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Cultivation method of glyphosate-resistant transgenic Glycine max(L.).Merr

A technology of genetically modified soybean and cultivation method, which is applied in the field of cultivation of glyphosate-resistant genetically modified soybean, can solve the problems of time-consuming and labor-intensive, low efficacy, inability to timely control large area, etc., and achieve the effect of improving planting benefit and reducing labor cost.

Active Publication Date: 2021-02-05
武汉天问生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditional manual weeding includes manual weeding and weeding with simple farm tools, etc., which is time-consuming and labor-intensive, with low efficacy and cannot prevent and control large areas in time
The use of chemical herbicides has greatly improved the control efficiency of weeds in farmland, but traditional herbicides such as chlorimuron-methyl and metsulfuron-methyl have serious residues in the soil and pollute the environment, which limits the scope of use of these herbicides

Method used

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  • Cultivation method of glyphosate-resistant transgenic Glycine max(L.).Merr
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  • Cultivation method of glyphosate-resistant transgenic Glycine max(L.).Merr

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1. Construction of plant expression vectors

[0033]The construction process of the transformation vector of the present invention is as follows: use XhoI enzyme digestion to remove the hpt gene in the pCAMBIA1300 vector, and then connect the UTR-CTP-EPSPs fragments with XhoI sticky ends at both ends to the carrier from which the hpt gene has been removed to obtain the final Expression vector pTWGM1 (map as figure 2 shown). The full length of the EPSPs gene is 1374bp, and a 100bp UTR sequence and a 228bp chloroplast localization signal peptide CTP are added at its 5' end. The plasmid of the final plant expression vector pTWGM1, whose nucleotide sequence is shown in SEQ ID NO: 1, was introduced into Escherichia coli DH5α by electroporation, and after sequencing verification, it was then introduced into Agrobacterium strain EHA105 to form a transformed engineering strain. Store in a -80°C refrigerator for later use.

Embodiment 2

[0034] Example 2. Agrobacterium-mediated genetic transformation of soybean

[0035] 2.1 Chlorine disinfection of soybean seeds

[0036] Select soybean seeds that are plump, free of disease spots and with intact seed coats, sterilize them with chlorine gas generated by the reaction of hydrochloric acid and sodium hypochlorite in a fume hood for 3 hours, then put them into an aseptic operating table to ventilate for 30 minutes, and then fully soak them in sterile water overnight.

[0037] 2.2 Soak the seeds overnight

[0038] The sterilized soybean seeds were selected and fully soaked in sterile water overnight.

[0039] 2.3 Isolation of explants

[0040] Soybean seeds soaked overnight were cut in half along the direction of embryo growth, the seed coat was discarded, part of the terminal bud was cut off, and a wound was vertically drawn near the cotyledon node along the cotyledon axis, so that each sterile Two cotyledonary node explants are available from soybean seeds for g...

Embodiment 3

[0066] Example 3. PCR positive detection of transgenic regenerated plants

[0067] The total DNA of soybean leaves was extracted by CTAB method (Document: Murry & Thompson, 1980). The design of PCR primers was completed using Primer Premier 5 software. The two primers used for the amplification of the I.variabilis-EPSPs* gene (subsequently abbreviated as IVA-EPSPs) are:

[0068] IVA-EPSPsF: 5'-ATTAGCGCTAGGGACGTGAG-3', with

[0069] IVA-EPSPsR: 5'-ATACGCTCCCCACATCCTGTC-3'.

[0070] The PCR product size is 593bp. PCR reaction system: 20ng template DNA, 2μl 10×PCR buffer, 0.3μl IdNTP (10mM), 0.3μl IVA-EPSPsF primer (10μM), 0.3μl IVA-EPSPsR primer (10μM), 1U rTaq, supplemented with ddH 2 0 to 20 μl. PCR reaction program: pre-denaturation at 94°C for 5 minutes, denaturation at 94°C for 30s, annealing at 57°C for 30s, extension at 72°C for 30s; repeat 32 cycles; 72°C, 5min; 25°C, 2min. The amplified products were detected by electrophoresis on 0.8% agarose gel. to all T 0 Th...

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Abstract

The invention provides a cultivation method of glyphosate-resistant transgenic Glycine max(L.).Merr, and relates to the field of plant cultivation by utilizing genetic engineering. The cultivation method comprises the following steps of constructing a vector pTWGM1 containing a target gene I.variabilis-EPSPS*, infecting and co-culturing an agrobacterium strain containing the expression vector pTWGM1 and a Glycine max(L.).Merr cotyledonary node explant, inducing cluster buds through glyphosate screening, and performing rooting culture when the induced buds grow to be about 3 cm to obtain transgenic T0-generation seedlings; in the growth period of T0-generation plants, screening positive plants, identifying the copy number of the positive plants through Southern blot, and collecting and planting the single-copy plants; and in the T1 generation, planting transgenic plants according to families, spraying glyphosate through field observation, and selecting the transgenic families with goodglyphosate tolerance and no obvious change of agronomic traits. The transgenic Glycine max(L.).Merr exogenous gene obtained by the method can be stably inherited in the later generations and has glyphosate herbicide tolerance, the labor cost in production management can be reduced, and the planting benefit is improved.

Description

【Technical field】 [0001] The invention relates to the technical field of plant genetic engineering, in particular to a method for cultivating glyphosate-resistant transgenic soybeans. 【Background technique】 [0002] Soybean (Glycine max (L.). Merr) originated in China, is an important oil crop and economic crop in the world, and is also the main source of edible vegetable oil and vegetable protein. As an important part of the agricultural ecosystem, weeds can compete with crops for resources such as light, water, and nutrients, thereby reducing crop yields. Therefore, the effective control of weeds in soybean field is one of the key factors for stable and high yield of soybean. Traditional manual weeding includes weeding by hand and weeding with simple agricultural tools, which is time-consuming and labor-intensive, with low efficacy and cannot prevent and control large areas in a timely manner. The use of chemical herbicides has greatly improved the control efficiency of ...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/82A01H5/00A01H6/54C12Q1/6895
CPCC07K14/415C12N15/8275C12Q1/6895
Inventor 凌飞刘宝红
Owner 武汉天问生物科技有限公司
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