Instantaneous conversion method for duckweed

An instantaneous transformation, duckweed technology, applied in biochemical equipment and methods, botanical equipment and methods, angiosperms/flowering plants, etc., can solve the problem of lack of duckweed instantaneous transformation system, etc. short time effect

Active Publication Date: 2021-10-01
TIANJIN NORMAL UNIVERSITY
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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 duckweed transient transformation method for the lack of duckweed transient transformation system in the prior art

Method used

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  • Instantaneous conversion method for duckweed
  • Instantaneous conversion method for duckweed
  • Instantaneous conversion method for duckweed

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] A kind of duckweed instantaneous transformation method, comprises the following steps:

[0035] Step 1, collection of duckweed: duckweed is collected from lake water in Xiqing District, Tianjin City, and it belongs to Lemnaturionifera 5511 of the Duckweed family.

[0036] Step 2, the cultivation of duckweed: culture duckweed on liquid medium, the culture condition is 23 ℃ temperature, photoperiod 16 hours / day, light intensity 95 μ mol m -2 the s -1 , subculture once a week.

[0037] Mass elements include:

[0038]

[0039] Trace elements include:

[0040]

[0041]

[0042] The above reagents were adjusted to pH 5.6-5.8 and used after autoclaving at 102.9kPa, 121°C for 20 minutes.

[0043] Step 3, activation and extraction of bacterial cells: activate Agrobacterium with a fluorescently labeled target gene (GCaMP in this example) from a glycerol cryopreservation tube for 3 times. First pick a single colony and activate the bacteria in a small volume for 30h ...

Embodiment 2

[0054] The wild-type duckweed was compared with the transiently infected duckweed obtained in Example 1.

[0055] Observing the transiently infected duckweed obtained in Example 1 and the wild-type duckweed collected under a fluorescent microscope, it can be observed that the green fluorescent signal of the duckweed root with the green fluorescent marker transient transgene is as follows: Figure 2-4 As shown, the collected wild-type duckweed had no obvious green fluorescence as figure 1 shown.

[0056] The method of transiently infecting duckweed with Agrobacterium in this invention has the characteristics of easy operation, short time (about 25 hours from treatment to cultivation), and low cost, and can be applied to duckweed molecular biology, bioreactor construction and fluorescent labeling It is an indispensable key operation in the analysis of duckweed cells and molecular biology in related theoretical and applied research.

Embodiment 3

[0058] Repeat the experiment for the second time according to the method and steps of Example 1, and after the transient infection of duckweed is obtained, the green fluorescent signal of the transformed duckweed root with the green fluorescent marker transient gene is observed as follows: Figure 5 As shown, the collected wild-type duckweed had no obvious green fluorescence as Image 6 shown.

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Abstract

The invention provides an instantaneous conversion method for duckweed. The instantaneous conversion method for duckweed comprises the following steps: collecting duckweed; carrying out subculture on the collected duckweed on a liquid culture medium; conducting activating and extracting agrobacterium tumefaciens with a fluorescence-labeled target gene; preparing a bacterium dissolving solution containing Silweet-775 and cane sugar; adding agrobacterium with the fluorescence-labeled target gene into the bacterium dissolving solution to completely dissolve thalli in the bacterium dissolving solution, and cracking the agrobacterium to release plasmids; selecting a complete duckweed, pricking a small hole in the back surface of a duckweed leaf by using a needle head, putting the duckweed into the bacterium dissolving solution for soaking and infecting, putting the infected duckweed into a duckweed liquid culture medium, and carrying out dark culture to obtain instantly infected duckweed. The method for instantly infecting the duckweed with the agrobacterium mainly adopts a needle punching method, has the characteristics of easiness in operation, short time, low cost and the like, and can be applied to related theories and application researches such as molecular biology of duckweed, bioreactor construction, fluorescence labeling and the like.

Description

technical field [0001] The invention relates to the technical field of plant transient transformation, in particular to a duckweed transient transformation method. Background technique [0002] Duckweed is a monocotyledon water surface floating plant, which belongs to the Duckweed family and is widely distributed all over the world. It has the ability to enrich heavy metal cadmium ions. However, the physiological and molecular mechanisms of duckweed enriching heavy metals have not yet been elucidated. By preparing duckweed transient transformation system and using technologies such as upright microscope observation, it will be of great value and significance for the ecological restoration of heavy metal-polluted water environment and the application of plant resistance to heavy metals. [0003] The conventional transformation method of duckweed is mainly Agrobacterium transformation method, and the infection time takes 1-2 months, which is a long time, and it is difficult ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/65A01H5/00
CPCC12N15/8205C12N15/65
Inventor 杨琳马旭王珅孙津歌
Owner TIANJIN NORMAL UNIVERSITY
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