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A method and application for inducing direct generation of somatic embryos

A technology of somatic embryos and cell embryos, which is applied in the field of direct induction of somatic embryos to achieve the effects of simple operation, short culture period and high occurrence frequency

Active Publication Date: 2019-11-08
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The initiation of somatic embryos is different from this. Plant somatic embryogenesis (somatic embryogenesis) means that plant somatic cells develop into a new individual by simulating sexual zygotic embryogenesis without fusion of sex cells. It does not need to go through the process of double fertilization and can be directly developed from somatic cells, but this process may require specific culture conditions, hormonal environment, or molecular regulation and other factors as initial signals

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  • A method and application for inducing direct generation of somatic embryos
  • A method and application for inducing direct generation of somatic embryos
  • A method and application for inducing direct generation of somatic embryos

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0098] Embodiment 1: RID3 and YUC10 gene cloning and plant expression vector construction

[0099] 1.1 Extraction of total RNA from Arabidopsis tissue

[0100] Total RNA was extracted from plant materials using the Trizol kit, and the steps were as follows:

[0101] (1) Add 1 mL of Trizol reagent to the centrifuge tube;

[0102] (2) Removal of cell wall residues, proteins, polysaccharides, and fats: Grind about 0.1 g of plant material in liquid nitrogen, transfer the powder to a centrifuge tube, mix with a shaker, and let stand at room temperature for 5 minutes, then 12,000 rpm, 4 Centrifuge for 10 minutes;

[0103] (3) Phase separation: transfer the supernatant to a new centrifuge tube, add 200 μL of chloroform, shake vigorously for 15 seconds, then let stand at room temperature for 2-3 minutes, centrifuge at 12000 rpm, 4 °C for 15 minutes;

[0104] (4) Precipitation and removal of polysaccharides: Transfer the colorless aqueous phase (about 600 μL) into a new centrifuge t...

Embodiment 2

[0128] Embodiment 2: the cloning of SERK1, RID3 and YUC10 promoter and the construction of plant expression vector

[0129] 2.1 Extraction of plant genomic DNA

[0130] The genomic DNA of Arabidopsis thaliana was extracted by the small amount method. The extraction method is as follows:

[0131] (1) Take fresh plant leaves and grind them into powder in liquid nitrogen;

[0132] (2) Add 500 μL of DNA extraction solution, shake and mix well, and place in a water bath at 60°C for 2 minutes;

[0133] (3) After taking it out, add 500 μL of phenol to the centrifuge tube, shake and mix well, centrifuge at 12000 rpm for 5 min;

[0134] (4) Take the supernatant, transfer it to a new 1.5mL centrifuge tube, add 500μL phenol / chloroform (1:1), shake and mix, and centrifuge at 12000rpm for 5min;

[0135] (5) Take the supernatant, transfer it to a new centrifuge tube, add 500 μL of chloroform, shake and mix, and centrifuge at 12000 rpm for 5 minutes;

[0136] (6) Transfer the supernatan...

Embodiment 3

[0169] Example 3: Agrobacterium-mediated transformation of Arabidopsis inflorescences and acquisition of resistant plants

[0170] The transformation steps of Arabidopsis thaliana are as follows:

[0171] (1) The preparation of Agrobacterium should be carried out one day before the transformation, and the Agrobacterium should be activated with 100mL of culture solution added with corresponding antibiotics. Arabidopsis can be transformed the next morning;

[0172](2) Configure liquid: 5% sucrose dissolved in ddH 2 O, add 0.03-0.05% Silwet L-77;

[0173] (3) Centrifuge at 5000-6000rpm for 5-8min, discard the supernatant, and collect the bacteria;

[0174] (4) Suspend the bacteria with the infection liquid;

[0175] (5) Soak the inflorescences of Arabidopsis thaliana in the infection solution and shake gently for about 30 seconds;

[0176] (6) Then Arabidopsis is placed horizontally in a cardboard box, covered with a film, and placed in the dark for one day;

[0177] (7) Pu...

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Abstract

The invention discloses a method and application for inducing direct somatic embryogenesis. According to the method and application, function genes RID3(AT3G49180) and YUC10(AT1G48910) capable of promoting direct somatic embryogenesis are found. Through ectopic expression of the function genes, inducing can be directly conducted on arabidopsis cotyledons to form the somatic embryo without any callus. The method for inducing direct somatic embryogenesis has the advantages of being simple in culture, short in inducing period, high in inducing frequency and the like, and can be used for study onaspects of differentiation, development and totipotency expression of botanical cells, crop variety improvement, production of artificial seeds, storage of high-quality germplasm resources, mutant screening and the like.

Description

technical field [0001] The invention relates to the technical field of plant genetic engineering, in particular to a method and application for inducing direct generation of somatic embryos. Background technique [0002] Angiosperms start from double fertilization to form zygotes to embryonic development and mature into seeds. This development process has successively experienced zygote activation, cell division and differentiation, establishment of polarity, pattern formation and organ formation, etc., and the development of embryos is controlled by Precise genetic regulation. Egg cells before fertilization are relatively static in metabolism, and can only be activated after fusion with sperm cells to start the process of embryonic development (Jiang Li et al., 2007). The initiation of somatic embryos is different. Plant somatic embryogenesis (somatic embryogenesis) means that plant somatic cells develop into a new individual by simulating sexual zygotic embryogenesis with...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C12N15/53C07K14/415C12N9/02C12N15/82A01H5/00A01H6/20A01H4/00
CPCA01H4/006C07K14/415C12N9/0073C12N15/8241C12Y114/13168
Inventor 张宪省唐丽萍苏英华翟立明张文杰殷佩佩
Owner SHANDONG AGRICULTURAL UNIVERSITY
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