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Method of screening transgene barley strain without antibiotic mediated by agrobacterium

An Agrobacterium-mediated, antibiotic-free technology, applied in botany equipment and methods, biochemical equipment and methods, genetic engineering, etc.

Inactive Publication Date: 2007-08-15
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method is the most commonly used method of PMI activity detection, all has application on transgenic wheat, corn and sweet orange detection (Boscariol RL, Almeida WA B, Derbyshire M T V C, Mour o Filho F A A, Mendes B M J. The use of the PMI / mannose selection system to recover transgenic sweet orange plants (Citrus sinensis L. Osbeck). Plant Cell Rep, 2003, 22:122-128; Wright M, Dawson J, Dunder E .Efficient biolistic transformation of maize (Zea mays L.) and wheat (Triticum aestivum L.) using the phosphomannose isomerase gene, pmi, as the selectable marker. Plant Cell Rep, 2001, 20: 429-436; Gao Z, Xie X, Yan L, Muthukrishnan S, Liang G H. Agrobacterium tumefaciens-mediated sorghum transformation using a mannose selection system. Plant Biotechnol J, 2005, 3: 591-599; Degenhardt J, Poppe A, Montag J, Szankowski I. The use of the phosphomannose isomerase / mannose selection system to recover transgenic apple plants. Plant Cell Rep, 2006, 25: 1149-1156; Lucca P, Ye X D, Potrykus I. Effective selection and regeneration of transgenic rice plants with mannose as selective agent. Mol Breeding, 2001, 7 :43-49; etc.), it has not been reported yet in the genetic transformation of barley

Method used

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  • Method of screening transgene barley strain without antibiotic mediated by agrobacterium
  • Method of screening transgene barley strain without antibiotic mediated by agrobacterium
  • Method of screening transgene barley strain without antibiotic mediated by agrobacterium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1: Agrobacterium-mediated genetic transformation of barley without antibiotics

[0066] 1. Induction of callus

[0067] Young barley embryos with a width of about 0.5 mm to 1.5 mm and translucent color (variety Zaoshu No. 3 - from Hubei Academy of Agricultural Sciences) were used as explants. First, the immature barley seeds were sterilized with 70% ethanol for 30 seconds, rinsed once with sterile water, then sterilized with 0.1% mercuric chloride for 8 minutes, and rinsed with sterile water three times, each time for 3 minutes. The explants were stripped, and the explants were inoculated on the induction medium (see the "Summary of the Invention") with the scutellum facing up, and cultured at 25° C. in the dark to induce callus.

[0068] 2. Pre-cultivation of callus

[0069] From the callus obtained in the first step, the vigorously growing callus was picked and inoculated onto the pre-culture medium (see the "Summary of the Invention" section), and cultivate...

Embodiment 2

[0082] Embodiment 2: Chlorophenol red chromogenic method (CPR) detection of transformed plant

[0083] Choose the newly grown leaf on the main stem of the transformed plant of Example 1, cut the leaf tip section with an area size of about 0.3cm * 0.5cm, and immerse the leaf section in the chlorophenol red liquid detection medium (see the composition of the medium for the test medium). In the preparation part of the chlorophenol red liquid detection medium in "Summary of the Invention", observe the color change of the medium after culturing at 25° C. in the dark for 4 days. Cut the leaf segment of untransformed plant according to same requirement simultaneously, immerse respectively in the chlorophenol red liquid detection medium and the chlorophenol red liquid contrast medium (referring to " the content of the invention " the preparation part of the chlorophenol red liquid contrast medium), specifically For the operation steps, refer to the "Summary of the Invention" section, ...

Embodiment 3

[0084] Embodiment 3: PCR detection to transformed plant

[0085] Take about 0.1 g of leaves of barley transgenic plants, grind them into powder in liquid nitrogen, and extract the total DNA of barley by conventional CTAB method (refer to the method disclosed by the applicant's patent application No. 200610019482.0). A pair of specific primers Primer1 (forward, 5'-GTTTCTTTTGTCGATGCTCACCC-3') and Primer2 (reverse, 5'-TCCGGCTTGTGGTTAGGATC-3') were designed 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 Primer1 (10pmol / ul), 0.5ul Primer2 (10pmol / ul), 1U Taq polymerase. The PCR reaction conditions were: 95°C for 5 min; 94°C for 1 min, 58°C for 50 sec, 72°C for 90 sec, 35 cycles; finally 72°C for 5 min, 10°C for 10 min. After the reaction was completed, 18ul of the PCR product was electrophoresed on a 1.2% agarose gel. In the gel imaging system, take pictures under ultraviolet light...

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Abstract

The invention discloses a method to screen and culture barley transfer-gene plant with non-antibiotic of agricillin dielectric conductance in plant transfer-gene technical domain, which comprises the following steps: leading reported gene into receptor barley cell through agricillin dielectric conductance; utilizing mannose to replace antibiotic as chosen agent; screening the reverting receptor cell; adjusting the genetic reforming process of barley through phosphomannose isomerase; further-screening transfer-gene barley with chlorophenol red colouration method and PCR method; getting the end product. The barley cultivating strain conversion rate can reach 12% with agricillin dielectric conductance and can reach 100% with chlorophenol red colouration method.

Description

technical field [0001] The invention belongs to the technical field of plant transgenics, and in particular relates to a method for cultivating barley transgenic plants through Agrobacterium-mediated antibiotic-free screening. The invention also relates to the application of the PMI (phosphomannose isomerase) / mannose screening system in the genetic transformation of human wheat, and the application of the chlorophenol red (CPR) chromogenic method in the identification of barley transgenic plants. Background technique [0002] Barley is one of the oldest crops in the world, and it is the fourth most important cereal crop after wheat, corn and rice. It is mainly used as feed, grain, raw material for beer industry, raw material for pharmaceutical industry and health food that have attracted attention in recent years. . Barley is also one of the model plants widely used in genetic research. [0003] Since Tingay et al first transformed barley with Agrobacterium-mediated transf...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11A01H1/00A01H5/00C12N15/31C12N15/82
Inventor 廖玉才李和平王韬黄涛
Owner HUAZHONG AGRI UNIV
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