Genetic transformation method of fraxinus mandshurica somatic embryo

Abstract

The invention discloses a fraxinus mandshurica somatic embryo genetic transformation method, which comprises the following steps: carrying out genetic transformation on fraxinus mandshurica embryonic cells through ultrasonic and vacuum assisted agrobacterium mediation, co-culturing for 3 days, removing bacteria, carrying out resistance screening, irradiating by using an LED red light source, illuminating for 16 hours every day, darkness for 8 hours every day, and culturing for 4-8 weeks at the temperature of 25 DEG C to obtain the somatic embryo. LED red and blue light combination (16 red and 9 blue) is used for irradiation, illumination is carried out for 16 h every day, darkness is carried out for 8 h every day, the temperature is 25 DEG C, resistant somatic embryo regeneration seedlings are obtained after 4-6 weeks, and GUS detection proves that genetic transformation succeeds. According to the method, ultrasonic and vacuum assistance is used in agrobacterium mediation, so that the addition of acetosyringone medicine is avoided, and the cost is saved. Red and blue light irradiation is utilized in somatic embryo induction and regeneration, light signals are accurately controlled, and compared with full-spectrum irradiation of white light, energy is saved, and cost is low. The invention provides a new way for genetic transformation of fraxinus mandshurica.

Description

Technical Field

[0001] The present invention relates to the field of plant biotechnology, and particularly to a method for genetic transformation of somatic embryos of Fraxinus mandshurica. Background Art

[0002] Genetic transformation, as the core technology of modern molecular breeding, refers to the introduction of foreign genes into the genome of a target organism to endow it with new genetic characteristics or change its original traits. In the field of plants, genetic transformation technology is a powerful tool for crop breeding and resistance improvement.

[0003] As a precious hardwood tree species in Northeast China, Fraxinus mandshurica has excellent wood mechanical properties and rich medicinal active ingredients, and also undertakes the key function of restoring degraded forest ecosystems. Therefore, the research on Fraxinus mandshurica is of great significance. However, due to the relatively long growth cycle of Fraxinus mandshurica, which usually takes decades to reach maturity, it brings great difficulties to its genetic improvement. Traditional genetic improvement methods, such as cross-breeding and selection breeding, although can improve some traits of Fraxinus mandshurica to a certain extent, due to its long growth cycle and complex genetic background, the breeding process is slow and the efficiency is low.

[0004] Currently, Liu Lin has established a genetic transformation system using the hypocotyls of zygotic embryos of Fraxinus mandshurica, induced buds from the hypocotyls, and found that the effect of infecting the hypocotyls by ultrasonic vacuum infiltration method is the best during the research process. However, it is limited by the fact that the hypocotyl materials can only be obtained at the initial stage of seed germination, and the spatio-temporal limitations are significant.

[0005] In recent years, somatic embryogenesis technology, as a new means of plant genetic improvement, has been widely applied in various plants. Somatic embryogenesis refers to the process by which plant somatic cells form embryonic structures with the potential to develop into complete plants through a series of cell divisions and differentiations under in vitro culture conditions. This method has the advantages of fast propagation speed and high genetic stability, and is particularly suitable for plants that are difficult to be genetically improved by traditional methods. However, although somatic embryogenesis technology has achieved remarkable results in other plants, the genetic transformation of somatic embryos of Fraxinus mandshurica is still in its infancy. The problems such as low induction rate of somatic embryos of Fraxinus mandshurica, instability of the somatic embryo development system, incomplete development of embryoids, and difficulty in regenerating plants are still the main bottlenecks restricting its genetic improvement.

[0006] Using the somatic embryos of Fraxinus mandshurica as transformation materials for genetic transformation overcomes the problems of single and limited genetic transformation materials in previous studies on Fraxinus mandshurica, as well as the problem of low genetic transformation efficiency of traditional genetic transformation methods in Fraxinus mandshurica, and lays a foundation for the genetic transformation of Fraxinus mandshurica. Summary of the invention

[0007] The present invention provides a method for genetic transformation of somatic embryos of ash, and obtains transgenic seedlings of ash somatic embryo regeneration pathway by using ultrasound, vacuum and red and blue light combination. The method overcomes the problem of single and limited genetic transformation materials of ash in previous studies, the problem of low genetic transformation efficiency of traditional genetic transformation methods on ash, and the problem of difficulty in somatic embryo regeneration of ash.

[0008] To achieve the above-mentioned object of the invention, the present invention includes the following method steps:

[0009] (1) Pre-culturing of embryonic cells: embryonic cells were inoculated on a pre-culture medium and pre-treated at 25° C. under dark conditions. The pre-culture medium was: WPM medium + 1.5 g / L activated carbon + 6 g / L agar + 51.29 g / L sucrose + 0.84 g / L hydrolyzed casein + 0.5 mg / L ZT + 0.2 mg / L NAA + 1.0 mg / L GA3, with a pH value of 6.0 to 6.2.

[0010] (2) Preparation of Agrobacterium infection solution: Use conventional methods to activate the strains and use a spectrophotometer to measure the OD of the Agrobacterium infection solution. 600 The concentration was adjusted to OD 600 =0.6, collect the cells by centrifugation, and add a special liquid culture medium to resuspend the cells. The special liquid culture medium is: WPM medium + 30g / L sucrose + 0.5mg / L ZT + 0.2mg / L NAA, + 1.0mg / L GA3. (3) Co-cultivation of Agrobacterium and cells: Place the embryonic cells pre-cultured in step (1) in the resuspended bacterial solution in step (2), ultrasonicate for 60s, vacuum for 10min, filter with sterile filter paper, collect the cells, inoculate the cells on the co-cultivation medium, and culture them at 25°C for 3-4d under dark conditions. The co-cultivation medium is: WPM medium + 1.5g / L activated carbon + 6g / L agar + 30g / L sucrose + 0.5mg / L ZT + 0.2mg / L NAA + 1.0mg / L GA3, with a pH value of 6.0-6.2.

[0011] (4) Degerming and screening of cells: After co-culture, the cells were washed with sterile distilled water, and 20-50 mg / L cephalosporin and 30-50 mg / L kanamycin were added to the culture medium.

[0012] (5) Light culture of cells: The cell embryos selected in step (4) are placed in a culture room and irradiated with an LED red light source with a light intensity of 50 μmol·m-2·s-1 and a peak wavelength of 660 nm. The cells are cultured for 4-8 weeks to obtain somatic embryos.

[0013] (6) Rooting culture of somatic embryos: Inoculate the somatic embryos obtained in step (5) on a rooting medium, and irradiate with a combination of LED red and blue light at a ratio of 16 red:9 blue, with 16 hours of light and 8 hours of darkness per day, at a temperature of 25 °C. Culture for 4 - 6 weeks to obtain somatic embryo regenerated seedlings. The rooting medium is: WPM medium + 1.5 g / L activated carbon + 6 g / L agar + 30 g / L sucrose + 0.5 mg / L ZT + 0.2 mg / L NAA + 1.0 mg / L GA3 + 0.4 mg / L 6 - BA + 0.5 mg / L IBA + 20 - 50 mg / L cephalosporin + 30 - 50 mg / L kanamycin, with a pH value of 6.0 - 6.2.

[0014] Advantages of the present invention:

[0015] The present invention provides a method for genetic transformation of Fraxinus mandshurica somatic embryos. The foreign gene is introduced into Fraxinus mandshurica embryogenic cell embryos by Agrobacterium - mediated method, and transgenic seedlings are obtained through resistance screening and regeneration culture. In the Agrobacterium - mediated process of the present invention, ultrasound and vacuum assistance are used, avoiding the addition of acetosyringone drug in genetic transformation, thus saving costs. In the induction and regeneration of somatic embryos, red and blue light irradiation is used to precisely control the light signal, saving energy compared with the full - spectrum irradiation of white light and having low cost. Description of the drawings

[0016] Figure 1 Fraxinus mandshurica embryogenic cells develop into somatic embryos during resistance screening.

[0017] Figure 2 Development process of Fraxinus mandshurica resistant somatic embryo culture.

[0018] Figure 3 GUS detection results of leaves of Fraxinus mandshurica resistant seedlings and resistant somatic embryos. A is the GUS detection result of non - transgenic seedling leaves, B is the GUS detection result of transgenic resistant seedling leaves, and C is the GUS detection result of resistant somatic embryos. Detailed implementation manners

[0019] To better understand the present invention, the content of the present invention is further clarified below in conjunction with embodiments. However, the content of the present invention is not limited only to the following embodiments.

[0020] Embodiment:

[0021] A method for genetic transformation of Fraxinus mandshurica somatic embryos includes:

[0022] The basic medium is WPM medium. The following culture media are sterilized at 121 °C and 1.1 Mpa for 20 minutes, with a pH value of 6.0 - 6.2.

[0023] (A) The pre-culture medium is: WPM medium + 1.5 g / L activated carbon + 6 g / L agar + 51.29 g / L sucrose + 0.84 g / L casein hydrolysate + 0.5 mg / L ZT + 0.2 mg / L NAA + 1.0 mg / L GA3.

[0024] (B) Special liquid medium: WPM medium + 30 g / L sucrose + 0.5 mg / L ZT + 0.2 mg / L NAA, + 1.0 mg / L GA3.

[0025] (C) The co-culture medium is: WPM medium + 1.5 g / L activated carbon + 6 g / L agar + 30 g / L sucrose + 0.5 mg / L ZT + 0.2 mg / L NAA + 1.0 mg / L GA3.

[0026] (D) The rooting medium is: WPM medium + 1.5 g / L activated carbon + 6 g / L agar + 30 g / L sucrose + 0.5 mg / L ZT + 0.2 mg / L NAA + 1.0 mg / L GA3 + 0.4 mg / L 6 - BA + 0.5 mg / L IBA + 20 mg / L cefotaxime + 30 mg / L kanamycin.

[0027] (1) Pre-cultivation of embryogenic cells: Inoculate Fraxinus mandshurica embryogenic cells on the pre-culture medium (A), and perform pre-treatment at 25°C under dark conditions.

[0028] (2) Preparation of Agrobacterium infection solution: Activate the bacterial strain using conventional methods, measure the OD value of the Agrobacterium liquid using a spectrophotometer, adjust the concentration to OD = 0.6, centrifuge to collect the bacterial cells, and resuspend the cells by adding the special liquid medium (B). (3) Co-culture of Agrobacterium and cells: Place the pre-cultured embryogenic cells in step (1) into the resuspended bacterial liquid in step (2), sonicate for 60 s, apply vacuum for 10 min, then filter with a sterile filter paper, collect the cells, inoculate the cells on the co-culture medium (C), and culture at 25°C under dark conditions for 3 - 4 d. 600 value, and adjust the concentration to OD 600 = 0.6, centrifuge to collect the bacterial cells, and resuspend the cells by adding the special liquid medium (B). (3) Co-culture of Agrobacterium and cells: Place the pre-cultured embryogenic cells in step (1) into the resuspended bacterial liquid in step (2), sonicate for 60 s, apply vacuum for 10 min, then filter with a sterile filter paper, collect the cells, inoculate the cells on the co-culture medium (C), and culture at 25°C under dark conditions for 3 - 4 d.

[0029] (4) Bacterial elimination and screening culture of cells: After co-culture, wash the cells with sterile distilled water, and add 20 mg / L cefotaxime and 30 mg / L kanamycin to the medium.

[0030] (5) Light culture of cells: Place the cell embryos screened in step (4) in the culture room, irradiate with an LED red light source, with a light intensity of 50 μmol·m -2 ·s -1 , a peak wavelength of 660 nm, switch to 8 h of dark conditions after 16 h of light per day, at a temperature of 25°C, and culture for 4 - 8 weeks to obtain somatic embryos ( Figure 1, Figure 2 )。

[0031] (6) Rooting culture of somatic embryos: Inoculate the somatic embryos obtained in step (5) on the rooting medium (D), and irradiate them with a combination of LED red and blue light, with a ratio of 16 red: 9 blue, 16 h of light and 8 h of darkness per day, at a temperature of 25 °C, and culture for 4 - 6 weeks to obtain somatic embryo regenerated seedlings.

[0032] (7) GUS detection of transgenic seedlings: Take out the leaves or somatic embryos of non-transgenic seedlings and resistant seedlings, and put them into the GUS detection solution (which can be purchased from a biological company). No blue color was observed in the leaves of non-transgenic seedlings ( Figure 3 A), while blue color was observed in the leaves of resistant seedlings ( Figure 3 B) and in the somatic embryos of resistant seedlings ( Figure 3 C), indicating successful genetic transformation.

[0033] Obviously, the above embodiments are only examples clearly described and not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all implementation manners here. And the obvious changes or modifications thus extended are still within the protection scope of the present invention.

Claims

1. The present invention provides a method for genetic transformation of somatic embryos of ash mandshurica, comprising the following steps: (1) Pre-culturing of embryonic cells: embryonic cells were inoculated on a pre-culture medium and pre-treated at 25° C. under dark conditions. The pre-culture medium was: WPM medium + 1.5 g / L activated carbon + 6 g / L agar + 51.29 g / L sucrose + 0.84 g / L hydrolyzed casein + 0.5 mg / L ZT + 0.2 mg / L NAA + 1.0 mg / L GA3, with a pH value of 6.0 to 6.

2. (2) Preparation of Agrobacterium infection solution: Use conventional methods to activate the strains and use a spectrophotometer to measure the OD of the Agrobacterium infection solution. 600 The concentration was adjusted to OD 600 =0.6, collect the bacteria by centrifugation, add special liquid culture medium to resuspend the bacteria, the special liquid culture medium is: WPM medium + 30g / L sucrose + 0.5mg / L ZT + 0.2mg / L NAA, + 1.0mg / L GA3. (3) Co-cultivation of Agrobacterium and cells: The embryonic cells pre-cultured in step (1) are placed in the resuspended bacterial solution in step (2), ultrasonicated for 60 seconds, vacuumed for 10 minutes, filtered with sterile filter paper, and the cells are collected. The cells are inoculated on a co-cultivation medium, and cultured at 25° C. for 3-4 days under dark conditions. The co-cultivation medium is: WPM medium + 1.5 g / L activated carbon + 6 g / L agar + 30 g / L sucrose + 0.5 mg / L ZT + 0.2 mg / L NAA + 1.0 mg / L GA3, and the pH value is 6.0-6.

2. (4) Degerming and screening of cells: After co-culture, the cells were washed with sterile distilled water, and 20-50 mg / L cephalosporin and 30-50 mg / L kanamycin were added to the culture medium. (5) Light culture of cells: The cells selected in step (4) are placed in a culture room for light culture, with 16 h of light and 8 h of darkness per day, at a temperature of 25° C., and cultured for 4-8 weeks to obtain somatic embryos. (6) Rooting culture of somatic embryos: The somatic embryos obtained in step (5) were inoculated on a rooting medium, with 16 h of light and 8 h of darkness per day, at a temperature of 25° C., and cultured for 4-6 weeks to obtain somatic embryo regenerated seedlings.

2. The method according to claim 1, characterized in that: The illumination conditions described in step (5) are to use an LED red light source for irradiation, with an illumination intensity of 50 μmol·m-2·s-1 and a peak wavelength of 660 nm.

3. The method according to claim 1, characterized in that The lighting condition described in step 6 is to use a combination of LED red and blue light, with a red-to-blue light ratio of 16:

9.

4. The method according to claim 1, characterized in that The rooting medium in step 6 is: WPM medium + 1.5g / L activated carbon + 6g / L agar + 30g / L sucrose + 0.5mg / L ZT + 0.2mg / L NAA + 1.0mg / L GA3 + 0.4mg / L6-BA + 0.5mg / L IBA + 20-50mg / L cephalosporin + 30-50mg / L kanamycin, with a pH value of 6.0-6.2.

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