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Method for betula luminifera xylem protoplast preparation and instantaneous conversion

A protoplast and instantaneous transformation technology, applied in the field of plant biology, can solve the problems of unreported protoplasts, short childhood, etc., and achieve the effect of easy separation, strong vitality and strong operability

Active Publication Date: 2016-11-30
ZHEJIANG FORESTRY UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Betula glabra grows fast, and most germplasms have a short childhood period (about 18 months). It is an excellent material for forest genetic research, but there is no report on protoplasts

Method used

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  • Method for betula luminifera xylem protoplast preparation and instantaneous conversion
  • Method for betula luminifera xylem protoplast preparation and instantaneous conversion
  • Method for betula luminifera xylem protoplast preparation and instantaneous conversion

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Embodiment 1

[0027] 1) Preparation of enzymatic hydrolysis solution and enzymatic separation conditions

[0028] Base solution: 0.4M mannitol, 20mM KCl, 20mM MES-KOH (pH 5.7), 10mM CaCl 2 , 0.1% bovine serum albumin (BSA, W / V). When preparing the enzyme solution, add mannitol, KCl, MES-KOH and enzyme first, and after the enzyme is completely dissolved, put it in a water bath at 55°C for 10 minutes, then add CaCl after cooling to room temperature 2 and BSA.

[0029] Enzyme solution for xylem cell protoplast separation: use basic solution to prepare.

[0030] 2) Material pretreatment

[0031] Select an annual plant of Betula glabra in good growth condition, take the stem section (thickness is about 0.5-0.8cm, length is 8cm), wash it with running water for 10min, put on disposable gloves, and peel off the bark .

[0032] 3) Isolation of xylem protoplasts

[0033] Place the pretreated stems of Betula glabra in a 50mL centrifuge tube filled with 20mL of enzymatic hydrolysis solution, wrap...

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Abstract

The invention provides a method for betula luminifera xylem protoplast preparation and instantaneous conversion. The method comprises a step 1) of selecting a betula luminifera annual plant which is good in growth conditions, taking a stem grown in the current year, using running water to flush the stem for 5-20 min, and removing bark; a step 2) of using enzymatic hydrolysate to perform enzymolysis on the betula luminifera stem xylem obtained through the step 1) in the dark; a step 3) of transferring the stem after the enzymolysis to MMG solution, dispersing protoplast in the MMG solution, using a cell sieve with a pore diameter of 70 [mu]m to perform filtration, collecting filtrate, centrifuging the filtrate, using the MMG solution to re-suspend the collected protoplast, and obtaining purified protoplast; a step 4) of causing the instantaneous conversion efficiency of the xylem protoplast to be highest when the plasmid concentration is 1500 ng / microliter and the PEG processing time is 10 min. The average density of the protoplast obtained according to the method is 4.5*10<6> per mL<1>, the average yield is 1.2*10<6> per g<1>.FW<1>, the activity is 96% at most, and the highest instantaneous conversion efficiency is averagely about 55%.

Description

technical field [0001] The invention belongs to the technical field of plant biology, and in particular relates to a preparation method of protoplasts of betula glabra. Background technique [0002] The earliest research on plant protoplasts can be traced back to 1892, when Klercker et al. successfully isolated protoplasts from an algal plant. Kuster and Miche first discovered the phenomenon of cell fusion, and the cell membrane and cytoplasm of two cells can fuse under certain conditions. In the early 1960s, Cocking enabled the successful preparation and isolation of protoplasts, and a large number of viable tomato root tip protoplasts were isolated by enzymatic hydrolysis. Protoplasts have no cell walls and are totipotent. They are ideal materials for research on cell wall regeneration, cell division, intake of foreign DNA, membrane permeability, and signal transduction. In recent years, protoplasts have been used to study light signals, stress, etc. And hormone action a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/04C12N15/82
CPCC12N5/04C12N15/8201
Inventor 董玉田林二培楼雄珍牛明月黄华宏童再康
Owner ZHEJIANG FORESTRY UNIVERSITY
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