METHOD FOR GENE EXPRESSION BY TRANSIENT TRANSFORMATION OF WILD RICE SEED USING AGROBACTERIUM

20250051787

18/652906

May 02, 2024

Disclosed is a method for gene expression by transient transformation of a wild rice seed using Agrobacterium. The method includes the following steps: subjecting a wild rice seed to induction culture and subculture in sequence, and selecting a resulting callus with a dense structure to allow pre-culture to obtain a pre-cultured callus; transforming a 1305Ubi-Ubi-GFP-H plasmid carrying a green fluorescent protein (GFP) gene into an Agrobacterium strain EHA105 or LBA4404 to obtain a positive Agrobacterium strain; subjecting the positive Agrobacterium strain to culture to obtain a bacterial suspension with an optical density (OD) value of 0.02 to 0.5 at a wavelength of 600 nm to obtain an infection bacterial solution; and infecting the pre-cultured callus with the infection bacterial solution to allow co-culture and recovery culture in sequence; observing an infection status of a recovered callus under a stereo fluorescence microscope, and calculating a transient expression rate.

Tobacco Research Institute of CAAS (Shandong, CN), Institute of Crop Sciences, CAAS (Beijing, CN)

1. A method for gene expression by transient transformation of a wild rice seed using Agrobacterium, comprising the following steps: (1) inoculating a sterilized wild rice seed into an induction medium to allow induction culture of a callus, conducting subculture, and selecting a resulting callus with a dense structure to allow pre-culture to obtain a pre-cultured callus; (2) transforming a 1305Ubi-Ubi-GFP-H plasmid carrying a green fluorescent protein (GFP) gene into an Agrobacterium strain EHA105 or LBA4404 to allow culture at 28° C. for 2 d, selecting a resulting single clone to allow PCR and electrophoresis detection to obtain a positive Agrobacterium strain; transferring the positive Agrobacterium strain into a yeast extract broth (YEB) liquid medium to allow culture, subjecting an obtained bacterial solution to centrifugation to collect a bacterial cell, resuspending the bacterial cell in an infection buffer until a resulting bacterial suspension has an optical density (OD) value of 0.02 to 0.5 at a wavelength of 600 nm to obtain an infection bacterial solution; and (3) infecting the pre-cultured callus with the infection bacterial solution, inoculating a resulting infected callus into a co-culture medium to allow co-culture in the dark at 22° C.±2° C. for 2 d to 4 d; transferring a resulting co-cultured callus into a recovery medium to allow recovery culture in the dark at 28° C.±2° C. for 3 d; observing an infection status of a recovered callus under a stereo fluorescence microscope, wherein the GFP gene serving as an exogenous target gene is successfully expressed in the callus of the wild rice seed if the recovered callus appears green fluorescence under ultraviolet light; and calculating a transient expression rate.

2. The method according to claim 1, wherein the Agrobacterium strain is any one selected from the group consisting of Agrobacterium strains EHA105 and LBA4404, the co-culture is conducted for 3 d, and the bacterial suspension has an OD value of 0.2 at a wavelength of 600 nm.

3. The method according to claim 1, wherein the Agrobacterium strain is any one selected from the group consisting of Agrobacterium strains EHA105 and LBA4404, the co-culture is conducted for 2 d, and the bacterial suspension has an OD value of 0.2 at a wavelength of 600 nm.

4. The method according to claim 1, wherein a preparation process of the sterilized wild rice seed in step (1) comprises: immersing a wild rice seed in 75% ethanol for 30 s to 60 s and in 20% sodium hypochlorite for 20 min in sequence, washing a resulting immersed wild rice seed with sterile water 5 to 7 times until an obtained washing solution is clear, and then immersing a resulting washed wild rice seed in the sterile water overnight; and an embryo of the sterilized wild rice seed is inoculated into the induction medium to allow germination, and the induction culture of the callus is conducted at 28° C.±2° C. for 4 to 5 weeks in the dark to obtain an embryogenic callus; an embryogenic callus with a light yellow appearance and a dense structure is selected to allow the subculture on the induction medium in the dark at 28° C.±2° C.; a resulting subcultured embryogenic callus with a dense structure is selected to allow the pre-culture in the dark at 28° C.±2° C. for 7 d to 9 d.

5. The method according to claim 1, wherein the induction medium in step (1) comprises: 4.43 g of Murashige & Skoog basal salt mixture (MS salt), 1 mL of 2 mg/mL 2,4-D, 0.5 g of casein hydrolysate, 30 g of sucrose, 0.1 g of inositol, and 3 g of phytagel per liter of the induction medium with a pH value of 5.7.

6. The method according to claim 1, wherein 50 mg/mL kanamycin and 50 mg/mL rifampicin are added into the YEB liquid medium, and the centrifugation is conducted at 1,000 rpm to 2,000 rpm for 5 min in step (2).

7. The method according to claim 1, wherein each liter of the infection buffer in step (2) comprises 4 g of trace element-containing CHU'S N6 BA SAL SALT MIX (N6) 1 mL of 200 μM/mL acetosyringone, 200 μL of 10 mg/mL 2,4-D, 1 g of casein hydrolysate, 30 g of sucrose, 0.1 g of inositol, and 10 g of glucose.

8. The method according to claim 1, wherein the pre-cultured callus is infected with the infection bacterial solution for 20 min, and the infected callus is co-cultured on the co-culture medium for 3 d in step (3).

9. The method according to claim 1, wherein the co-culture medium in step (3) comprises: 4 g of trace element-containing N6, 1 mL of 200 μM/mL acetosyringone, 200 μL of 10 mg/mL 2,4-D, 1 g of casein hydrolysate, 30 g of sucrose, 0.1 g of inositol, 10 g of glucose, and 3 g of phytagel per liter of the co-culture medium with a pH value of 5.7.

10. The method according to claim 1, wherein the recovery medium in step (3) comprises: 4.43 g of MS salt, 0.5 g of casein hydrolysate, 2 mL of 2 mg/mL 2,4-D, 100 μL of 1 mg/mL 6-BA, 2 mL of 200 mg/mL Timentin, 0.1 g of inositol, 30 g of sucrose, and 3 g of phytagel per liter of the recovery medium with a pH value of 5.7.

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