Method for conversing switchgrass conducted by agrobacterium

An Agrobacterium-mediated, Agrobacterium-based technology, applied to other methods of inserting foreign genetic materials, horticultural methods, botanical equipment and methods, etc., can solve the problems of late transformation, difficulty, and low transformation efficiency, and achieve differentiation and transformation Efficiency improvement, significant effect, and the effect of avoiding a large number of escapes

Inactive Publication Date: 2008-02-13
NORTHWEST A & F UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, due to the late start of the genetic transformation research of switchgrass and the difficulty of transformation, there is only one successful report of transgenic in the world (M.N.Somleva, Z.Tomaszewski, and B.V.Conger. Agrobacterium-mediated genetic transformation of switchgrass. Crop Sci. 2002, 42:2080-2087), and the conversion efficiency is low (about 2%)

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Two switchgrass varieties, Alamo and Blackwell, were transformed with EHA105 Agrobacterium pCAMBIA1301 plasmid containing hygromycin and kanamycin selection markers. The specific conversion process is:

[0066] (1) Artificial induction of young panicles

[0067] a. When Alamo and Blackwell grow to have 6 nodes, the uppermost stem node is manually cut, and the cut part includes the upper and lower sections with a length of 1.5cm each. Cut out 2000 stem nodes from different plants respectively;

[0068] b. Disinfect the surface with 70% alcohol by volume for 1 minute;

[0069] c. Sterilize with 20% volume concentration of commercial sodium hypochlorite (adding 0.1% Tween 100) for 12 minutes;

[0070] d. Put 2 stem nodes into each dish on the MBP medium to artificially induce young ears. The formula of the MBP medium is: MS inorganic salt and vitamin mixture 4.43g / L, maltose 30g / L, 6-BA (6-benzyl aminopurine) 3mg / L, agar powder 7.5g / L, PH=5.8;

[0071] e. 28°C, light ...

Embodiment 2

[0092] Leaf senescence suppressor gene P SAG12 - IPT's Agrobacterium EHA105 transformed GA-001A and NFSGOS-12 switchgrass varieties. The specific conversion process is:

[0093] (1) Artificial induction of young panicles

[0094] a. When GA-001A and NFSGOS-1 have developed to 6 nodes, the uppermost stem node is manually cut, and the cut part includes the upper and lower sections with a length of 1.5cm each. Cut out 2000 stem nodes from different plants respectively;

[0095] b. sterilize the surface of the stem node of the plant with alcohol of 70% volume concentration for 1 minute;

[0096] c. Sterilize with 20% volume concentration of commercial sodium hypochlorite (with 0.1% Tween 100 added) for 15 minutes. The stalks of GA-001A and NFSGOS-1 are thicker, and the sterilization time is extended to 15 minutes;

[0097] d. put 2 stem nodes into each dish on the MBP medium to artificially induce young ears;

[0098] e. Culture at 28°C under light for 20 days until the leng...

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Abstract

The invention discloses an agrobacterium-mediated versatile-switch-grass transformation method. During the transformation, a man-induced young spike is taken as an explant; embryonic callus is induced on screening SMB culture medium; agrobacterium transformation and co-culture are adopted, and screening is conducted on culture media SM1 and SM2 successively to achieve resistant callus; the resistant callus is transferred to split-up culture media SMK1 and SMK2 successively to achieve regenerated plants; the regenerated plants are arranged in the rooting culture medium SMO and cultivated into seedlings; the seedlings are transplanted into a greenhouse or in a crop field for molecular biology-based testing; and the transgene plants expressed by extraneous objective gene is achieved according to molecular biologic method and objective character screening. The versatile-switch-grass transformation method has transformation efficiency about 10 percent, and the method lays a foundation for genetic improvement of the bio-energy (versatile switch grass) by transgene.

Description

technical field [0001] The invention relates to the field of genetic engineering utilization of bioenergy plant switchgrass, and further relates to a method for transforming switchgrass mediated by Agrobacterium. Background technique [0002] With the sharp rise of international oil prices and the continuous deterioration of the ecological environment caused by the massive consumption of petroleum products, bioenergy characterized by fuel ethanol and biodiesel is of increasing strategic significance for sustainable economic and social development. Switchgrass (English name Switchgrass, Latin name Panicumvirgatumlinn) is internationally recognized as the preferred bioenergy plant for the production of fuel ethanol because of its simple cultivation techniques, wide adaptability, strong windbreak and sand fixation ability, tolerance to barrenness, and high biological yield. A lot of manpower and material resources for research and development. Among them, the use of genetic en...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/87C12N5/04A01H4/00
Inventor 奚亚军刘曙东
Owner NORTHWEST A & F UNIV
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