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Agrobacterium tumefaciens-mediated barley stem apex transformation method

A technology of Agrobacterium tumefaciens and shoot tips, which is applied in the preparation and processing of barley shoot tip materials, and in the field of cultivating transgenic plants, which can solve problems such as limitations, low transformation frequency, and long growth cycle of barley

Inactive Publication Date: 2012-01-04
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although transgenic plants have been obtained, the experiment is difficult and costly
At the same time, in the Agrobacterium-mediated transformation of barley, the outstanding problem at present is that the immature embryo about 15 days after flowering is used as the transformation recipient, the model variety Golden promise is used for transformation, and the transformation research is carried out with cultivated barley varieties. Limited and infrequent conversions
In actual work, due to the long growth cycle of barley, it takes about 150 days from sowing to the formation of immature embryos, and the field can only take materials once a year for transformation. Therefore, the defects and deficiencies of the existing technical systems must be improved

Method used

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  • Agrobacterium tumefaciens-mediated barley stem apex transformation method
  • Agrobacterium tumefaciens-mediated barley stem apex transformation method
  • Agrobacterium tumefaciens-mediated barley stem apex transformation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Using barley shoot tips as explants, Agrobacterium-mediated gene transformation ( figure 1 ):

[0056] 1. Barley seed sterilization and germination:

[0057] Select healthy and plump barley caryopsis (barley variety Edamai No. 9, Edamai 32122 from Hubei Academy of Agricultural Sciences), remove the lemma slices, soak in 70% ethanol for 3min, and then wash with 0.1% HgCl 2 Soak 13-15mm, rinse with sterile water 4 times, 2min each time. The sterilized seeds were soaked in sterile water, germinated at room temperature for 16 hours, the mature embryos were stripped, placed on the MS1 medium (see "Summary of the Invention") with the scutellum down, and 30 mature embryos were placed in each dish.

[0058] 2. Cutting and pretreatment of stem tips:

[0059] Take sterile seedlings from mature embryos that germinate and grow for 4 days at 22°C, remove the roots, scutellum and young leaves, leave about 5mm of the shoot tip, and prick the growth point with a needle. The shoot t...

Embodiment 2

[0069] Construction of vector PMBL-9 containing bar gene and GFP gene.

[0070] 1) The intermediate vector pMBL-3 was double digested with BamHI and BglII, and the large fragment was recovered by electrophoresis.

[0071] 2) The intermediate vector PUC18-GFP was digested with BamHI and BglII, and the 710bp GFP fragment was recovered by electrophoresis.

[0072] 3) The linearized pMB3 vector obtained in 1) and the GFP fragment obtained in 2) were ligated with T4 ligase.

[0073] 4) Transform the ligation product into Escherichia coli DH5α and extract the plasmid.

[0074] 5) HindIII digestion was used to identify recombinants. A band of about 3000bp was obtained by electrophoresis detection, and the vector PMBL-9 was successfully constructed.

Embodiment 3

[0076] to T 0 PCR detection of generation transformed plants:

[0077] Take about 0.1g of T 0 The leaves of the barley transgenic plants were ground into powder in liquid nitrogen, and the total DNA of barley was extracted by conventional CTAB method (refer to the method disclosed by the applicant's patent application number 200610019482.0). Design a pair of primers Ubi-P5 primer (forward, 5'-CATCTCTGTATATGCATCAG-3') and Ubi-P6 primer (reverse, 5'-CGGTAGTTCTACTTCTGTTC-3' for PCR amplification. In 25ul PCR reaction solution, containing 300ng Template DNA, 2.5ul PCR buffer, 1.5ul 25mM MgCl 2 , 2ul 1.25mMdNTPs, 0.5ul Ubi-P5 primer (10pmol / ul), 0.5ul Ubi-P6 primer (10pmol / ul), 1U Taq polymerase. The PCR reaction program was: pre-denaturation at 95°C for 4 min; denaturation at 94°C for 1 min, annealing at 58°C for 1 min, extension at 72°C for 1 min, 36 cycles; extension at 72°C for 10 min. Agarose Gel Detection of PCR Products After the reaction was completed, 18ul of PCR produ...

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Abstract

The invention discloses an agrobacterium tumefaciens-mediated barley stem apex transformation method. The transformation method comprises the following steps: A) culturing barley stem apex and constructing a carrier, wherein the specific steps are as follows: 1) selecting barley caryopses, removing hulls, stripping mature embryos and downwards placing scutella on a germination culture medium for germination; and 2) obtaining a maize ubiquitin promoter and the related sequence from an intermediate carrier pMBL-3 and a green fluorescent protein gene (GFP) through enzyme digestion and a PCR (polymerase chain reaction) method; and B) mediating the genetic transformation of the barley stem apex by utilizing agrobacterium tumefaciens, wherein the specific steps are as follows: 1) obtaining the barley stem apex; 2) dip-dyeing the stem apex; 3) using the stem apex and the agrobacterium tumefaciens GV3101 to co-culture the culture medium: taking out the stem apex which is dip-dyed with the agrobacterium tumefaciens and sucking up surface bacterial liquid with sterile filter paper; 4) performing restoring culture on the stem apex by using a restoring culture medium; 5) screening the stem apex on a screening the culture medium; and 6) vernalizing and cultivating a regenerated plant so as to obtain a candidate transformed plant. The method is easy to operate and simple to operate and has the advantages of short cycle, high efficiency and simple procedure, and the result is reliable.

Description

technical field [0001] The invention belongs to the technical field of plant transgenesis, in particular to a method for cultivating transgenic plants using barley shoot tips as materials mediated by Agrobacterium tumefaciens. The invention relates to a method for preparing and processing barley shoot tip materials, which is used for genetic transformation of barley cultivars in combination with Agrobacterium tumefaciens. Background technique [0002] Barley is one of the most important crops in the world. Its planting area and output are the fourth most important cereal crops after wheat, corn and rice. It is mainly used as food, feed, raw materials for beer industry and raw materials for pharmaceutical industry. and health food. [0003] In 1997, Tingay et al. successfully transformed barley embryos by Agrobacterium-mediated method for the first time to obtain transgenic barley plants. Subsequently, Agrobacterium-mediated transformation method gradually developed into an ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/66A01H4/00A01H5/00
Inventor 廖玉才李和平刘正位高春生黎冬华
Owner HUAZHONG AGRI UNIV
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