Method and medium formula for improving gene gun transient transformation efficiency of young wheat embryo

Abstract

The invention discloses a method and medium formula for improving the gene gun transient transformation efficiency of a young wheat embryo, belonging to the field of plant transformation. Based on experimental results, the invention provides a hypertonic medium containing mannitol with a concentration of 0.2 to 0.4 M and sorbitol with a concentration of 0.2 to 0.4 M; and the hypertonic medium allows the gene gun transient transformation efficiency of the young wheat embryo to be increased to 53.5 to 65.4%. The invention also establishes a high-efficiency transient transformation system for the young wheat embryo, so a good foundation is laid for subsequent functional genomics research and molecular breeding of wheat.

Description

technical field

[0001] The invention belongs to the field of plant transformation, and in particular relates to a method for improving the instantaneous transformation efficiency of wheat immature embryo gene gun. Background technique

[0002] Wheat is the last important cereal crop successfully transformed. On the one hand, wheat is a hexaploid crop with a complex genetic background and a relatively large genome (about 17Gb), which is 7 times that of corn and 40 times that of rice; , during the process of genetic transformation of wheat, the frequency of DNA introduction is low and the regenerative ability after transformation is not high, which has a strong genotype dependence. Since the first case of transgenic wheat was successfully obtained in 1992, people have tried to transform wheat using various transformation methods and various explants, including Agrobacterium, gene gun, pollen tube passage, ultrasonic method, ion beam implantation, laser Microbeam puncture meth...

Examples

Embodiment 1

[0033] Embodiment 1, immature embryo is drawn material

[0034] Cut the young ears of wheat that have grown vigorously and have less pests and diseases and have bloomed for 10-14 days in the field or in the growth room, strip the seeds of the young ears, and disinfect them with 75% ethanol solution by volume for 1 minute; rinse them with sterile water for 3 times; Then sterilize with 1% silver nitrate solution for 15-20min; rinse with sterile water 3 times. The translucent immature embryos (ie young wheat embryos) with a size of 0.8-2.0 mm were peeled off after the embryos were excised with a scalpel in the ultra-clean workbench.

Embodiment 2

[0035] Embodiment 2, the transient transformation of immature embryo

[0036] Take the immature embryos stripped in Example 1, pre-culture for 6-8 days, inoculate the scutellum up on the hyperosmotic medium prepared by the following method, and place them in a 60×15 mm petri dish with a central diameter of 2.0 —In a circular area of ​​3.0 cm, seal with Parafilm, and culture in hypertonicity at 22°C for 4-6 hours in the dark. Biolistic transformation is a method of introducing exogenous DNA into plant cells by physical means. Hyperosmotic treatment of receptor material before bombardment by gene gun can reduce the damage to recipient cells during the bombardment process.

[0037] The formula and preparation method of hypertonic medium used in this embodiment are: CaCl 2 2.99mM, KNO 3 18.79mM, NH 4 NO 3 20.61mM, KH 2 PO 4 1.25mM, MgSO 4 1.50mM, MnSO 4 1mM, ZnSO 4 30 μM, H 3 BO 3 100 μM, KI 5 μM, NaMoO 4 1 μM, CuSO 4 0.1 μM, CoCl 6 0.1 μM, FeSO 4 100 μM...

Embodiment 3

[0061] Embodiment 3, the detection of instantaneous conversion efficiency

[0062] Because the concentration of hypertonic medium and hypertonic treatment time applicable to different receptor materials are different, the inventors compared the transient transformation efficiency of the plasmid in the ratio of different concentrations of mannitol and sorbitol in the above-mentioned medium. , the hyperosmotic treatment time is 4-6 hours, and the formula of MSV4 medium is: CaCl 2 2.99mM, KNO 3 18.79mM, NH 4 NO 3 20.61mM, KH 2 PO 4 1.25mM, MgSO 4 1.50mM, MnSO 4 1mM, ZnSO 4 30 μM, H 3 BO 3 100 μM, KI 5 μM, NaMoO 4 1 μM, CuSO 4 0.1 μM, CoCl 6 0.1 μM, FeSO 4 100 μM, Na 2 -EDTA 100μM, niacin 8μM, inositol 0.56mM, thiamine hydrochloride 30μM, pyridoxine hydrochloride 4.9μM, glycine 0.027mM, glutamine 0.2mM, hydrolyzed casein 200mg / L, sucrose 30g / L. The results are shown in Table 1 below. The acceptor material treated with 0.3M mannitol+0.3M sorbitol has the ...

technical field

[0001] The invention belongs to the field of plant transformation, and in particular relates to a method for improving the instantaneous transformation efficiency of wheat immature embryo gene gun. Background technique

[0002] Wheat is the last important cereal crop successfully transformed. On the one hand, wheat is a hexaploid crop with a complex genetic background and a relatively large genome (about 17Gb), which is 7 times that of corn and 40 times that of rice; , during the process of genetic transformation of wheat, the frequency of DNA introduction is low and the regenerative ability after transformation is not high, which has a strong genotype dependence. Since the first case of transgenic wheat was successfully obtained in 1992, people have tried to transform wheat using various transformation methods and various explants, including Agrobacterium, gene gun, pollen tube passage, ultrasonic method, ion beam implantation, laser Microbeam puncture meth...

Examples

Embodiment 1

[0033] Embodiment 1, immature embryo is drawn material

[0034] Cut the young ears of wheat that have grown vigorously and have less pests and diseases and have bloomed for 10-14 days in the field or in the growth room, strip the seeds of the young ears, and disinfect them with 75% ethanol solution by volume for 1 minute; rinse them with sterile water for 3 times; Then sterilize with 1% silver nitrate solution for 15-20min; rinse with sterile water 3 times. The translucent immature embryos (ie young wheat embryos) with a size of 0.8-2.0 mm were peeled off after the embryos were excised with a scalpel in the ultra-clean workbench.

Embodiment 2

[0035] Embodiment 2, the transient transformation of immature embryo

[0036] Take the immature embryos stripped in Example 1, pre-culture for 6-8 days, inoculate the scutellum up on the hyperosmotic medium prepared by the following method, and place them in a 60×15 mm petri dish with a central diameter of 2.0 —In a circular area of ​​3.0 cm, seal with Parafilm, and culture in hypertonicity at 22°C for 4-6 hours in the dark. Biolistic transformation is a method of introducing exogenous DNA into plant cells by physical means. Hyperosmotic treatment of receptor material before bombardment by gene gun can reduce the damage to recipient cells during the bombardment process.

[0037] The formula and preparation method of hypertonic medium used in this embodiment are: CaCl 2 2.99mM, KNO 3 18.79mM, NH 4 NO 3 20.61mM, KH 2 PO 4 1.25mM, MgSO 4 1.50mM, MnSO 4 1mM, ZnSO 4 30 μM, H 3 BO 3 100 μM, KI 5 μM, NaMoO 4 1 μM, CuSO 4 0.1 μM, CoCl 6 0.1 μM, FeSO 4 100 μM...

Embodiment 3

[0061] Embodiment 3, the detection of instantaneous conversion efficiency

[0062] Because the concentration of hypertonic medium and hypertonic treatment time applicable to different receptor materials are different, the inventors compared the transient transformation efficiency of the plasmid in the ratio of different concentrations of mannitol and sorbitol in the above-mentioned medium. , the hyperosmotic treatment time is 4-6 hours, and the formula of MSV4 medium is: CaCl 2 2.99mM, KNO 3 18.79mM, NH 4 NO 3 20.61mM, KH 2 PO 4 1.25mM, MgSO 4 1.50mM, MnSO 4 1mM, ZnSO 4 30 μM, H 3 BO 3 100 μM, KI 5 μM, NaMoO 4 1 μM, CuSO 4 0.1 μM, CoCl 6 0.1 μM, FeSO 4 100 μM, Na 2 -EDTA 100μM, niacin 8μM, inositol 0.56mM, thiamine hydrochloride 30μM, pyridoxine hydrochloride 4.9μM, glycine 0.027mM, glutamine 0.2mM, hydrolyzed casein 200mg / L, sucrose 30g / L. The results are shown in Table 1 below. The acceptor material treated with 0.3M mannitol+0.3M sorbitol has the ...

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