Method for introducing substance into plant

A technology for introducing plants and substances, applied in botany equipment and methods, biochemical equipment and methods, plant cells, etc., can solve problems such as objects that are not suitable for gene introduction, and achieve good reproducible results

Inactive Publication Date: 2019-09-13
KANEKA CORP +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Based on this situation, it can be speculated that egg cells, sperm cells, and fertilized egg cells are not suitable as targets for gene introduction, and there have been no reports of gene introduction and cell division using methods such as the PEG method.

Method used

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  • Method for introducing substance into plant
  • Method for introducing substance into plant
  • Method for introducing substance into plant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0172] The separation of the egg cell of embodiment 1 rice and sperm cell

[0173] In the present example, the separation of egg cells and sperm cells from rice flowers ( figure 1 ).

[0174] Isolation of oocytes was performed as described below. The unflowered flowers obtained by the ears of rice were dissected, and the ovary was collected, and the ovary was put into 3 mL of 6% mannitol solution (370 mosmol / kg H 2 O) in a 3.5cm plastic petri dish. Sink the ovary with the stigma removed into 3 mL of 6% mannitol solution (370 mosmol / kg H 2 O), the lower part of the ovary was cut off using a laser blade (FEATHER Safety Razor company, FA-10) at the bottom of the petri dish. The oocytes protruding from the incision were confirmed by microscopic observation, and the oocytes were separated with a microcapillary. About 10-15 oocytes were obtained from about 30-40 ovaries. The diameter of each egg cell is 40 to 50 μm. Use a microcapillary to move isolated oocytes into droplets ...

Embodiment 2

[0180] Example 2 Production of rice fertilized egg cells by gamete fusion

[0181] In this example, rice fertilized egg cells (rice fertilized egg cells in vitro) were produced in vitro by gamete fusion based on electrofusion ( figure 2 ).

[0182] Sperm cells and egg cells separated by the method shown in Example 1 were moved one by one into droplets on a cover glass. Then, these cells were arrayed on an electrode (CUY5100-100Ti from NEPAGENE) under alternating current (1 MHz, 0.4 kV / cm, ECFG21 from NEPAGENE). Use a microcapillary to add half to the same amount of mannitol solution (520 mosmol / kg H 2 O). Then, by applying a DC pulse (50 μs, 12 to 15 kV / cm, distance between electrodes 50 to 150 μm), the gametes were fused to produce fertilized egg cells.

Embodiment 3

[0183] Example 3 Introduction of nucleic acid into rice fertilized egg cells

[0184] In this example, the nucleic acid was introduced into the rice in vitro fertilized egg cells produced in Example 2.

[0185] According to this example, the fertilized egg cells produced in Example 2 were treated so that the introduction of the substance was completed at the time of 120 minutes from the gamete fusion treatment. Move the resulting fertilized egg cells to MMG solution (15mM MgCl 2 , 4mM MES (pH5.7), 450mosmol / kg H 2 O mannitol) in droplets (about 2 μL), and then moved to the MMG containing the base sequence to be introduced, 35S promoter::signal sequence::GFP::endoplasmic reticulum retention signal (HDEL): : In the droplet of the plasmid (plasmid DNA for GFP expression, about 6000 bp) of the NOS terminator. It should be noted that this plasmid was prepared according to the description in Non-Patent Document 17. Next, the droplets containing fertilized egg cells were mixed wi...

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Abstract

The present invention relates to a method for introducing a substance into a plant. The method involves introducing a substance into plant gametes that have incomplete cell walls.

Description

technical field [0001] The present invention relates to methods for introducing substances into plants. Background technique [0002] For the gene recombination technology of plants, especially monocotyledonous plants, the application of the method using Agrobacterium in rice and corn was developed in the 1990s, and its application has spread rapidly. Various transformation methods have been developed so far. However, it is known that most of them require dedifferentiation and redifferentiation of plant tissues, so the transformation efficiency varies greatly among species and varieties. Depending on the species and variety, the transformation efficiency is low, and reproducible transformed plants cannot be obtained. For example, for the line B73, which is very important in maize breeding, a general transformation method that can efficiently obtain transformed plants has not yet been developed. [0003] In addition, in recent years, it has gradually become possible to per...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01H1/00C12N5/04
CPCA01H3/04C12N15/8206C12N15/8213C12N15/8201C12N15/82
Inventor 加藤纪夫市川雅子冈本龙史古矶成美木羽隆敏户田绘梨香
Owner KANEKA CORP
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