Coriander genetic transformation culture medium and application thereof in coriander genetic transformation

Abstract

The invention belongs to the technical field of plant genetic transformation, and particularly relates to a coriander genetic transformation culture medium and application thereof in coriander genetic transformation. The culture medium suitable for coriander genetic transformation is formed by adjusting the composition and concentration of each component in the culture medium. Based on the coriander genetic transformation culture medium, the coriander genetic transformation method is successfully established by limiting coriander genetic transformation conditions, the coriander genetic transformation efficiency is improved, and coriander transgenic seedlings are efficiently and stably obtained. According to the coriander genetic transformation method, the genetic improvement efficiency of coriander can be greatly improved, powerful technical support is provided for coriander variety development, disease resistance improvement, quality improvement and the like, and the yield and resistance of coriander are improved; an important tool is provided for fundamental researches such as gene function research, metabolic pathway analysis and the like of the coriander.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic transformation, and particularly relates to a coriander genetic transformation culture medium and application thereof in coriander genetic transformation. Background Art

[0002] Coriandrum sativum L. is not only a seasoning with unique flavor, but also has a variety of bioactive ingredients such as cilantro, cilantro acid, etc., which have antioxidant, antibacterial and anticancer effects. Therefore, how to improve the quality, yield and stress resistance of coriander has become an important topic in the field of agricultural research.

[0003] In recent years, with the development of plant genetic engineering technology, genetic transformation technology has provided a new way to improve crop resistance, improve quality, and increase yield. Genetic transformation technology introduces foreign genes into plants to give them new traits or improve existing traits, and has achieved remarkable results in a variety of crops. However, there are relatively few studies on the genetic transformation technology of coriander, mainly because coriander is a difficult crop to transform and its genetic transformation efficiency is low. Summary of the invention

[0004] The object of the present invention is to provide a coriander genetic transformation culture medium and application thereof in coriander genetic transformation, on the basis of the coriander genetic transformation culture medium, the genetic transformation of coriander can be achieved, and at the same time, the gene transformation efficiency of coriander can be effectively improved.

[0005] The present invention provides a coriander genetic transformation culture medium, comprising one or more of a co-culture medium, a callus culture medium, a bud induction culture medium and a rooting culture medium;

[0006] The co-culture medium is based on B5 culture medium and further includes 28-30 g / L maltose, 6-8 g / L agar, 1.6-1.7 mg / L thidiazuron (TDZ), 0.2-0.3 mg / L naphthaleneacetic acid (NAA), 0.04-0.06 g / L adenine hemisulfate, 0.5-0.8 g / L 2-morpholineethanesulfonic acid and 180-200 μmol / L acetosyringone (AS);

[0007] The callus tissue culture medium is based on B5 culture medium and further includes 28-30 g / L maltose, 6-8 g / L agar, 1.6-1.7 mg / L thidiazuron, 0.2-0.3 mg / L naphthaleneacetic acid, 0.04-0.06 g / L adenine hemisulfate, 0.5-0.8 g / L 2-morpholineethanesulfonic acid, 280-300 mg / L timentin and 180-200 μmol / L acetosyringone;

[0008] The bud induction medium is based on B5 medium and further comprises 28-30 g / L maltose, 6-8 g / L agar, 1.6-1.7 mg / L thidiazuron, 0.2-0.3 mg / L naphthaleneacetic acid, 0.04-0.06 g / L adenine hemisulfate, 0.5-0.8 g / L 2-morpholineethanesulfonic acid, 30-35 mg / L kanamycin and 280-300 mg / L carbenicillin;

[0009] The rooting culture medium is based on 1 / 2B5 culture medium and further comprises 28-30 g / L maltose, 2-3 g / L activated carbon, 6-8 g / L agar and 0.6-0.8 mg / L naphthaleneacetic acid.

[0010] Preferably, the coriander genetic transformation culture medium also includes a preculture medium, which uses B5 culture medium as a basic culture medium and also includes 28-30 g / L maltose, 6-8 g / L agar, 1.6-1.7 mg / L thidiazuron and 0.2-0.3 mg / L naphthaleneacetic acid.

[0011] The present invention also provides the use of the coriander genetic transformation culture medium described in the above technical solution in coriander tissue culture and / or coriander genetic transformation.

[0012] The present invention also provides a method for genetic transformation of coriander based on the coriander genetic transformation medium described in the above technical solution, comprising the following steps:

[0013] Infecting coriander hypocotyls with Agrobacterium infection solution containing the target gene to obtain infected coriander hypocotyls;

[0014] Inoculating the infected coriander hypocotyls into a co-culture medium for dark culture to obtain co-cultured coriander hypocotyls;

[0015] Inoculating the co-cultured coriander hypocotyls into a callus culture medium for callus induction culture to obtain coriander callus;

[0016] Inoculating the coriander callus into a bud induction medium for adventitious bud induction culture to obtain coriander adventitious buds;

[0017] The coriander adventitious buds are inoculated into a rooting medium for rooting culture to obtain coriander genetically transformed seedlings.

[0018] Preferably, the OD of the Agrobacterium infection solution is 600 It is 0.3~0.5.

[0019] Preferably, the infection method comprises: immersing the coriander hypocotyls in an Agrobacterium infection solution containing the target gene for ultrasonic treatment, and subjecting the ultrasonically treated hypocotyls to vacuum negative pressure treatment;

[0020] The power of the ultrasonic treatment is 170-190W, the frequency is 35-45kHz, and the time is 45-60s; the vacuum intensity of the vacuum negative pressure treatment is 0.04-0.06MPa, and the time is 10-12min.

[0021] Preferably, the dark culture temperature is 25-30°C and the time is 2-3 days;

[0022] The callus induction culture is dark culture at a temperature of 25-30° C. for 2-3 days.

[0023] Preferably, the temperature of the adventitious bud induction culture is 25-30°C, the light duration is 12-14h / d, and the light intensity is 2000-2500lx; the adventitious bud induction culture is terminated when the coriander adventitious bud grows to 2-3cm.

[0024] The rooting culture temperature is 25-30°C, the illumination time is 12-14h / d, and the illumination intensity is 2000-2500lx.

[0025] Preferably, the method further comprises domesticating and transplanting the genetically transformed coriander seedlings to obtain coriander seedlings.

[0026] Preferably, the domestication method includes: when the genetically transformed coriander seedlings grow 6 to 8 roots and the seedling height is 7 to 8 cm, the genetically transformed coriander seedlings are further cultured for 2 to 3 days, and then transplanted into a sterilized plant matrix for culture for 7 to 9 days.

[0027] Beneficial effects:

[0028] The present invention provides a coriander genetic transformation culture medium, including one or more of a co-culture medium, a callus culture medium, a bud induction culture medium and a rooting culture medium; the present invention forms a culture medium suitable for the genetic transformation of coriander by adjusting the composition and concentration of each component in the culture medium. Based on the coriander genetic transformation culture medium, the present invention successfully establishes a genetic transformation method for coriander by limiting the conditions for the genetic transformation of coriander (such as limiting the explant for the genetic transformation of coriander to the hypocotyl), thereby improving the genetic transformation efficiency of coriander and obtaining transgenic coriander seedlings efficiently and stably. The coriander genetic transformation method of the present invention can greatly improve the genetic improvement efficiency of coriander, provide strong technical support for the development of coriander varieties, improvement of disease resistance, quality improvement, etc., and improve the yield and resistance of coriander; and provide an important tool for basic research such as gene function research and metabolic pathway analysis of coriander. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0030] Figure 1 The PCR detection results of the transgenic coriander VvPIP1;1 gene in Example 3, wherein M: Marker; -: negative control; +: positive control; 1, 2, 3, 4, 5, 6, 8: plants containing the VvPIP1;1 drought resistance gene; 7: plants without the VvPIP1;1 gene;

[0031] Figure 2 The effect of the infection solution concentration on the differentiation rate of coriander resistance buds in Examples 13-14 and Comparative Examples 62-63;

[0032] Figure 3 The effect of infection time on the differentiation rate of coriander resistance buds in Examples 15 to 17 and Comparative Examples 64 to 65;

[0033] Figure 4 The effect of the concentration of acetosyringone in the co-culture medium and callus culture medium on the differentiation rate of coriander resistance buds in Examples 18 to 20 and Comparative Examples 66 to 67;

[0034] Figure 5 The effect of co-cultivation time on the differentiation rate of coriander resistance buds in Examples 21-22 and Comparative Examples 68-69;

[0035] Among them, Figures 2 to 5 Different letters indicate significant differences (p<0.05). DETAILED DESCRIPTION

[0036] The present invention provides a coriander genetic transformation culture medium, comprising one or more of a co-culture medium, a callus culture medium, a bud induction culture medium and a rooting culture medium;

[0037] The co-culture medium is based on B5 culture medium and further includes 28-30 g / L maltose, 6-8 g / L agar, 1.6-1.7 mg / L thidiazuron, 0.2-0.3 mg / L naphthaleneacetic acid, 0.04-0.06 g / L adenine hemisulfate, 0.5-0.8 g / L 2-morpholineethanesulfonic acid and 180-200 μmol / L acetosyringone;

[0038] The callus tissue culture medium is based on B5 culture medium and further includes 28-30 g / L maltose, 6-8 g / L agar, 1.6-1.7 mg / L thidiazuron, 0.2-0.3 mg / L naphthaleneacetic acid, 0.04-0.06 g / L adenine hemisulfate, 0.5-0.8 g / L 2-morpholineethanesulfonic acid, 280-300 mg / L timentin and 180-200 μmol / L acetosyringone;

[0039] The bud induction medium is based on B5 medium and further comprises 28-30 g / L maltose, 6-8 g / L agar, 1.6-1.7 mg / L thidiazuron, 0.2-0.3 mg / L naphthaleneacetic acid, 0.04-0.06 g / L adenine hemisulfate, 0.5-0.8 g / L 2-morpholineethanesulfonic acid, 30-35 mg / L kanamycin and 280-300 mg / L carbenicillin;

[0040] The rooting culture medium is based on 1 / 2B5 culture medium and further comprises 28-30 g / L maltose, 2-3 g / L activated carbon, 6-8 g / L agar and 0.6-0.8 mg / L naphthaleneacetic acid.

[0041] As an embodiment, the coriander genetic transformation medium includes a co-culture medium, a callus tissue culture medium, a bud induction culture medium and a rooting culture medium.

[0042] As an embodiment, the co-culture medium is based on B5 medium and also includes 29g / L maltose, 7g / L agar, 1.7mg / L thidiazuron, 0.3mg / L naphthaleneacetic acid, 0.05g / L adenine hemisulfate, 0.7g / L 2-morpholineethanesulfonic acid and 190μmol / L acetosyringone.

[0043] As an embodiment, the callus culture medium is based on B5 medium and further includes 29 g / L maltose, 7 g / L agar, 1.7 mg / L thidiazuron, 0.3 mg / L naphthaleneacetic acid, 0.05 g / L adenine hemisulfate, 0.7 g / L 2-morpholineethanesulfonic acid, 290 mg / L timentin and 190 μmol / L acetosyringone.

[0044] As an embodiment, the bud induction medium is based on B5 medium and also includes 29g / L maltose, 7g / L agar, 1.7mg / L thidiazuron, 0.2mg / L naphthaleneacetic acid, 0.05g / L adenine hemisulfate, 0.6g / L 2-morpholineethanesulfonic acid, 32mg / L kanamycin and 290mg / L carbenicillin.

[0045] As an embodiment, the rooting medium uses 1 / 2B5 medium as the basic medium and also includes 29g / L maltose, 2-3g / L activated carbon, 7g / L agar and 0.7mg / L naphthaleneacetic acid.

[0046] As one embodiment, the coriander genetic transformation culture medium also includes a preculture medium, which uses B5 culture medium as a basic culture medium and also includes 28-30 g / L maltose, 6-8 g / L agar, 1.6-1.7 mg / L thidiazuron and 0.2-0.3 mg / L naphthaleneacetic acid; as another embodiment, the preculture medium uses B5 culture medium as a basic culture medium and also includes 29 g / L maltose, 7 g / L agar, 1.7 mg / L thidiazuron and 0.3 mg / L naphthaleneacetic acid.

[0047] The invention forms a culture medium suitable for coriander genetic transformation by limiting the components and ratio concentrations in each culture medium to improve the efficiency of coriander genetic transformation. The concentration setting of thidiazuron and naphthylacetic acid in the coriander genetic transformation culture medium of the invention can improve the induction rate of coriander adventitious buds and adventitious roots.

[0048] The present invention also provides the use of the coriander genetic transformation culture medium described in the above technical solution in coriander tissue culture and / or coriander genetic transformation.

[0049] The present invention also provides a method for genetic transformation of coriander based on the coriander genetic transformation medium described in the above technical solution, comprising the following steps:

[0050] Infecting coriander hypocotyls with Agrobacterium infection solution containing the target gene to obtain infected coriander hypocotyls;

[0051] Inoculating the infected coriander hypocotyls into a co-culture medium for dark culture to obtain co-cultured coriander hypocotyls;

[0052] Inoculating the co-cultured coriander hypocotyls into a callus culture medium for callus induction culture to obtain coriander callus;

[0053] Inoculating the coriander callus into a bud induction medium for adventitious bud induction culture to obtain coriander adventitious buds;

[0054] The coriander adventitious buds are inoculated into a rooting medium for rooting culture to obtain coriander genetically transformed seedlings.

[0055] As an embodiment, the present invention sterilizes coriander seeds to obtain sterilized coriander seeds. As an embodiment, the sterilization method of coriander seeds comprises: soaking coriander seeds in 50-55°C warm water for 15-20 minutes, and then soaking them in 25-30°C warm water for 4-6 hours to obtain soaked coriander seeds; soaking the soaked coriander seeds in an ethanol solution with a volume concentration of 75% for 1-1.5 minutes, and then soaking the coriander seeds in a sodium hypochlorite solution with a mass fraction of 3% for 6-8 minutes, and rinsing with sterile water for 3-5 times to obtain the sterilized coriander seeds.

[0056] After obtaining the sterilized coriander seeds, as an embodiment, the present invention performs germination culture on the sterilized coriander seeds to obtain sterile coriander seedlings. As an embodiment, the temperature of the germination culture is 20-25°C; as another embodiment, the temperature of the germination culture is 22-24°C. As an embodiment, the light intensity of the germination culture is 2000-2500lx, and the light time is 12-14h / d; as another embodiment, the light intensity of the germination culture is 2200-2400lx, and the light time is 13h / d. As an embodiment, when the cotyledons of the sterile coriander seedlings are fully opened and 2-3 true leaves grow, the germination culture is stopped.

[0057] After obtaining the sterile coriander seedlings, as an embodiment, the present invention inoculates the hypocotyls of the sterile coriander seedlings into a preculture medium for dark culture to obtain precultured coriander hypocotyls. As an embodiment, the length of the hypocotyls of the sterile coriander seedlings is 0.4 to 0.6 cm; as another embodiment, the length of the hypocotyls of the sterile coriander seedlings is 0.5 cm. As an embodiment, the temperature of the dark culture is 25 to 30°C, and the time is 2 to 3 days; as another embodiment, the temperature of the dark culture is 25°C, and the time is 2 days. The preculture medium of the present invention has been defined in the above technical solution and will not be repeated here.

[0058] After obtaining the pre-cultured coriander hypocotyls, the present invention infects the pre-cultured coriander hypocotyls with an Agrobacterium infection solution containing the target gene to obtain infected pre-cultured coriander hypocotyls. As an embodiment, the OD of the Agrobacterium infection solution is 600 is 0.3 to 0.5; as another embodiment, the OD 600is 0.4; the setting of the concentration of the Agrobacterium infection solution has the advantages of low explant cell damage and high conversion rate. As an embodiment, the infection method includes: immersing the pre-cultured coriander hypocotyls in the Agrobacterium infection solution containing the target gene for ultrasonic treatment, and subjecting the ultrasonically treated hypocotyls to vacuum negative pressure treatment. As an embodiment, the power of the ultrasonic treatment is 170 to 190 W; as another embodiment, the power of the ultrasonic treatment is 180 W. As an embodiment, the frequency of the ultrasonic treatment is 35 to 45 kHz; as another embodiment, the frequency of the ultrasonic treatment is 40 kHz. As an embodiment, the time of the ultrasonic treatment is 45 to 60 s; as another embodiment, the time of the ultrasonic treatment is 50 s. As an embodiment, the vacuum intensity of the vacuum negative pressure treatment is 0.04 to 0.06 MPa; as another embodiment, the vacuum intensity of the vacuum negative pressure treatment is 0.05 MPa. As an embodiment, the vacuum negative pressure treatment time is 10 to 12 minutes; as another embodiment, the vacuum negative pressure treatment time is 11 minutes; the vacuum negative pressure treatment time has the advantages of low explant necrosis rate and high transformation efficiency. The present invention does not specifically limit the type of the target gene, and it can be selected according to research. In the process of Agrobacterium infecting explants, the negative pressure treatment of the present invention will form many small wounds on the coriander explants, and more phenolic substances will be secreted from the wounds, which will help to improve the transformation activity of Agrobacterium. At the same time, the high pressure and high temperature shock waves generated by the rupture of cavitation bubbles caused by ultrasonic waves in ultrasonic treatment cause local rupture of the cytoplasmic membrane. Before the cytoplasmic membrane recovers, it is possible to absorb surrounding substances to cause transformation. The present invention has the advantage of high transformation efficiency using the hypocotyl of sterile coriander seedlings as explants.

[0059] After obtaining the infected pre-cultured coriander hypocotyls, the present invention inoculates the infected pre-cultured coriander hypocotyls into a co-culture medium for dark culture to obtain co-cultured coriander hypocotyls. As an embodiment, the dark culture temperature is 25 to 30° C., and the time is 2 to 3 days; as another embodiment, the dark culture temperature is 25° C., and the time is 2 days.

[0060] After obtaining the co-cultured coriander hypocotyls, the present invention inoculates the co-cultured coriander hypocotyls into a callus tissue culture medium for callus induction culture to obtain coriander callus. As an embodiment, the callus induction culture is dark culture. As an embodiment, the temperature of the callus induction culture is 25-30°C, and the time is 2-3 days; as another embodiment, the temperature of the callus induction culture is 25°C, and the time is 2 days.

[0061] After obtaining the coriander callus, the present invention inoculates the coriander callus into a bud induction medium for adventitious bud induction culture to obtain coriander adventitious buds. As an embodiment, the temperature of the adventitious bud induction culture of the present invention is 25-30°C, the illumination time is 12-14h / d, and the light intensity is 2000-2500lx; the adventitious bud induction culture ends when the coriander adventitious bud grows to 2-3cm. As another embodiment, the temperature of the adventitious bud induction culture is 25°C, the illumination time is 14h / d, and the light intensity is 2500lx.

[0062] After obtaining the coriander adventitious buds, the present invention inoculates the coriander adventitious buds into a rooting medium for rooting culture to obtain coriander genetic transformation seedlings. As an embodiment, the rooting culture temperature is 25-30°C, the illumination time is 12-14h / d, and the illumination intensity is 2000-2500lx; as another embodiment, the rooting culture temperature is 25°C, the illumination time is 14h / d, and the illumination intensity is 2500lx.

[0063] After obtaining the coriander genetic transformation seedlings, as an embodiment, the present invention acclimates and transplants the coriander genetic transformation seedlings to obtain coriander seedlings. As an embodiment, the acclimation method includes: when the coriander genetic transformation seedlings grow 6 to 8 roots and the seedling height is 7 to 8 cm, the coriander genetic transformation seedlings are further cultured for 2 to 3 days, and then transplanted into a sterilized plant matrix for 7 to 9 days. The present invention does not specifically limit the type of the plant matrix, and a conventional plant matrix in the art can be used.

[0064] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0065] Example 1

[0066] A coriander genetic transformation medium, comprising a co-culture medium, a callus tissue culture medium, a bud induction culture medium and a rooting culture medium:

[0067] The co-culture medium was B5 medium + 30 g / L maltose + 8 g / L agar + 1.7 mg / L thidiazuron + 0.3 mg / L naphthaleneacetic acid + 0.06 g / L adenine hemisulfate + 0.8 g / L 2-morpholineethanesulfonic acid + 200 μmol / L acetosyringone;

[0068] The callus culture medium was B5 medium + 30 g / L maltose + 8 g / L agar + 1.7 mg / L thidiazuron + 0.3 mg / L naphthaleneacetic acid + 0.06 g / L adenine hemisulfate + 0.8 g / L 2-morpholineethanesulfonic acid + 300 mg / L timentin + 200 μmol / L acetosyringone;

[0069] The bud induction medium was B5 medium + 30 g / L maltose + 8 g / L agar + 1.7 mg / L thidiazuron + 0.3 mg / L naphthaleneacetic acid + 0.06 g / L adenine hemisulfate + 0.8 g / L 2-morpholineethanesulfonic acid + 35 mg / L kanamycin + 300 mg / L carbenicillin;

[0070] The rooting medium was 1 / 2B5 medium + 30 g / L maltose + 3 g / L activated carbon + 8 g / L agar + 0.8 mg / L naphthaleneacetic acid;

[0071] The preparation steps of the co-culture medium, callus culture medium, bud induction culture medium and rooting culture medium are to add corresponding amounts of other components to B5 culture medium or 1 / 2B5 culture medium, and then sterilize to obtain the corresponding culture medium.

[0072] Example 2

[0073] A coriander genetic transformation medium, comprising a pre-culture medium, a co-culture medium, a callus tissue medium, a bud induction medium and a rooting medium;

[0074] The pre-culture medium was B5 medium + 30 g / L maltose + 8 g / L agar + 1.7 mg / L thidiazuron + 0.3 mg / L naphthaleneacetic acid;

[0075] The compositions of co-cultivation, callus medium, shoot induction medium and rooting medium are the same as those in Example 1;

[0076] The preparation steps of each culture medium in Example 2 are the same as those in Example 1.

[0077] Example 3

[0078] A method for genetic transformation of coriander, comprising the following steps:

[0079] 1. Preparation of infection solution

[0080] Construction of pCambia1304-VvPIP1;1 vector: VvPIP1;1 (gene ID sequence number in the grape reference genome database (http: / / plants.ensembl.org / Vitis_vinifera / Info / Index): DQ834694.1) is a gene related to drought resistance in grape. The construction of pCambia1304-VvPIP1;1 vector refers to the following literature [Yu, Jun, Jia Li, Zhangfen Hong, Qing Zeng, Yizheng Fu, Rouxue Deng, Ke Xu, Zhenyu Huang and Mingtao Zhu. VvPIP1;1 plays a role in grape berry cracking by regulating wateruptake. Horticultural Plant Journal, 2025. DOI: 10.1016 / j.hpj.2024.07.010].

[0081] A single colony of Agrobacterium tumefaciens EHA105 containing the pCambia1304-VvPIP1;1 plasmid was selected and transferred to LB liquid medium containing 50 mg / L kanamycin (Kanamycin, Kan) and 20 mg / L rifampicin (Rif) and cultured with shaking (28°C, 200 r / min) until OD 600 =0.8, then centrifuge the bacterial solution at room temperature (3000×g) for 15 min, discard the supernatant, collect the bacterial cells, add suspension (B5 medium + 200μmol / L acetosyringone + 30g / L maltose + 0.02% Tween-20) to resuspend the bacterial cells, and adjust the OD 600 =0.5 for backup.

[0082] 2. Obtaining sterile coriander seedlings

[0083] Select mature, full-grained coriander seeds and soak them in 50-55°C warm water for 20 minutes, stirring constantly, then soak them in 30°C water for 6 hours, then use sterile filter paper to absorb the surface moisture of the seeds, soak them in 75% alcohol by volume for 1.5 minutes, then take out the seeds and rinse them with sterile water, then soak them in 3% sodium hypochlorite solution for 8 minutes, rinse them with sterile water 5 times, use sterile filter paper to absorb the surface moisture of the seeds, inoculate them in B5 culture medium, and place them in a light incubator for culture (25°C, light time 14h / day, light intensity 2500lx). When the cotyledons are fully opened and two true leaves grow, they are ready for use.

[0084] 3. Pre-culture

[0085] The hypocotyls of the sterile seedlings obtained above were cut into 0.5 cm pieces, inoculated into a pre-culture medium (B5 medium + 30 g / L maltose + 8 g / L agar + 1.7 mg / L thidiazuron + 0.3 mg / L naphthaleneacetic acid) and cultured in the dark at 25°C for 2 days.

[0086] 4. Explant Infection

[0087] The pre-cultured coriander hypocotyls were immersed in the Agrobacterium infection solution prepared in step 1, and ultrasonicated for 60 seconds (ultrasonic power 180 W, ultrasonic frequency 40 kHz) during the infection process, and then vacuumed with a vacuum pump for 12 minutes (vacuum strength 0.05 MPa).

[0088] 5. Co-culture

[0089] The infected hypocotyls were blotted dry with sterile filter paper, inoculated into co-culture medium (B5 medium + 30 g / L maltose + 8 g / L agar + 1.7 mg / L thidiazuron + 0.3 mg / L naphthaleneacetic acid + 0.06 g / L adenine hemisulfate + 0.8 g / L 2-morpholineethanesulfonic acid + 200 μmol / L acetosyringone), and cultured in the dark at 25°C for 3 days.

[0090] 6. Callus Induction

[0091] After the co-cultivation, the infected hypocotyls were transferred to callus culture medium (B5 medium + 30 g / L maltose + 8 g / L agar + 1.7 mg / L thidiazuron + 0.3 mg / L naphthaleneacetic acid + 0.06 g / L adenine hemisulfate + 0.8 g / L 2-morpholineethanesulfonic acid + 300 mg / L timentin + 200 μmol / L acetosyringone) and cultured in the dark at 25 °C for 30 days.

[0092] 7. Adventitious bud induction

[0093] The callus tissue obtained above was inoculated onto adventitious bud induction medium (B5 medium + 30 g / L maltose + 8 g / L agar + 1.7 mg / L thidiazuron + 0.3 mg / L naphthaleneacetic acid + 0.06 g / L adenine hemisulfate + 0.8 g / L 2-morpholineethanesulfonic acid + 35 mg / L kanamycin + 300 mg / L carbenicillin), the culture temperature was 25°C, the lighting time was 14 h / day, and the light intensity was 2500 lx.

[0094] 8. Adventitious bud rooting

[0095] When the adventitious buds grow to 3 cm, they are transferred to rooting medium (1 / 2B5 medium + 30 g / L maltose + 3 g / L activated carbon + 8 g / L agar + 0.8 mg / L naphthaleneacetic acid) to induce rooting. The culture temperature is 25°C, the lighting time is 14 h / day, and the light intensity is 2500 lx.

[0096] 9. Domestication and transplantation of transgenic plants

[0097] When the adventitious buds in the above tissue culture bottle grow 8 roots and the seedlings are 8 cm tall, open the bottle cap and culture them in the open for 2 days (25°C, light time 14 h / day, light intensity 2500 lx), then wash away the culture medium at the roots of the transgenic seedlings with clean water, transplant them into a sterilized matrix, and transplant them after one week of culture in the greenhouse.

[0098] 10. DNA molecular level detection of transgenic plants

[0099] The DNA of the leaves of the transgenic plants was extracted and PCR amplified using the specific primers of the VvPIP1;1 gene (F: 5'-GAGCCTGGAGAGCTGTGTTC-3' (SEQ ID NO: 1); R: 5'-GACGAAGGTGCCA ACAATCT-3' (SEQ ID NO: 2)). The wild-type leaf DNA was used as a negative control, and the vector plasmid carrying the VvPIP1;1 gene was used as a positive control. A band of about 441 bp could be detected in the strains successfully transformed with the VvPIP1;1 gene, while no band was detected in the strains that were not successfully transformed ( Figure 1 ).

[0100] In the experiment, the inventor used the genetic transformation technology to infect 120 explants, and performed PCR detection on the genetically transformed strains, obtaining a total of 58 positive transgenic strains with a genetic transformation rate of 48.33% (genetic transformation rate = number of positive plants / total number of infected explants × 100%).

[0101] Example 1, Examples 4 to 6 and Comparative Examples 1 to 61

[0102] The genetic transformation of coriander was carried out according to the method in Example 3 using the coriander genetic transformation medium in Example 1, except that the concentrations of thidiazuron (TDZ) and naphthaleneacetic acid (NAA) in the co-culture medium, callus tissue culture medium and bud induction culture medium were adjusted at the same time, as shown in Table 1, and the effects of different hormone concentration combinations in the coriander genetic transformation culture medium on the induction rate of adventitious buds of coriander were detected, and the results are shown in Table 1. Table 1 Effects of different hormone concentration combinations in the coriander genetic transformation culture medium on the induction rate of adventitious buds in Example 1, Examples 4 to 6 and Comparative Examples 1 to 61

[0103]

[0104]

[0105]

[0106] Note: Different letters in Table 1 indicate significant differences (p < 0.05), the same as in Table 2.

[0107] It can be concluded from Table 1 that the induction rate of coriander adventitious buds can be significantly increased by using the concentrations of TDZ and NAA in the genetic transformation medium of the present invention and using hypocotyls as explants for induction of adventitious buds.

[0108] Example 1, Examples 7-8 and Comparative Examples 62-63

[0109] The genetic transformation medium of coriander in Example 1 was used to carry out genetic transformation of coriander according to the method in Example 3, except that the concentration of naphthaleneacetic acid (NAA) in the rooting medium was adjusted, as shown in Table 1, and the effects of different hormone concentration combinations in the genetic transformation medium of coriander on the rooting rate of coriander were detected, and the results are shown in Table 2.

[0110] Table 2 Effects of different hormone concentration combinations in coriander genetic transformation medium on coriander rooting rate in Example 1, Examples 7-8 and Comparative Examples 62-63

[0111] serial number NAA concentration (mg / L) Rooting rate (%) Comparative Example 62 0.5 77.31b Example 7 0.6 94.29a Example 8 0.7 93.52a Example 1 0.8 92.79a Comparative Example 63 0.9 78.27b

[0112] It can be concluded from Table 2 that the rooting rate of coriander can be significantly improved by using the concentration of NAA in the rooting medium of the present invention.

[0113] Example 3, Example 9 and Comparative Examples 64-65

[0114] The genetic transformation medium of coriander in Example 1 was used to carry out genetic transformation of coriander according to the method in Example 3, except that the concentration of the infection solution (bacterial solution) in Example 3 (i.e., OD 600 Value), OD of the infection solution in Comparative Example 64, Example 9, Example 3 and Comparative Example 65 600 The values ​​were 0.1, 0.3, 0.5 and 0.7 respectively, and the differentiation rate of coriander resistance buds (adventitious buds) was detected. The results are as follows Figure 2 shown.

[0115] Depend on Figure 2 It can be concluded that the concentration of the infection solution in the present invention can significantly increase the differentiation rate of coriander resistant buds.

[0116] Example 3, Examples 10-11 and Comparative Examples 66-67

[0117] The genetic transformation of coriander was carried out according to the method in Example 3 using the coriander genetic transformation medium in Example 1, except that the infection time of the Agrobacterium infection solution (i.e., the time of vacuum negative pressure treatment with a vacuum pump) was adjusted. The infection time of the Agrobacterium infection solution in Comparative Example 66, Examples 10 to 11, Example 3, and Comparative Example 67 was 9, 10, 11, 12, and 13 minutes, respectively, and the differentiation rate of coriander resistance buds was detected. The results are as follows: Figure 3 shown.

[0118] Depend on Figure 3 It can be concluded that the infection time of the infection solution in the present invention can significantly improve the differentiation rate of coriander resistant buds.

[0119] Example 1, Examples 12-13 and Comparative Examples 68-69

[0120] The coriander genetic transformation medium in Example 1 was used according to the method in Example 3 to carry out coriander genetic transformation, except that the concentrations of acetosyringone in the co-culture medium and the callus culture medium were adjusted at the same time. The concentrations of acetosyringone in the co-culture medium and the callus culture medium in Comparative Example 68, Examples 12 to 13, Example 1 and Comparative Example 69 were 170, 180, 190, 200 and 210 μmol / L, respectively, and the differentiation rate of coriander resistant buds was detected. The results are as follows: Figure 4 shown.

[0121] Depend on Figure 4 It can be concluded that the concentration of acetosyringone in the co-culture medium and callus culture medium of the present invention can significantly improve the differentiation rate of coriander adventitious buds.

[0122] Example 3, Example 14 and Comparative Examples 70 to 71

[0123] The genetic transformation medium of coriander in Example 1 was used according to the method in Example 3 to carry out genetic transformation of coriander, except that the dark culture time in the co-culture in step 5 was adjusted, and the co-culture time in Comparative Example 70, Example 14, Example 3 and Comparative Example 71 was 1, 2, 3 and 5 days respectively, and the differentiation rate of coriander resistance buds was detected. The results are as follows Figure 5 shown.

[0124] Depend on Figure 5 It can be concluded that the co-cultivation time set in the present invention can significantly improve the differentiation rate of coriander resistant buds.

[0125] It can be concluded from the above embodiments that the coriander genetic transformation medium and genetic transformation method of the present invention can achieve the genetic transformation of coriander and at the same time, can improve the genetic transformation efficiency of coriander.

[0126] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A coriander genetic transformation medium, characterized in that: comprising one or more of a co-culture medium, a callus culture medium, a bud induction culture medium and a rooting culture medium; The co-culture medium is based on B5 culture medium and further includes 28-30 g / L maltose, 6-8 g / L agar, 1.6-1.7 mg / L thidiazuron, 0.2-0.3 mg / L naphthaleneacetic acid, 0.04-0.06 g / L adenine hemisulfate, 0.5-0.8 g / L 2-morpholineethanesulfonic acid and 180-200 μmol / L acetosyringone; The callus tissue culture medium is based on B5 culture medium and further includes 28-30 g / L maltose, 6-8 g / L agar, 1.6-1.7 mg / L thidiazuron, 0.2-0.3 mg / L naphthaleneacetic acid, 0.04-0.06 g / L adenine hemisulfate, 0.5-0.8 g / L 2-morpholineethanesulfonic acid, 280-300 mg / L timentin and 180-200 μmol / L acetosyringone; The bud induction medium is based on B5 medium and further comprises 28-30 g / L maltose, 6-8 g / L agar, 1.6-1.7 mg / L thidiazuron, 0.2-0.3 mg / L naphthaleneacetic acid, 0.04-0.06 g / L adenine hemisulfate, 0.5-0.8 g / L 2-morpholineethanesulfonic acid, 30-35 mg / L kanamycin and 280-300 mg / L carbenicillin; The rooting culture medium is based on 1 / 2B5 culture medium and further comprises 28-30 g / L maltose, 2-3 g / L activated carbon, 6-8 g / L agar and 0.6-0.8 mg / L naphthaleneacetic acid.

2. The coriander genetic transformation medium according to claim 1, characterized in that: The coriander genetic transformation culture medium also includes a preculture medium, which uses B5 culture medium as a basic culture medium and also includes 28-30 g / L maltose, 6-8 g / L agar, 1.6-1.7 mg / L thidiazuron and 0.2-0.3 mg / L naphthaleneacetic acid.

3. Use of the coriander genetic transformation medium according to claim 1 or 2 in coriander tissue culture and / or coriander genetic transformation.

4. A method for genetic transformation of coriander based on the coriander genetic transformation medium according to claim 1 or 2, characterized in that: The steps include: Infecting coriander hypocotyls with Agrobacterium infection solution containing the target gene to obtain infected coriander hypocotyls; Inoculating the infected coriander hypocotyls into a co-culture medium for dark culture to obtain co-cultured coriander hypocotyls; Inoculating the co-cultured coriander hypocotyls into a callus culture medium for callus induction culture to obtain coriander callus; Inoculating the coriander callus into a bud induction medium for adventitious bud induction culture to obtain coriander adventitious buds; The coriander adventitious buds are inoculated into a rooting medium for rooting culture to obtain coriander genetically transformed seedlings.

5. The method according to claim 4, characterized in that The OD of the Agrobacterium infection solution 600 It is 0.3~0.

5.

6. The method according to claim 4, characterized in that The infection method comprises: immersing the coriander hypocotyl in an Agrobacterium infection solution containing a target gene for ultrasonic treatment, and subjecting the ultrasonically treated hypocotyl to vacuum negative pressure treatment; The power of the ultrasonic treatment is 170-190W, the frequency is 35-45kHz, and the time is 45-60s; the vacuum intensity of the vacuum negative pressure treatment is 0.04-0.06MPa, and the time is 10-12min.

7. The method according to claim 4, characterized in that The dark culture temperature is 25-30°C and the time is 2-3 days; The callus induction culture is dark culture at a temperature of 25-30° C. for 2-3 days.

8. The method according to claim 4, characterized in that The temperature of the adventitious bud induction culture is 25-30°C, the light duration is 12-14h / d, and the light intensity is 2000-2500lx; the adventitious bud induction culture ends when the coriander adventitious bud grows to 2-3cm. The rooting culture temperature is 25-30°C, the illumination time is 12-14h / d, and the illumination intensity is 2000-2500lx.

9. The method according to any one of claims 4 to 8, characterized in that: The method also includes domesticating and transplanting the genetically transformed coriander seedlings to obtain coriander seedlings.

10. The method according to claim 9, characterized in that The domestication method comprises: when the genetically transformed coriander seedlings grow 6 to 8 roots and the seedling height is 7 to 8 cm, the genetically transformed coriander seedlings are further cultured for 2 to 3 days, and then planted in a sterilized plant matrix for culture for 7 to 9 days.

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