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Sugarcane callus protoplast separation and purification method

A technology of protoplast and callus, applied in the direction of plant cells, can solve the problems of insufficient quality and quantity, and achieve the effect of good uniformity, high quality and sufficient quantity

Inactive Publication Date: 2013-01-30
FUJIAN AGRI & FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to provide a kind of fast, efficient, easy separation and purification method of sugarcane callus protoplast, to overcome the existing quality and quantitative problems of sugarcane callus protoplast separation and purification method, for Sugarcane exogenous gene expression, promoter analysis, gene silencing and other transgene transient expression studies provide high-quality, high-yield sugarcane callus protoplasts

Method used

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  • Sugarcane callus protoplast separation and purification method
  • Sugarcane callus protoplast separation and purification method
  • Sugarcane callus protoplast separation and purification method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Embodiment 1: a kind of separation and purification method of sugarcane callus protoplast comprises the following steps:

[0025] 1. Sugarcane callus culture: select sugarcane tips with normal growth and no pests and diseases, remove the peripheral old leaves until the highest visible hypertrophic leaves and excess leaves growing from the top, and retain 25 cm long heart leaf tissue; use volume ratio After scrubbing and disinfecting the surface with 75% alcohol solution, the sugarcane heart leaf tissue was cut into 1.5 cm thick slices in a sterile ultra-clean workbench, cultured on MS3 solid medium at 28 ℃ for 1.5 months in the dark, every Subculture once every 2 weeks;

[0026] 2. Suspension cell culture of sugarcane: select 5 grams of dense, golden yellow, and granular embryogenic callus, mash it, transfer it into 100 mL MS3 liquid medium, and culture it in the dark at 28°C for 2 weeks. The culture solution was filtered with gauze, and the filtrate was allowed to sta...

Embodiment 2

[0030] Example 2: Using protoplasts isolated and purified from heart leaf callus of sugarcane variety CP72-1210, the activities of silencing suppressors encoded by different plant viruses were studied.

[0031] 1. PEG genetic transformation

[0032] 1.1 Put the prepared sugarcane CP72-1210 callus protoplasts in ice bath for 30 min, remove the W5 solution, and suspend the protoplasts with 1-2 mL MMG solution to a concentration of 2×10 per ml 5 indivual.

[0033] 1.2 Take 5 μL of pUbi-EYFP-Nos and pUbi-P19-Nos plasmid DNA (1 μg / μl), 5 μL of pUbi-EYFP-Nos and pUbi-γb-Nos plasmid DNA (1 μg / μl), 5 μL pUbi-EYFP-Nos and pUbi-Nos plasmid DNA (1 μg / μl) into 2 mL centrifuge tubes.

[0034] 1.3 Add 100 μL sugarcane CP72-1210 protoplasts, mix, add 110 μL 40% polyethylene glycol solution (PEG-4000), mix well, and incubate at room temperature for 5-15 min.

[0035] 1.4 Add 440 uL W5 solution, mix well, stop genetic transformation, and centrifuge at 100 g for 2 min. The supernatant was d...

example 3

[0040] Example 3: Using the protoplasts isolated and purified from the heart leaf callus of sugarcane variety CP72-1210, the activities of different promoters were studied.

[0041] 1. Transient expression of transgene

[0042] 1.1 Put the prepared sugarcane CP72-1210 callus protoplasts in ice bath for 30 min, remove the W5 solution, and suspend the protoplasts with 1-2 mL MMG solution to a concentration of 2×10 per ml 5 indivual.

[0043] 1.2 Take 10 μL of plasmid DNA (pPr4-GUS-NOS, pUbi-GUS-NOS, p35S-GUS-NOS, 10 μg) into 2 mL centrifuge tubes, and set empty vector (pUbi-NOS) and water as blank control.

[0044] 1.3 Add 100 μL of protoplasts, after mixing, add 110 μL of 40% polyethylene glycol conversion solution, mix well, and incubate at room temperature for 5-15 min.

[0045] 1.4 Add 440 μL of W5 solution and mix gently to terminate the genetic transformation, and centrifuge at 100 g for 2 min. The supernatant was discarded, and the protoplasts were suspended in 1 mL WI...

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Abstract

The invention relates to a sugarcane callus protoplast separation and purification method, which comprises sugarcane callus culture, sugarcane suspended cell culture, sugarcane protoplast separation, and sugarcane protoplast purification. By the method, suspended cell lines can be obtained by sugarcane embryogenic callus culture and an adequate amount of complete protoplasts with high quality and uniformity can be obtained by enzyme digestion, separation and purification; therefore, ideal experimental materials can be provided for study transgenetic transient expression such as sugarcane exogenous gene expression, promoter analysis and gene silencing.

Description

technical field [0001] The invention relates to a method for separating and purifying plant protoplasts, in particular to a method for separating and purifying protoplasts from sugarcane callus, and belongs to the field of biotechnology. Background technique [0002] Plant protoplasts overcome the barrier of the cell wall, and are ideal materials for theoretical research on plant physiology, cell biology, and somatic cell genetics, and have important application prospects in genetic engineering and the cultivation of new varieties of crop improvement. In recent years, with the development of plant molecular biology, protoplasts, as a single-cell system, have been widely used in the research of gene localization, gene expression regulation, RNA silencing mechanism, cellular calcium signal transduction and its regulation mechanism, etc. [0003] The research on sugarcane callus protoplasts began in the 1970s, and the isolation, cultivation and plant regeneration of protopl...

Claims

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Application Information

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IPC IPC(8): C12N5/04
Inventor 高三基米尔科夫艾瑞克朴钟元陈如凯傅华英付为林林艺华吴小斌
Owner FUJIAN AGRI & FORESTRY UNIV
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