LaCl3 in Comparative Example 4 can enhance the tolerance of S. sutchuenensis to Agrobacterium, thereby increasing the Agrobacterium infection concentration to a normal level, and finally obtained the optimization method represented by Example 3.

[0156] In addition, the present invention also uses the sugarcane seedlings constructed in Example 3 and Comparative Example 1 as examples to verify their herbicide resistance. Specifically, the transgenic VN2 cut-hand dense seedlings constructed in Example 3 were sprayed with 400 mg / L of glufosinate-ammonium herbicide at 6 weeks of age and the phenotype was observed after 1 week. The non-transgenic VN2 cut-hand dense seedlings constructed in Comparative Example 1 were used as a control.

[0157] The results are as follows Figure 3 As shown, after the transgenic VN2 cut-hand dense seedlings constructed by the present invention were sprayed with glufosinate herbicide, the leaves did not dry up, while the leaves of the control turned yellow and dried up.

[0158] In summary, the method of the present invention is systematic and is based on the above iterative exploration process, and the results are obtained by continuous hypothesis and verification.

[0159] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for Agrobacterium-mediated genetic transformation of Psoralea corylifolia, characterized in that: The following steps are involved: (1) Using the heart leaf at the top growth point of the cut palm as an explant, induction culture was performed to obtain embryonic callus tissue; (2) heat shocking the embryonic callus in a LaCl3 solution, then ultrasonically treating the embryonic callus, and then transferring the embryonic callus to an Agrobacterium infection solution for infection; transferring the infected embryonic callus to a co-culture medium for co-cultivation, and then performing a sterilization treatment using a LaCl3 solution, an antioxidant solution, and a Timentin solution; (3) transferring the embryonic callus after sterilization treatment to a recovery medium for dark culture; (4) the embryonic callus after recovery culture is transferred to a callus screening medium and a differentiation screening medium in sequence for differentiation screening to obtain differentiated seedlings; (5) transferring the differentiated seedlings to a root induction medium to induce rooting, thereby obtaining resistant seedlings; (6) Hardening, transplanting and transgenic identification of resistant seedlings to obtain positive transgenic cut-off plants; In step (1), during the induction culture, the induction medium components are: 1 / 2MS + 2,4-D 2.0 mg / L + citric acid 150 mg / L + cysteine ​​100 mg / L + polyvinyl pyrrolidone 500 mg / L + sucrose 30 g / L + agar powder 8.0 g / L; In step (2), the embryonic callus is heat-shocked in a LaCl3 solution at 45°C for 3-5 minutes, wherein the concentration of the LaCl3 solution is 10-20 mM; the ultrasonic treatment conditions are: 40 kHz ultrasonic oscillation for 1-3 minutes; The composition of the dyeing liquid is: 1 / 2MS + 2,4-D 0.5mg / L + sucrose 10-20g / L + glucose 10-20g / L + acetosyringone 20-30mg / L; the dyeing treatment conditions are: vacuum treatment at room temperature 28-44kPa for 5-15min, and then standing in the dark for 15-20min; The co-culture medium components are: 1 / 2MS+2,4-D 1.0 mg / L+citric acid 150 mg / L+cysteine ​​100 mg / L+sucrose 10-15 g / L+glucose 15-20 g / L+acetosyringone 20 mg / L; During the sterilization treatment, the concentration of the LaCl3 solution is 10-13.5 mM; the antioxidant solution is an aqueous solution containing 150 mg / L citric acid, 100 mg / L cysteine, and 50 mM 2-morpholineethanesulfonic acid, with a pH value of 5.8; the concentration of the timentin solution is 300 mg / L; In step (3), the recovery medium composition is: 1 / 2MS + 2,4-D 2.0 mg / L + citric acid 150 mg / L + cysteine ​​100 mg / L + polyvinyl pyrrolidone 500 mg / L + sucrose 30 g / L + agar powder 8.0 g / L + timentin 200 mg / L; In step (4), the callus screening medium components are: 1 / 2MS + 2,4-D 2.0 mg / L + citric acid 150 mg / L + cysteine ​​100 mg / L + polyvinyl pyrrolidone 500 mg / L + sucrose 30 g / L + agar powder 8.0 g / L + timentin 200 mg / L + glufosinate 1.0 mg / L; The differentiation screening medium comprises: 1 / 2MS+6-BA 1.0 mg / L+citric acid 150 mg / L+cysteine ​​100 mg / L+polyvinylpyrrolidone 500 mg / L+sucrose 30 g / L+agar powder 8.0 g / L+timentin 200 mg / L+glufosinate 1.0 mg / L; In step (5), the root induction medium composition is: 1 / 2MS+NAA 2.0mg / L+activated carbon 1.5g / L+sucrose 30g / L+agar powder 8.0g / L+timentin 200mg / L+glufosinate 0.5mg / L; The method for obtaining the explant is as follows: taking the shoot of a 6-8 month old plant of the cut hand dense plant, peeling off the outer old leaves and leaf sheaths, sterilizing, and standing in the dark at 18-22° C. for 14-36 hours, then aseptically obtaining a 2-7 cm section above the top growth point of the cut hand dense shoot, peeling off the outer old leaves layer by layer, and retaining the innermost 2-3 heart leaves, which are the explant.

2. The method according to claim 1, characterized in that In step (1), during the induction culture, the culture conditions are: dark culture at 28°C, and subculture once every 3 weeks.

3. The method according to claim 1, characterized in that In step (2), the co-cultivation conditions are: 22° C. in the dark for 3 days.

4. The method according to claim 1, characterized in that In step (2), the embryonic callus tissue after co-cultivation is first washed with sterile water until clear, and then soaked in LaCl3 solution, antioxidant solution, and Timentin solution for 20 minutes respectively.

5. The method according to claim 1, characterized in that In step (3), the culture conditions are: 28°C in the dark for 2 weeks.

6. The method according to claim 1, characterized in that In step (4), during callus screening, the culture conditions are: dark culture at 28°C, subculture every 3 weeks; During differentiation screening, the culture conditions were: temperature 28°C, light intensity 60-70 μmol / s / m 2 The photoperiod was 12 h light / 12 h dark, and subculture was performed every 3 weeks.

7. The method according to claim 1, characterized in that In step (5), the culture conditions are: temperature 28°C, light intensity 60-70 μmol / s / m 2 The photoperiod was 12 h light / 12 h dark, and subculture was performed every 3 weeks.

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