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Enzymolysis method for rapidly removing cell walls of Populus alba L or Eucalyptus robusta Smith, and application

A cell wall and enzymatic hydrolysis technology, applied in the field of plant genetic engineering, can solve the problem of no report

Pending Publication Date: 2022-01-07
BEIJING FORESTRY UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The enzymatic hydrolysis method invented in the later period has created a new technology for isolating protoplasts, but how to quickly remove the cell walls of poplar and eucalyptus to obtain protoplasts of poplar and eucalyptus with a completeness of 90% and above has not been reported so far

Method used

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  • Enzymolysis method for rapidly removing cell walls of Populus alba L or Eucalyptus robusta Smith, and application
  • Enzymolysis method for rapidly removing cell walls of Populus alba L or Eucalyptus robusta Smith, and application
  • Enzymolysis method for rapidly removing cell walls of Populus alba L or Eucalyptus robusta Smith, and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment one: the cultivation of poplar, eucalyptus tissue culture seedling aseptic seedling

[0038] The Populus tomentosa tissue culture seedlings are asexually propagated through the following medium respectively:

[0039] The medium formula used is as follows:

[0040] Callus induction medium: MS+KT 0.5mg / L+0.1mg / L 2,4-D+6.5g / L agar

[0041] Differentiation budding medium: MS+TDZ 0.05mg / L+6-BA 0.5mg / L+6.5g / L agar

[0042] Shoot elongation medium: MS+6-BA0.5mg / L+6.5g / L agar

[0043] Rooting medium: 1 / 2MS+NAA0.5mg / L+6g / L agar

[0044] (1) Exocarp disinfection: first the stem section of the Populus tomentosa plant grown in the wild is cut off, gently scrubbed with a hairbrush, rinsed with running water for 12 hours, dried, and the branch is built into a 3cm long stem section, which is then cleaned in ultra-clean work Soak the table with 75% alcohol for 30 seconds, use sterile water 3 times for 1 minute each time, then disinfect with 2% sodium hypochlorite (NaClO)...

Embodiment 2

[0054] Embodiment two: plasmolysis pretreatment

[0055] (8) Configure 0.8M mannitol solution: prepare a disposable petri dish with a diameter of 9cm, weigh 2.912g mannitol and dissolve it in 20ml ddH 2 O, fully dissolve mannitol and mix well.

[0056] (9) Cut the blades of poplar and eucalyptus into thin strips with a width of about 1 mm with scissors or blades, soak in the mannitol solution prepared in the previous step, and ensure that the blade thin strips are all immersed in the solution.

[0057] (10) Stand at room temperature for 20 minutes.

Embodiment 3

[0058] Example 3: Enzymatic hydrolysis to remove cell walls

[0059] Use a 10ml centrifuge tube to configure 10ml of the enzymatic hydrolysis solution with the following formula:

[0060] 0.15g Cellulase R-10

[0061] 0.04g isolated enzyme R-10

[0062] 0.05g pectinase Y-23

[0063] 1.092g mannitol

[0064] 1ml 200mM KCl

[0065] 1ml 200mM MES

[0066] Add 5ml of ddH 2 O, incubate at 55°C for 10min, after cooling to room temperature, add

[0067] 100 μl 1M CaCl 2

[0068] 66.67 μl 7.5 mM β-mercaptoethanol

[0069] 1ml 1%BSA

[0070] Dilute to 10ml for use.

[0071] (11) Take out the thin strips of poplar and eucalyptus leaves treated with mannitol, drain them, put them into the enzymatic solution (try not to bring out the mannitol solution), and soak them loosely and evenly in a 10ml centrifuge tube containing 10ml of enzymatic solution middle.

[0072] (12) Under dark conditions, place on a horizontal shaker at 23° C., 10 rpm, and perform enzymatic hydrolysis for...

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Abstract

The invention relates to an enzymolysis method for quickly removing the cell walls of Populus alba L or Eucalyptus robusta Smith. The enzymolysis method comprises the following steps of utilizing a specifically prepared enzymolysis solution, putting Populus alba L or Eucalyptus robusta Smith leaves subjected to plasmolysis pretreatment into the specifically prepared enzymolysis solution, and treating at the room temperature so as to obtain Populus alba L or Eucalyptus robusta Smith protoplast of which the integrity is 90%. According to the enzymolysis method, the cell walls of the Populus alba L or Eucalyptus robusta Smith are rapidly removed, and the enzymolysis method has a great application value for cultivating new excellent varieties of Populus alba L or Eucalyptus robusta Smith through the field of genetic engineering.

Description

technical field [0001] The invention relates to the field of plant genetic engineering, in particular to an enzymatic hydrolysis method for rapidly removing cell walls of poplar and eucalyptus. [0002] technical background [0003] Poplar is the main plantation tree species in the Yellow River Basin in my country. Because of its rapid growth, drought resistance, barren tolerance, and beautiful tree shape, it is widely planted in northern my country. It is an important tree species for windbreak and sand fixation, and for returning farmland to forests. Poplar also plays an important role in forestry industries such as wood processing and papermaking. At present, the total area of ​​poplar plantations in my country is 8.5 million hectares, which has exceeded 100 million mu (2018 data), ranking first in the world. However, the traditional poplar seedling breeding has the disadvantages of long breeding cycle and difficulty in diversifying and integrating excellent traits. At the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/04C12N1/06C12N15/82
CPCC12N5/04C12N1/06C12N15/8214
Inventor 夏新莉王厚领刘晓牛梦雪于晓倩张涵
Owner BEIJING FORESTRY UNIVERSITY
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