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Corn transforming method

A corn, suspension cell technology, applied in biochemical equipment and methods, horticultural methods, botanical equipment and methods, etc., can solve the problems of long cycle, seasonal restrictions and high cost

Pending Publication Date: 2017-03-22
BEIJING DABEINONG BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to provide a method for transforming corn, which can effectively overcome the technical defects of the prior art such as high cost, seasonal restrictions on material selection, long cycle, and unstable state.

Method used

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  • Corn transforming method
  • Corn transforming method
  • Corn transforming method

Examples

Experimental program
Comparison scheme
Effect test

no. 1 example

[0113] The first embodiment, construction of recombinant expression vector and transformation of recombinant expression vector into Agrobacterium

[0114] 1. Construct a recombinant cloning vector containing the target gene

[0115] The PAT nucleotide sequence was connected to the cloning vector pGEM-T (Promega, Madison, USA, CAT: A3600), and the operation steps were carried out according to the instructions of the pGEM-T vector produced by Promega Company to obtain the recombinant cloning vector DBN01-T, and its construction process Such as figure 1 Shown (wherein, Amp represents the ampicillin resistance gene; f1 represents the replication origin of phage f1; LacZ is the LacZ initiation codon; SP6 is the SP6 RNA polymerase promoter; T7 is the T7 RNA polymerase promoter; PAT is glufosinate Acetyltransferase gene nucleotide sequence (SEQ ID NO: 1); MCS is a multiple cloning site).

[0116] Then, the recombinant cloning vector DBN01-T was transformed into Escherichia coli T1 ...

no. 2 example

[0124] The second embodiment, the acquisition of transgenic corn plants

[0125] 1. Extraction of young corn embryos

[0126] Sow the seeds of corn variety Qi 319 (Q319) in a greenhouse or field, and harvest corn ears 7-12 days after the corn plants grow into pollination, soak them in alcohol with a concentration of 75% (v / v) for 5 minutes, and peel them off. Take immature embryos, and select transparent immature embryos with a length of 0.6-1.2 mm for subsequent use.

[0127] 2. Screen callus induction medium

[0128]Inoculate the above-mentioned stripped corn immature embryos on the following five different callus induction media for callus induction, and culture them in dark for 10-14 days at a temperature of 28°C, preferably 14 days, and count the callus Induction rate of wound tissue and observation of callus morphology, the results are shown in Table 2. Each callus induction medium was replicated three times, and 100 immature embryos were inoculated in each replicate....

no. 3 example

[0160] The third embodiment, using TaqMan to verify transgenic maize plants

[0161] About 100 mg of the leaves of 8 transgenic maize plants were taken as samples, and the genomic DNA was extracted with Qiagen's DNeasy PlantMaxi Kit, and the copy number of the PAT gene was detected by fluorescent quantitative PCR with Taqman probes. At the same time, wild-type maize plants were used as a control, and detection and analysis were carried out according to the above method. The experiment was repeated 3 times, and the average value was taken.

[0162] The specific method for detecting the copy number of PAT gene is as follows:

[0163] Step 11, take 100 mg of leaves of 8 kinds of transgenic corn plants and wild-type corn plants respectively, grind them into homogenate with liquid nitrogen in a mortar, and take 3 replicates for each sample;

[0164] Step 12, using the DNeasy Plant Mini Kit of Qiagen to extract the genomic DNA of the above sample, the specific method refers to its...

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Abstract

The present invention relates to a corn transforming method, which comprises: obtaining primary callus by using a corn callus inducing method, obtaining a corn suspending cell line by using a corn suspending cell line obtaining method, and infecting the corn suspending cell aggregate in the corn suspending cell line through Agrobacterium. According to the present invention, the suspending cell line is rapidly obtained firstly from the primary callus induced by the corm immature embryo, such that the experimental period is shortened to 40-50 days, the operation is simple and convenient, the reproducibility is good, the genetic transformation of the corn is not affected by the season, and the annual stable transforming requirements are met; the corn suspending cell line is infected and transformed by using the Agrobacterium and then the seedling is generated, such that the transforming efficiency can achieve about 30%, the transgene positive rate can achieve about 90%, and the single copy rate is about 50%; and the corn explant (immature embryo) supply pressure is significantly reduced so as to substantially reduce the manpower and material resource consumption required by the explants culture and achieve the large-scale genetic transformation.

Description

technical field [0001] The invention relates to a method for plant transformation, in particular to a method for transforming maize suspension cell lines to obtain transgenic plants. Background technique [0002] Maize (Zea mays L.) is one of the three major food crops in the world, and it is also an important industrial raw material and bioenergy material, and plays a pivotal role in the food production in my country and the world. Among the many factors that increase the yield of corn, the role of new variety selection accounts for 40%. With the rapid development of molecular biology technology, transgenic technology has begun to play an important role in the breeding of new maize varieties. GM corn has become the second largest GM crop in the world after GM soybean. [0003] A good receptor system is an important part of maize genetic transformation, which is related to the provision of suitable receptor materials for transformation and whether the transformed cells can...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/04C12N5/10C12N15/82A01H5/00A01H4/00
CPCA01H4/001A01H4/005C12N5/04C12N15/8205
Inventor 周文祥陈莉莉张世平
Owner BEIJING DABEINONG BIOTECHNOLOGY CO LTD
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