Adventitious bud transformation method for sweet potatoes mediated by agrobacterium

Abstract

The invention discloses an adventitious bud transformation method for sweet potatoes mediated by agrobacterium. The method comprises the following steps: (1) acquisition and culture of aseptic seedlings; (2) pre-culture of stem segments of the sweet potato; (3) preparation of activating agent bacteria solutions of strains; (4) infection and co-culture of the agrobacterium; (5) bacteria-elimination of the agrobacterium and adventitious bud regeneration; (6) segmentation and rootage of gemmules of the sweet potato; (7) selection of transgenic plants; and (8) detection and transplantation. The invention provides a new method of genetic transformation of the sweet potatoes, which aims to use the stem segments of the sweet potato as an explant and obtain the transgenic plants through infectingand inducing the adventitious bud, therefore, the invention is suitable for various kinds of gene types with fast regeneration speed, and is a reliable system for sweet potato transgenic breeding.

Description

technical field

[0001] The invention relates to a method for transforming adventitious buds of sweet potato mediated by Agrobacterium, belonging to the field of plant genetic engineering. Background technique

[0002] Sweet potato is a vine herbaceous plant belonging to Convolvulaceae Ipomoea genus. It has high yield, its root tubers are rich in nutrients, and its starch content is as high as 12%-20%. It is an important food crop and industrial raw material. However, there is incompatibility in interspecific and intraspecific hybridization of sweet potato, which limits the combination of parents, and the efficiency of mutagenesis breeding is often low and chimerism is serious. Therefore, it is of great significance to improve sweet potato germplasm through genetic engineering technology.

[0003] Agrobacterium is a class of Gram-negative bacteria, including Agrobacterium tumefaciens (containing tumor-causing Ti plasmid) and Agrobacterium rhizogenes (containing root-causing R...

Examples

Embodiment 1

[0039] Using Jishu 22 as the recipient material, the transformed strain was Agrobacterium strain AGL104 carrying the pKGWFS7 vector (the vector was purchased from Invitrogen).

[0040] (1) Acquisition and cultivation of sterile vaccines

[0041] Cut off the part of the sweet potato plant above 10 cm from the ground, wash it with tap water for 30 minutes, cut off the leaves, cut the stem into small pieces of about 2 cm, disinfect with 70% ethanol for 1 minute, and wash with 0.1% HgCl after removing the ethanol. 2Disinfect for 10 minutes, rinse with sterile water 4 times, absorb the water with sterile filter paper, put the stems on the solid medium, and cultivate under light at 25°C. Wherein the formula of the solid medium is: MS+1.0 mg / L NAA (naphthalene acetic acid)+20 g / L sucrose+7 g / L agar, pH 5.8.

[0042] (2) Pre-cultivation of sweet potato stem section

[0043] Cut off the leaves and roots of the aseptic sweet potato seedlings grown for 30 days, cut the stems into 0.5-1...

Embodiment 2

[0059] Jishu 21 was used as the recipient material, and the transformed strain was Agrobacterium AGL104 carrying the snac1 gene (p2K7GW7 vector). The pDONR and p2K7GW7 vectors were purchased from Invitrogen.

[0060] (1) Acquisition and cultivation of sterile vaccines

[0061] Cut off the part of the sweet potato plant above 10 cm from the ground, wash it with tap water for 30 minutes, cut off the leaves, cut the stem into small pieces of about 2 cm, disinfect with 70% ethanol for 1 minute, and wash with 0.1% HgCl after removing the ethanol. 2 Disinfect for 10 minutes, rinse with sterile water 4 times, absorb the water with sterile filter paper, put the stems on the solid medium, and cultivate under light at 25°C. Wherein the formula of the solid medium is: MS+0.5mg / L NAA (naphthalene acetic acid)+20g / L sucrose+7g / L agar, pH5.8.

[0062] (2) Pre-cultivation of sweet potato stem section

[0063] Cut off the leaves and roots of the aseptic sweet potato seedlings grown for 30 ...

technical field

[0001] The invention relates to a method for transforming adventitious buds of sweet potato mediated by Agrobacterium, belonging to the field of plant genetic engineering. Background technique

[0002] Sweet potato is a vine herbaceous plant belonging to Convolvulaceae Ipomoea genus. It has high yield, its root tubers are rich in nutrients, and its starch content is as high as 12%-20%. It is an important food crop and industrial raw material. However, there is incompatibility in interspecific and intraspecific hybridization of sweet potato, which limits the combination of parents, and the efficiency of mutagenesis breeding is often low and chimerism is serious. Therefore, it is of great significance to improve sweet potato germplasm through genetic engineering technology.

[0003] Agrobacterium is a class of Gram-negative bacteria, including Agrobacterium tumefaciens (containing tumor-causing Ti plasmid) and Agrobacterium rhizogenes (containing root-causing R...

Examples

Embodiment 1

[0039] Using Jishu 22 as the recipient material, the transformed strain was Agrobacterium strain AGL104 carrying the pKGWFS7 vector (the vector was purchased from Invitrogen).

[0040] (1) Acquisition and cultivation of sterile vaccines

[0041] Cut off the part of the sweet potato plant above 10 cm from the ground, wash it with tap water for 30 minutes, cut off the leaves, cut the stem into small pieces of about 2 cm, disinfect with 70% ethanol for 1 minute, and wash with 0.1% HgCl after removing the ethanol. 2Disinfect for 10 minutes, rinse with sterile water 4 times, absorb the water with sterile filter paper, put the stems on the solid medium, and cultivate under light at 25°C. Wherein the formula of the solid medium is: MS+1.0 mg / L NAA (naphthalene acetic acid)+20 g / L sucrose+7 g / L agar, pH 5.8.

[0042] (2) Pre-cultivation of sweet potato stem section

[0043] Cut off the leaves and roots of the aseptic sweet potato seedlings grown for 30 days, cut the stems into 0.5-1...

Embodiment 2

[0059] Jishu 21 was used as the recipient material, and the transformed strain was Agrobacterium AGL104 carrying the snac1 gene (p2K7GW7 vector). The pDONR and p2K7GW7 vectors were purchased from Invitrogen.

[0060] (1) Acquisition and cultivation of sterile vaccines

[0061] Cut off the part of the sweet potato plant above 10 cm from the ground, wash it with tap water for 30 minutes, cut off the leaves, cut the stem into small pieces of about 2 cm, disinfect with 70% ethanol for 1 minute, and wash with 0.1% HgCl after removing the ethanol. 2 Disinfect for 10 minutes, rinse with sterile water 4 times, absorb the water with sterile filter paper, put the stems on the solid medium, and cultivate under light at 25°C. Wherein the formula of the solid medium is: MS+0.5mg / L NAA (naphthalene acetic acid)+20g / L sucrose+7g / L agar, pH5.8.

[0062] (2) Pre-cultivation of sweet potato stem section

[0063] Cut off the leaves and roots of the aseptic sweet potato seedlings grown for 30 ...

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