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Rice transformation process lead-in by agrobacterium induced

An Agrobacterium-mediated, Agrobacterium-mediated technology, applied in the field of plant genetics and plant genetic engineering, can solve problems such as the inability to adapt to the requirements of BIBAC system transformation

Inactive Publication Date: 2005-01-05
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The conventional Agrobacterium-mediated rice transformation system (Hiei et al. Plant J.1994, 6: 271-282) cannot meet the requirements of BIBAC system transformation

Method used

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  • Rice transformation process lead-in by agrobacterium induced
  • Rice transformation process lead-in by agrobacterium induced
  • Rice transformation process lead-in by agrobacterium induced

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0044] 1. Induction of callus

[0045] Select fresh and disease-free mature seeds of the cultivated japonica rice line H1493, remove the shell, soak with 70% ethanol for 1 min, and then wash with 0.1% HgCl 2 Disinfect the surface for 15-20 minutes (shaking at 100r / min), and then fully soak with sterile water (change the water at least 4 times). Put the sterilized seeds on the sterilized filter paper and blot dry, then transfer to induction medium (N6I) [N6 (Chu et al., Scientia Sinica 1975, 18: 659-668), proline 1.0g / l, hydrolyzed casein Protein 0.4g / l, 2,4-D 2.0g / l, sucrose 45g / l, tissue culture gel (phytagel) 3.0g / l, pH 5.9]. Put 10-12 seeds in each dish, and cultivate them in the dark at 26-28°C for 2 weeks.

[0046] 2. Subculture and pre-culture

[0047] After 2 weeks, the fresh, bright yellow scutellum calli were removed and transferred to a new N6I medium for subculture for 2 weeks. After 2 weeks, the well-growing embryogenic callus was transferred to the pre-medium ...

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Abstract

The invention is an Agrobacterium Tumefacies mediated rice transforming method, concretely using a binary bacterium artificial chromosome (BIBAC) carrier, mediating by LBA4404 Agrobacterium Tumefacies strains containing a toxic helper plasmid pCH32, adopting an optimized transforming system to lead a big fragment of exogenous DNA coming from a gene group 120kb of medicinal wild rice into cultivated rice H1493. The identification and verification of transgenic plants by a reporter gene (gus), polyase chain reaction (PCR) detection, Southern molecular hybridization, etc show that the transformation of big fragmentof DNA is implemented in the rice. The method opens a new way for plant gene cloning and function analysis, properties improvement, etc.

Description

technical field [0001] The invention relates to the technical fields of plant genetics and plant genetic engineering, and more specifically relates to an Agrobacterium-mediated rice transformation method. Background technique [0002] Agrobacterium (Agrobacterium) is a Gram-negative soil bacteria, divided into Agrobacterium tumefaciens (Agrobacterium tumefaciens) and Agrobacterium rhizogenes (A.rhizogenes). Agrobacterium tumefaciens contains a tumor-inducing plasmid (Tumor-inducing plasmid), referred to as Ti plasmid. The Agrobacterium plasmid DNA fragment on the Ti plasmid that can be integrated into the plant genome is named transfer DNA (Transfer DNA), referred to as T-DNA. There is a border of 25bp repeat sequences at both ends of T-DNA, which are called left border (LB) and right border (RB) respectively. RB is necessary for the transfer of T-DNA, but LB has a certain effect on the size of the transferred fragment. Significant; the exogenous ...

Claims

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Application Information

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IPC IPC(8): A01H4/00C12N5/04C12N5/10C12N15/82
Inventor 何光存何瑞锋祝莉莉
Owner WUHAN UNIV