A non-tissue culture method for regenerating non-heading Chinese cabbage

Abstract

The invention discloses a non-tissue culture method for regenerating non-heading cabbage, which comprises the following steps: placing non-heading cabbage seeds treated with germination in a plug tray for growth; when the seedlings grow to the cotyledon expansion stage or the two-leaf one-heart stage, cutting off the top meristem of the seedlings with a new blade, ensuring that the cotyledons are cut off together, leaving only the hypocotyl, and injecting the Agrobacterium suspension into the wound of the seedlings with a micro-syringe; the Agrobacterium cells in the suspension contain the pMKV057 binary plasmid vector; placing an absorbent cotton ball dipped in the Agrobacterium suspension on the wound of the seedlings to keep them moist, removing the absorbent cotton ball after being placed in the dark for two days, and continuing to culture the seedlings to obtain regenerated plants. The method of the invention avoids the stage of forming regenerated buds from callus tissue through dedifferentiation and redifferentiation in the traditional method, and provides a new solution for the subsequent gene editing of genetically transformed stubborn vegetable crops using non-tissue culture methods.

Description

Technical Field

[0001] The invention belongs to the technical field of plant regeneration, and in particular relates to a non-tissue culture method for regenerating non-heading Chinese cabbage. Background Art

[0002] Genetic transformation technology has played an important role in improving crop traits, and the improved excellent traits include yield, quality, and resistance to biological and abiotic stresses. Among them, gene editing technology based on CRISPR / Cas9 has become an emerging technology for crop genetic improvement due to its advantages such as simple operation, high editing efficiency, support for multi-target editing, and diverse editing forms. It has played an important role in improving many crop traits. However, this method is a genetic transformation method based on tissue culture, and the process is heavily dependent on the regeneration and transformation system of crops. For crops with relatively mature genetic transformation systems, such as wheat, rice, and tomatoes, the application of gene editing technology is relatively easy to achieve. However, for most vegetable crops, the genetic transformation system is not mature, which has become a key bottleneck problem limiting the application of CRISPR / Cas9 gene editing technology in vegetable crops. The process of inducing differentiation from explants to form regenerated buds in vegetable crops is very difficult, which is affected by many factors such as genotype, explant state, culture medium type, growth regulator type and content, culture environment, or the efficiency of T-DNA system transformation and insertion into the recipient genome is extremely low. Therefore, establishing a non-tissue culture genetic transformation system is a key link in achieving vegetable gene editing, and a technological breakthrough is urgently needed. Summary of the invention

[0003] In order to overcome the shortcomings and deficiencies of the prior art, the object of the present invention is to provide a non-tissue culture method for regenerating non-heading Chinese cabbage.

[0004] The present invention is achieved by a non-tissue culture method for regenerating non-heading Chinese cabbage, the method comprising the following steps:

[0005] (1) placing non-heading cabbage seeds that have been subjected to germination treatment in a plug tray for growth;

[0006] (2) When the seedlings grow to the cotyledon expansion stage or the two-leaf and one-heart stage, the apical meristem of the seedlings is cut off with a new blade, ensuring that the cotyledons are cut off together, leaving only the hypocotyl, and the Agrobacterium suspension is injected into the wound of the seedlings with a microinjector; wherein the Agrobacterium cells in the suspension contain the pMKV057 binary plasmid vector, and the pMKV057 binary plasmid vector contains a gene encoding a growth regulator;

[0007] (3) placing a cotton ball dipped in the Agrobacterium suspension on the wound of the seedling to keep it moist, and removing the cotton ball after two days in the dark. The seedling is further cultured under a photoperiod of 16 hours light / 8 hours dark and a temperature of 25° C. to 28° C. to obtain a regenerated plant.

[0008] Preferably, the OD of the Agrobacterium suspension is 600 =0.8.

[0009] Preferably, the growth regulatory factors are WUS and IPT.

[0010] Plant meristems are determined by several major developmental regulators (DRs), such as WUS (WUSCHEL), IPT (ISOPENTENYL TRANSFERASE), STM (SHOOT MERISTEMLESS) and MP (MONOPTEROS). By specifically expressing DRs in somatic cells, meristem regeneration can be directly induced. Based on the above theory, the present invention uses the pMKV057 binary plasmid vector containing the wus and ipt genes to directly obtain regenerated plants by inducing the formation of meristems.

[0011] Compared with the shortcomings and deficiencies of the prior art, the present invention has the following beneficial effects: the innovative point of the method constructed by the present invention is that it does not induce the formation of regenerated buds by optimizing the exogenous hormone formula in the traditional way, but rather constructs the growth regulatory factor on the pMKV057 binary plasmid vector, transforms the vector into Agrobacterium, and directly induces the formation of regenerated buds with the Agrobacterium suspension, thus avoiding the stage of forming regenerated buds from callus tissue through dedifferentiation and redifferentiation in the traditional method. The method is easy to operate and has a high success rate, and can provide a new solution for the subsequent gene editing of genetically transformed stubborn vegetable crops using non-tissue culture methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 It is a schematic diagram of the structure of the pMKV057 vector involved in the present invention;

[0013] Figure 2 It is a schematic diagram of the Agrobacterium injection method in the method of the present invention;

[0014] Figure 3 It is a diagram of the regeneration process of non-heading Chinese cabbage in an embodiment of the present invention. DETAILED DESCRIPTION

[0015] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0016] 1. Material preparation

[0017] 1. pMKV057 binary plasmid vector was purchased from Miaoling Biotechnology http: / / www.miaolingbio.com / , the wus and ipt genes are cloned on this vector, such as Figure 1 shown.

[0018] 2. The pMKV057 plasmid containing wus and ipt was transformed into Agrobacterium GV3101

[0019] (1) Take the competent Agrobacterium stored at -80°C and place it at room temperature or in the palm of your hand for a while until it partially melts. When it is in a mixed state of ice and water, insert it into ice;

[0020] (2) Add 0.01-1 μg of plasmid per 100 μL competent medium, stir the bottom of the tube by hand to mix, and place on ice for 5 minutes, in liquid nitrogen for 5 minutes, in a 37°C water bath for 5 minutes, and in an ice bath for 5 minutes;

[0021] (3) Add 700 μL of LB liquid medium without antibiotics and culture at 28°C with shaking for 2 to 3 hours;

[0022] (4) Collect the bacteria by centrifugation at 6000 rpm for one minute, and take about 100 μL of the supernatant. Gently blow and resuspend the bacteria, and spread it on an LB plate containing 50 μg / mL Kan and 20 μg / mL Rif antibiotics. Invert and place it in a 28°C incubator for 2 to 3 days. Perform PCR verification on the grown single colonies, and save the positive colonies obtained.

[0023] 3. Preparation process of Agrobacterium suspension

[0024] The positive colonies were inoculated into 5 mL LB liquid medium (containing 50 μg / mL Kan, 20 μg / mL Rif antibiotics), cultured overnight at 28°C, 200 rpm, and the bacterial solution was transferred to 50 mL LB liquid medium (containing 50 μg / mL Kan, 20 μg / mL Rif antibiotics, 20 μM acetosyringone) at a ratio of 1%. The Agrobacterium concentration was continued to reach OD 600 = 0.4-0.6, 5000 rpm, room temperature, centrifuge for 10 min to collect the bacteria, resuspend the bacteria in MS liquid medium (containing 10 mM MgCl2, 150 μM acetosyringone), and adjust the Agrobacterium concentration to OD 600 =0.8, and allowed to stand at 28°C for 2 h before injection.

[0025] 2. Regeneration of non-heading cabbage by non-tissue culture

[0026] (1) placing the non-heading cabbage seeds that have been subjected to germination treatment in a plug tray for growth, and waiting for the seedlings to grow to the cotyledon expansion stage or the two-leaf one-heart stage;

[0027] (2) When the seedlings reach the stage of cotyledon expansion or two leaves and one heart, cut off the apical meristem of the seedlings with a new blade, making sure to cut off the cotyledons as well, leaving only the hypocotyl (e.g. Figure 2 As shown), the Agrobacterium suspension was injected into the wound of the seedling with a microsyringe, and the injection amount was based on not overflowing or seeping out of the wound of the seedling;

[0028] (3) Place a cotton ball soaked in the suspension on the wound to keep it moist. After two days in the dark, continue culturing under a photoperiod of 16 hours (light) / 8 hours (darkness) at a temperature of 25°C until the plant regenerates (e.g. Figure 3 shown), from Figure 3 It can be seen that the regeneration of non-heading cabbage is very good.

[0029] The innovative point of the method constructed by the present invention is that it does not induce the formation of regenerated buds by optimizing the traditional exogenous hormone formula, but rather constructs the growth regulatory factor on the pMKV057 binary plasmid vector, transforms the vector into Agrobacterium, and directly induces the formation of regenerated buds with the Agrobacterium suspension, thus avoiding the stage of forming regenerated buds from callus tissue through dedifferentiation and redifferentiation in the traditional method. The method is easy to operate and has a high success rate, and can provide a new solution for the subsequent gene editing of genetically transformed stubborn vegetable crops using non-tissue culture methods.

[0030] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for regenerating non-heading Chinese cabbage by non-tissue culture, characterized in that: The method comprises the following steps: (1) Placing the non-heading cabbage seeds that have been germinated in a plug tray for growth; (2) When the seedlings grow to the cotyledon expansion stage or the two-leaf and one-heart stage, the apical meristem of the seedlings is cut off with a new blade, ensuring that the cotyledons are cut off together and only the hypocotyl is left, and the Agrobacterium suspension is injected into the wound of the seedlings with a microinjector; wherein the Agrobacterium cells in the suspension contain the pMKV057 binary plasmid vector, and the pMKV057 binary plasmid vector contains a gene encoding a growth regulator; (3) placing a cotton ball dipped in the Agrobacterium suspension on the wound of the seedling to keep it moist, and removing the cotton ball after two days in the dark. The seedling is then cultured under a photoperiod of 16 hours of light / 8 hours of darkness and a temperature of 25°C to 28°C to obtain a regenerated plant; The OD of the Agrobacterium suspension 600 =0.8; The growth regulatory factors are WUS and IPT.

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