Non-antibiotic method for sifting wheat transgenic plant

A non-antibiotic screening and transgenic plant technology, applied in the field of plant transgenic technology, can solve problems such as herbicides cannot be killed, pathogenic bacteria develop drug resistance, and ecological imbalance

Inactive Publication Date: 2010-12-22
HUAZHONG AGRI UNIV
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

[0003] The potential hazards of resistance marker genes are mainly manifested in the following aspects: (1) transgenic plants with antibiotic or herbicide resistance marker genes are resistant to a variety of plant pathogens and herbicides, and may turn into harmful weeds. (2) If there are related wild weeds that can hybridize with the transgenic plant planting area, the herbicide resistance marker gene in the transgenic plant may be transferred to these related wild weeds through natural hybridization, thereby forming "Super weeds" that cannot be killed by existing herbicides; (3) If the marker gene spreads to other organisms, it may cause ecological imbalance, such as the production of super pests; (4) Resistance markers present in genetically modified foods Genes and their products may be harmful to human or animal health; (5) resistance marker genes may be transferred to human or animal intestinal parasites, making these pathogenic bacteria resistant to drugs and reducing the therapeutic effect of antibiotics
[0004] Chinese invention patent application (Application No.: 97190983.0, Monsanto Company of the United States) proposed a patent named "Agrobacterium-mediated transformation method for producing stable transformed fertile wheat and related components". The main points of the invention are To transform wheat under the mediation of Agrobacterium, it is necessary to add antibiotics in the whole screening program to obtain transgenic plants; the disadvantage is that antibiotic genes are integrated in the genome of transgenic plants obtained, and the accumulated antibiotics are expressed in wheat grains, directly Enter the food chain; at the same time, the promotion and planting of transgenic plants may spread antibiotic genes to other biological groups

Method used

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  • Non-antibiotic method for sifting wheat transgenic plant
  • Non-antibiotic method for sifting wheat transgenic plant
  • Non-antibiotic method for sifting wheat transgenic plant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Callus Induction of Wheat Varieties

[0079] The young ears of the wheat variety Zheng 9023 (this variety comes from the Henan Academy of Agricultural Sciences) at the floret primordia formation stage were induced to callus under the following conditions: at the floret primordium formation stage, the young ears of wheat were taken after being disinfected with alcohol Strip the young ear, select the young ear with a length of 1 cm and cut it into about 1 mm, and place it on the L7 medium to induce callus (the composition of the L7 medium: 250 mg / L NH 4 NO 3 , 1500mg / L KNO 3 , 200mg / L KH 2 PO 4 , 350mg / L MgSO 4 ·7H 2 O, 450mg / L CaCl 2 2H 2 O, 11.61mg / L MnSO 44H 2 O, 5mg / L H 3 B O 3, 7.5mg / LZnSO 4 ·7H 2 O, 0.75mg / L KI, 0.25mg / L Na 2 MoO 4 2H 2 O, 0.025mg / L CuSO 4 ·5H 2 O, 0.025mg / L CoCl 2 ·6H 2 O, 27.8mg / L FeSO 4 ·7H 2 O, 37.3 mg / L Na 2 ·EDTA·2H 2 O, 750mg / L Glutamine, 150mg / L Proline, 100mg / L Asparagine, 200mg / L Inositol, 2mg / L 2,4-D, 30g / L Maltose...

Embodiment 2

[0082] Genetic Transformation and Screening of Wheat Varieties

[0083] The transformation, cultivation and screening conditions of wheat young ears and immature embryos are the same. Will soon contain the expression vector pMBL5 ( figure 2 ) Agrobacterium tumefaciens cells C58, inoculated on YEB liquid medium at a ratio of 1:100 (v / v), cultured overnight at 28°C with shaking (250r / min) (to OD 600 Value around 0.8), centrifuged (5000xg) 10min to collect bacteria, resuspended in MS liquid medium, cultured at 25°C for 2h. Place the callus to be transformed in a 50ml Erlenmeyer flask, pour 20ml of bacterial solution into it, and dip for 30 minutes. The dipping medium is 1 / 10 of the L7 medium with 100mg / L ascorbic acid added, 0.02% Fluronic F68, 1.95g / L B Sulfonic acid, 200μM / L acetosyringone, 10g / L glucose, pH5.2. Take out the callus and put it on the filter paper to blot dry the bacterial liquid on the surface of the callus, then place the soaked callus on the co-culture medi...

Embodiment 3

[0086] Identification of Wheat Transgenic Plants

[0087] Take the transgenic wheat leaves obtained from the culture medium (from low to high gradient test of mannose), extract the total DNA of transgenic plant leaves by SDS method, and use primer PMIP2 (5'-AAGGATCCTTACAGCTTGTTGAAACACG-3') and primer PMIP4 (5'-CGCTGGAAGTGATGGCAAAC-3') for PCR amplification, the reaction system is: 10×PCR Buffer 2.5μl, 50mmol / LMgCl 2 0.75μl, 2mmol / L dNTP 2μl, 10μmol / L primer 0.5μl each, Taq enzyme (1U / μl) 0.5μl, template DNA 20-50ng, add ultrapure water to a total volume of 25μl. The PCR reaction program was: 95°C for 5 min; 94°C for 1 min, 55°C for 50 s, 72°C for 1 min, 35 cycles; 72°C for 5 min. The results of PCR product electrophoresis showed that in the 2 screened transgenic wheat plants with young panicles as explants and 4 transgenic wheat plants with immature embryos as explants, a specific DNA band with a fragment size of 400 bp was amplified , exactly consistent with the expected s...

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Abstract

The invention discloses a sieving method of transgenic wheat mediated by agricillin through non-antibiotics, which comprises the following steps: designing primer; doing PCR augumentation; constructing the carrier and genetic transformation; sieving and identifying the transgenic plant. The invention leads manA gene into the wheat through agricillin with mannose instead of antibiotics as selective agent, which is safe for environment without antibiotics or herbicide gene.

Description

technical field [0001] The invention belongs to the technical field of plant transgenics, and in particular relates to a method for non-antibiotic screening of transgenic plants, especially a method for transgenic wheat. The genetically modified plants also do not contain antibiotic or herbicide genes, so they are safe for the environment and are a green method of genetically modified plants. Background technique [0002] Since the first transgenic plant came out in 1983, plant genetic engineering has achieved rapid development. Because transgenes can introduce beneficial genes, so that transgenic plants have multiple advantages such as high yield, high quality, and resistance to adversity, the types and yields of transgenic crops are increasing rapidly all over the world. However, with the commercialization of genetically modified crop products, the problem that genetically modified plants may cause adverse effects and damage to the environment and human health has become ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68A01H1/00
Inventor 廖玉才李和平姚明镜
Owner HUAZHONG AGRI UNIV
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