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Method for establishing transgenic system of high-sugar variety beet

A method of establishment and transgenic technology, which is applied in the field of establishment of the sugar beet transgenic system, can solve the problems of the reporting of the transgenic method and the unreported method of the sugar beet transgenic method, and achieve the effect of accelerating the transfer and improving the value of the ecological environment quickly and effectively

Inactive Publication Date: 2020-05-08
LUDONG UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although sugar beet tissue culture and rapid propagation techniques have matured, and callus induction has also been reported, there is no report on the transgenic method of sugar beet, and there is no report on the transgenic method in the genus Beta.

Method used

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  • Method for establishing transgenic system of high-sugar variety beet
  • Method for establishing transgenic system of high-sugar variety beet
  • Method for establishing transgenic system of high-sugar variety beet

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Germination and growth of sugar beet seeds

[0044] 1) Select 60 sugar beet seeds harvested in the same year, place them in a 250ml Erlenmeyer flask and add tap water to immerse them, culture them in a shaker at 37°C for 16 hours, wash them with running water for 24 hours, then sow them in a culture bottle containing moist gauze, and place them at 22°C , cultured in a culture room with a light duration of 16h / 8h, see figure 1 .

[0045] 2) When the beet seeds germinate small buds, start to record the number of days of growth, and it can be used for experiments when it grows for about 10 days.

[0046] See figure 2 .

Embodiment 2

[0047] Example 2: Transformation of transgenic sugar beets

[0048] a. Expanded culture of Agrobacterium

[0049] ① With LB solid and liquid medium containing kanamycin; the concentration of kanamycin used is 50mg / L;

[0050] ② In the workbench, use an inoculation loop to draw the preserved strain on the solid medium containing kanamycin, and culture it in the dark at 28°C for 48 hours;

[0051] ③ Take out the bacteria plate, pick a single colony with a sterilized yellow tip and place it in 20ml LB liquid medium containing kanamycin, shake the bacteria at 220rpm at 28°C for 24h;

[0052] ④ Pour the shaken bacterial solution into 200ml LB liquid medium containing kanamycin, shake at 28°C, 220rpm for 8-12h (note: volume ratio: V medium:V bacterial solution=100:1);

[0053] b. Agrobacterium-infected sugar beet

[0054] ① For the beet seedlings that have grown cotyledons, cut off a cotyledon and terminal bud with a scalpel, and put the cut beet upside down in a beaker;

[0055...

Embodiment 3

[0066] Implementation 3: GM sugar beet detection

[0067] 1. Fluorescence detection of young leaves of transgenic sugar beet

[0068] Fluorescence detection was carried out on the newly grown fresh leaves obtained in Example 2. The fluorescence detection steps were as follows: take fresh leaf dorsal epidermis tissue and place it on a glass slide, and drop sterile water to press it for fluorescence detection.

[0069] 2. PCR detection of transgenic sugar beets

[0070] The PCR method was used to detect the fresh leaves, and the primers were designed according to the GFP gene sequence carried on the carrier. The theoretical amplification product size was 516 bp, and the primer sequences were as follows:

[0071] Primer 1 (upstream primer): 5'-ATCCCGCCGGGAATGGTGATTACCGA

[0072] Primer 2 (downstream primer): 5'-GTCGTGCACCATCAGCACGTTATCGA

[0073] The total DNA of fresh sugar beet leaves was extracted by SDS method, and used as a template, under the guidance of primer 1 and pri...

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Abstract

The invention discloses a method for establishing a transgenic system of a high-sugar variety beet. The method comprises the steps of: beet seed germination, beet seedling growth, agrobacterium infection, gene identification or resistance screening, etc. The method for transgenic beet provided by the invention provides a technical support for basic research depending on a transgenic technology related to improvement of the important alkaloid content of the beet and the ornamental quality and the economic value of the beet, and has important reference significance for establishment of transgenic methods of plants of beet genus.

Description

technical field [0001] The invention relates to a method for establishing a sugar beet transgenic system. Background technique [0002] Beet (Beta vulgaris L.) is a class of herbaceous plants with high economic and ornamental value. Beet belongs to Chenopodiaceae and is a biennial herbaceous plant. The aboveground part is basal leaves, the leaves are huge, and the roots are conical to spindle-shaped and juicy. The stem is erect, the basal leaves are oblong, long petiole, the top is shrunken and uneven, and the bottom has thick and protruding veins, entire or slightly wavy, the petiole is strong, the cauline leaves are alternate, small, clustered flowers, perianth lobes Bar-shaped or narrowly oblong, the upper part of the utricle is slightly fleshy. The seeds are lenticular, reddish-brown, bloom in May-June, and bear fruit in July. [0003] Native to the western and southern coasts of Europe and transplanted from Sweden to Spain, this species is widely cultivated and highl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82A01H5/00A01H6/02
CPCC12N15/8205C12N15/8243C12N15/8245
Inventor 宋志忠高雅超张少英张洪霞
Owner LUDONG UNIVERSITY
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