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Transformation system for Spirodelapolyrrhiza callus induction and capable of realizing transgene stable inheritance

A transgenic and stable technology, which is applied in the field of Ziping callus to obtain stable genetic regenerated plants, can solve the problems of poor growth and achieve high-efficiency expression

Inactive Publication Date: 2014-08-20
GUANGDONG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, so far there is no report on callus induction and transformation system of Ziping (Spirodela polyrrhiza)
The main reason is that Ziping is very sensitive to auxin, as the result obtained by Stomp et al. "Ziping's thallus does not survive on higher concentration of 2,4-D, but does not grow well at lower concentration"

Method used

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  • Transformation system for Spirodelapolyrrhiza callus induction and capable of realizing transgene stable inheritance
  • Transformation system for Spirodelapolyrrhiza callus induction and capable of realizing transgene stable inheritance
  • Transformation system for Spirodelapolyrrhiza callus induction and capable of realizing transgene stable inheritance

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Screening of different phytohormone combinations and concentrations:

[0058] Each phytohormone combination contains an auxin and a plant cytokinin.

[0059] Plant auxins include: 2,4-D, 2,4-Dichlorophenoxyacetic Acid (2,4-D, 2,4-Dichlorophenoxyacetic Acid), 1-Naphthylacetic Acid (NAA, 1-Naphthylacetic Acid), Dicamba (DCB, Dicamba) and p-Chlorophenoxy acetic acid (PCA, p-Chlorophenoxy acetic acid ).

[0060] Plant cytokinins include: thiadizuron (TDZ, thidiazuron), 6-benzylaminopurine (6-BA, 6-Benzylaminopurine), N 6 -Isopentenyl adenine (2iP, N 6 -(2-Isopentenyl) adenine) and kinetin (KT, kineting).

[0061] The phytohormone combinations and their concentrations are shown in the table below. Eight concentrations of each phytohormone were tested. such as D 1 T 8 represents the combination of 2,4-D (0.1 mg / L) and TDZ (1.6 mg / L), M 5 I 4 Represents the combination of DCB (2 mg / L) and 2iP (0.1 mg / L), and so on.

[0062] The front (adaxial face, adaxial face) of ...

Embodiment 2

[0067] Effects of different media on callus induction of Ziping:

[0068] In order to test whether different media can affect the callus induction of Ziping, the following six media were tested: MS (Murashige and Skoog), N6 (Zhu Zhiqing), B5 (Gamborg B5), WP (McCown Woody Plant), SH (Schenk and Hildebrandt), NN (Nitsch JP and Nitsch C) media. Prepare 50 ml of each culture medium (containing 3% sucrose, 0.7% Agar, D 4 T 6 , pH 5.8), sterilized at 121°C for 20 minutes, and evenly divided into two glass Petri dishes with a diameter of 9 cm. Put 6 clumps of rooted Zipain fronds on each petri dish, the front side (adaxial face, adaxial face) of Zihuai fronds is in contact with the callus induction medium, and the back side (off-axis face, abaxial face) is upward, and the pseudo The roots were suspended in the air and cultured in the dark at 25°C for 2 weeks. The 6 culture media can induce callus to varying degrees, and the callus induction rate ranges from 30% to 100%. Relativ...

Embodiment 3

[0070] Effects of different light conditions on callus induction of Ziping

[0071] In order to test whether different light conditions have an effect on the callus induction of Ziping, three light conditions were tested: total darkness,

[0072] Semi-dark, illuminated.

[0073] Total darkness: put the petri dish in an airtight carton and put it in an illumination of 2000 Lux (about 25 μmolm -2 the s -1 ) plant lighting incubator, the photoperiod is 16h light / 8h dark.

[0074] Semi-darkness: Place the petri dish in a carton with an open top, partially cover the top of the carton with white thin paper so that the illuminance inside the carton is around 400 Lux (about 5 μmolm -2 the s -1 ). The light intensity of the plant light incubator is 2000 Lux, and the light cycle is 16h light / 8h dark.

[0075] Illumination: Illumination is 2000 Lux, and the photoperiod is 16h light / 8h dark.

[0076] Prepare 300 ml of MS medium (containing 3% sucrose, 0.7% Agar, D 4 T 6 , pH 5.8)...

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Abstract

The invention relates to a transformation system for Spirodelapolyrrhiza callus induction and capable of realizing transgene stable inheritance, and belongs to the field of biotechnology. The method is as below: first, inducing the formation of Spirodelapolyrrhiza callus through different plant culture medium and plant hormone combination by using the high dedifferentiation ability of new rhizoid meristematic zone or a binding site of new rhizoid and thallus meristematic zone; and then using Agrobacterium or particle bombardment method to realize integration of exogenous DNA fragments into Spirodelapolyrrhiza genome and stable inheritance of the transformed exogenous DNA fragments in Spirodelapolyrrhiza. The system of the invention can integrate any exogenous DNA fragment to Spirodelapolyrrhiza genome. The technology requires short time, has high efficiency, can induce callus in 2 weeks, and reaches an induction rate of 100%; and transgenic plants can be obtained in the shortest time of about 3 months.

Description

technical field [0001] The invention relates to a method for inducing callus of Zipingia spirodela polyrrhiza, in particular to a method for obtaining regenerated plants capable of stable heredity by using Zipingia spirodela polyrrhiza callus through different combinations of culture media and plant hormones, and belongs to the field of biotechnology. Background technique [0002] Duckweed belongs to Magnoliophyta - Liliopsida - Arecidae - Alismatales - Araceae - Lemnaceae. Early taxonomic systems classified Duckweedae as a family under the order Araceae, while more recent classification systems incorporated Duckweedaceae into Araceae and classified Araceae as a family under the order Alisinales. Duckweed subfamily has 5 genera and 38 species. The five genera are: Spirodela (Zipia), containing 2 species; Landoltia (Banza), containing 1 species; Lemna (Qingping), containing 14 species; Wolffia (Genus), Contains 11 species; Wolffiella (Platina genus), contains 10 species. I...

Claims

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Application Information

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IPC IPC(8): C12N15/84C12N15/90A01H5/00A01H4/00
Inventor 黄明星
Owner GUANGDONG UNIV OF TECH
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