Transformation method of eucalyptus grandis

A technology of Eucalyptus gigantea and gene, applied in the field of Eucalyptus transformation

Inactive Publication Date: 2013-12-18
BEIJING FORESTRY UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to provide a safe and efficient method for genetic transformation of Eucalyptus magnanimum, so as to solve the problems encountered in the transformation of Eucalyptus by the Agrobacterium-mediated method described in the prior art

Method used

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  • Transformation method of eucalyptus grandis
  • Transformation method of eucalyptus grandis
  • Transformation method of eucalyptus grandis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] The target gene AmCBL1 was connected to the expression vector pCAMBIA1301 containing 6-phosphomannose isomerase (PMI), and the vector was introduced into Agrobacterium tumefaciens EHA105. Shake the Agrobacterium in YEB medium containing 100 mg / mL kanamycin and 50 mg / mL rifampicin at 28°C at 180 rpm in the dark until the OD value is 0.5-0.6; 8000 rpm , centrifuged for 10 minutes, pelleted and collected bacteria. Use sucrose-free MS liquid medium containing AS 50mg / L, MES 150mg / L and galactose 1.8g / L to suspend the bacteria, and the OD value of the suspended bacteria is still adjusted to 0.5-0.6; Stem segments, cut to about 0.5 cm, were inoculated in pre-cultured callus induction medium containing 0.5 mg / L of Thidiazuron (hereinafter referred to as TDZ), 1-naphthlcetic acid (hereinafter referred to as NAA) ) 0.1mg / L, sucrose 30g / L and agar 7g / L MS medium, in a culture room with a temperature of 25±2°C, culture in dark for 1 day for pre-cultivation; For the pre-cultivate...

Embodiment 2

[0067]The target gene AmCBL1 was connected to the expression vector pCAMBIA1301 containing 6-phosphomannose isomerase (PMI), and the vector was introduced into Agrobacterium tumefaciens EHA105. Shake the Agrobacterium in YEB medium containing 100 mg / mL kanamycin and 50 mg / mL rifampicin at 28°C, 180 rpm, in the dark until the OD value is 0.4-0.5; 8000 rpm , centrifuged for 10 minutes, pelleted and collected bacteria. Use sucrose-free MS liquid medium containing AS 50mg / L, MES 150mg / L and galactose 1.8g / L to suspend the bacteria, and the OD value of the suspended bacteria is still adjusted to 0.4-0.5; Stem segments, cut to about 0.5 cm, were inoculated in pre-cultured callus induction medium containing 0.5 mg / L of Thidiazuron (hereinafter referred to as TDZ), 1-naphthlcetic acid (hereinafter referred to as NAA) ) 0.1mg / L, sucrose 30g / L and agar 7g / L MS medium, in a culture room with a temperature of 25±2°C, culture in dark for 1 day for pre-cultivation; For the pre-cultivated ...

Embodiment 3

[0069] The target gene AmCBL1 was connected to the expression vector pCAMBIA1301 containing 6-phosphomannose isomerase (PMI), and the vector was introduced into Agrobacterium tumefaciens EHA105. Shake the Agrobacterium in YEB medium containing 100 mg / mL kanamycin and 50 mg / mL rifampicin at 28°C, 180 rpm, in the dark until the OD value is 0.4-0.5; 8000 rpm , centrifuged for 10 minutes, pelleted and collected bacteria. Use sucrose-free MS liquid medium containing AS 50mg / L, MES 150mg / L and galactose 1.8g / L to suspend the bacteria, and the OD value of the suspended bacteria is still adjusted to 0.4-0.5; Stem segments, cut to about 0.5 cm, were inoculated in pre-cultured callus induction medium containing 0.5 mg / L of Thidiazuron (hereinafter referred to as TDZ), 1-naphthlcetic acid (hereinafter referred to as NAA) ) 0.1mg / L, sucrose 30g / L and agar 7g / L MS medium, in a culture room with a temperature of 25±2°C, culture in dark for 1 day for pre-cultivation; The pre-cultured Eucal...

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Abstract

The invention provides a safe and efficient genetic transformation method of eucalyptus grandis in order to solve problems encountered during the process of transforming eucalyptus by using an agrobacterium-mediated method in the prior art. The method is to successfully transfer a target gene AmCBL1 into eucalyptus grandis by constructing an expression vector with a safety marker gene PMI (6-phosphomannose isomerase) as a selection marker, making a plurality of tissue culture experiments and improving genetic transformation methods. The target gene comes from super-xerophyte ammopiptanthus mongolicus (Ammopiptanthus mongolicus (Maxim.) Cheng F.), and is driven by its specific promoter AmCBL1P.

Description

technical field [0001] The invention relates to a method for transforming eucalyptus, in particular to a method for transforming Eucalyptus magnanimum. Background technique [0002] Eucalyptus is a collective name for Eucalyptus plants of Myrtaceae Myrtaceae, an evergreen tree, and one of the world's three largest fast-growing afforestation tree species. Eucalyptus is native to Australia and is widely distributed in tropical and subtropical regions. It has the characteristics of fast growth, high yield and wide adaptability. In my country, eucalyptus is mainly distributed in Hainan, Fujian and Guangdong and Guangxi, and is the most widely used afforestation and economic tree species in southern my country. However, in the mid-subtropical region, low temperature and freezing damage are the main limiting factors for tree-based introduction, expanded cultivation, and fast growth and high yield. Therefore, the selection of cold-resistant varieties of trees, especially the sele...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82C12Q1/68A01H4/00A01H5/00
Inventor 夏新莉尹伟伦庞涛
Owner BEIJING FORESTRY UNIVERSITY
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