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Cultivation method for lily with pollen abortion and herbicide-resistance

A technology against herbicides and pollen abortion, applied in chemical instruments and methods, botany equipment and methods, biochemical equipment and methods, etc., can solve the problems of low artificial weeding efficiency, high labor costs, and high production efficiency

Inactive Publication Date: 2021-01-12
ZHUZHOU INST OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The application of fertilizers and pesticides in the lily planting process can be carried out by mechanized devices, with high production efficiency and relatively low labor costs; in contrast, weed removal in the lily planting process cannot be effectively carried out by mechanization, manual weeding The efficiency is low, and weeding needs to be carried out many times during the lily planting cycle, which results in high labor costs
In addition, transgenic plants formed by genetic engineering can easily lead to gene drift through pollen mediation, allowing foreign genes to enter non-transgenic plants, causing biosafety issues such as genetic pollution

Method used

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  • Cultivation method for lily with pollen abortion and herbicide-resistance
  • Cultivation method for lily with pollen abortion and herbicide-resistance
  • Cultivation method for lily with pollen abortion and herbicide-resistance

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] 1.1 Synthesis of TA29-Barnase-Nos element

[0034] The TA29-Barnase-NOs element sequence information was searched in the NCBI database, and SpeI and PstI restriction sites were added between the TA29 promoter and the Barnase regulatory element to facilitate replacement of the promoter in subsequent experiments. Send the sequence information to Huada Genomics Co., Ltd. to synthesize the TA29-Barnase-NOs element, and the sequence is shown in SEQ ID NO:1. BGI inserted the synthesized element into the pUC57-simple vector, and the vector obtained after insertion was named pUC57-simple-TA29-bar (Pucs7-bar for short), such as figure 1 shown.

[0035] 1.2 Preparation of LB medium

[0036] The composition of 150mL solid liquid medium is as follows:

[0037] Table 1.1 LB medium

[0038]

[0039] 1.3 Plasmid amplification and extraction

[0040] The Pcambia3301 and Pucs7-bar plasmids were extracted using Shanghai Sangong Sanprep column plasmid extraction kit.

[0041] 1.4...

Embodiment 2

[0066] Example 2 Agrobacterium-mediated system establishment and optimization

[0067] 2.1 Pre-cultivation

[0068] Choose healthy lily seedlings, cut lily scales size, inoculated in M1 medium. The scales that were not directly pre-cultured were used for pre-culture for 1d, 2d, 3d and 4d in the dark at 25°C, with the same co-cultivation time, the same concentration of Agrobacterium treatment and the same selection culture conditions. After 40 days, the pre-incubation time was determined by counting and comparing the regeneration rate of different resistant shoots. The results of the study showed that pre-cultivation for three days was the most favorable for transformation (see Figure 8 ).

[0069] 2.2 Preparation of Agrobacterium

[0070] 200uL containing the carrier (referring to obtain such as Figure 7 The bacterial solution of the indicated recombinant vector) was added to 5 mL of LB liquid medium, and cultured at 28° C. for 20-25 hours on a shaker at 180 r / min. A...

Embodiment 3

[0085] Example 3 Molecular Detection of Resistant Plants

[0086] 3.1 PCR detection

[0087] From the screening medium of step 2.4, select the plants with normal growth to carry out PCR detection and GUS identification results such as Figure 12 As shown, figure a is the result of PCR detection, figure b is the result of GUS identification, where M: Marker DL2000; P: positive plasmid; 0: untransformed lily; 1-16 in figure a: transformed lily; 1-13: Transform Lily. The results of PCR identification showed that the detected plants (No. 1-16) were all positive, and GUS identification showed that the detected plants (No. 1-13) were all positive, which preliminarily proved that the target gene had been transformed into the lily plant genome.

[0088] 3.2 Recovery of PCR positive fragments for sequencing

[0089] PCR positive fragments were recovered, and the sequencing results were completely consistent with the target gene sequence Figure 13 , which fully proves that the targ...

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Abstract

The invention provides a cultivation method of lily with pollen abortion and herbicide resistance. The method mainly comprises the following steps: S1. constructing a recombinant vector, loading a promoter TA29 and a terminator Nos onto Barnase to obtain TA29-Barnase-NOs, inserting the TA29-Barnase-NOs into a vector I, conducting propagating, and extracting recombinant plasmids; S2. recovering target DNA from the recombinant plasmid obtained in the step S1, conducting PCR amplification, and connecting the amplified DNA to a vector II to obtain a recombinant vector; S3. transferring the constructed recombinant vector into lily scales by using an agrobacterium-mediated method, obtaining positive plants by using a herbicide screening method, optimizing an agrobacterium-mediated method system,and sequentially transferring the positive plants into rooting and germination induction culture media for culturing, so as to obtain the positive transgenic lily. The obtained lily plants are successfully transferred into pollen abortion genes, no pollen is generated during flowering. Herbicide resistance is achieved, herbicides can be thus conveniently used for efficient weeding in the plantingprocess, so that labor force can be effectively reduced, and production benefits are improved.

Description

technical field [0001] The invention relates to the field of plant cultivation, in particular to transferring a target gene into a lily plant through genetic engineering and making it obtain pollen abortion and herbicide resistance characteristics through screening. Background technique [0002] Lily plants are relatively common agricultural garden plants, and their bulbs can be used as food or medicine, and lilies are popular as common ornamental flowers. In order to meet the diverse needs, the requirements for the cultivation of new germplasm of lily have been increased accordingly. The application of fertilizers and pesticides in the lily planting process can be carried out by mechanized devices, with high production efficiency and relatively low labor costs; in contrast, weed removal in the lily planting process cannot be effectively carried out by mechanization, manual weeding The efficiency is low, and weeding needs to be carried out many times during the lily plantin...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/29A01H5/00A01H6/56
CPCC12N15/8205C12N15/8274C12N15/827C07K14/415
Inventor 郑思乡廖晓珊荣朵艳周俊张翔谭益民汤建新宋志伟邓雅文林庆丹吴龙云
Owner ZHUZHOU INST OF AGRI SCI
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