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Breeding method using protocorm based on germinated phalaenopsis seeds as receptor

A technology of protocorm and Phalaenopsis, which is applied in the field of breeding with protocorm as receptor, can solve the problems of low antibiotic sensitivity, low induction rate, low transfection rate, etc., and achieves good breeding effect, mature technical means and wide source. Effect

Active Publication Date: 2015-07-15
HENAN ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For the transgenic breeding of Phalaenopsis, theoretically, somatic embryos, protocorms and protocorms can be used as ideal recipient materials for genetic transformation of Phalaenopsis, but the current way to obtain somatic embryos and protocorms is mainly through the shoot tip , stems, leaves, root tips, root segments, pedicel axillary buds and other explants, but in the induction process, there are often problems such as browning and low induction rate. Therefore, in the practice of Phalaenopsis transgenic breeding, somatic cells Embryos or protocorm-like receptor materials often have certain limitations
In addition, when somatic embryos or protocorms are used as recipient materials for transgenic breeding, when gene guns or Agrobacterium dipping are used for transgenic operations, there are also defects of low transfection efficiency, and in the process of Agrobacterium dipping, There are also disadvantages such as low sensitivity to antibiotics, so further improvement is urgently needed

Method used

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  • Breeding method using protocorm based on germinated phalaenopsis seeds as receptor
  • Breeding method using protocorm based on germinated phalaenopsis seeds as receptor
  • Breeding method using protocorm based on germinated phalaenopsis seeds as receptor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Due to the construction of recombinant Agrobacterium strain GV3101, the plasmid expression vector pCAMBIA1301 -RcOOMT2 , pCAMBIA1303 -VwF3'5'H Recombination into the original Agrobacterium strain GV3101 is a relatively important link in the present invention, so the acquisition of the recombinant Agrobacterium strain GV3101 is introduced as follows.

[0072] Such as figure 1 , figure 2 As shown, the process of obtaining the recombinant Agrobacterium strain GV3101 is as follows: first, transform the pCAMBIA1301 and pCAMBIA1303 vectors, respectively add the CAMV35S promoter and the CAMV35S terminator to the multiple cloning sites of the vectors; Rose Fragrance Gene RcOOMT2 , pansy blue petal key genes VwF3'5'H ; Finally, the transformed plasmid expression vector pCAMBIA1301 -RcOOMT2 , pCAMBIA1303 -VwF3'5'H Transform to Agrobacterium strain GV3101. The detailed process is described below.

[0073] (1) Transformation of pCAMBIA1301 and pCAMBIA1303 vectors—...

Embodiment 2

[0143] This example focuses on the construction of genetic material based on protocorms germinated from Phalaenopsis seeds as receptors before transgenic breeding in Agrobacterium dipping, and a brief introduction is as follows.

[0144] (1) Under sterile conditions, induce the germination and growth of Phalaenopsis seeds in the medium to obtain protocorms;

[0145] Specifically: Cut the mature Phalaenopsis fruit from the mother plant, wash it with soapy water, and rinse it under clean water for 30 minutes; put the rinsed fruit into a large sterile beaker on an ultra-clean workbench, add 70% Sterilize with ethanol for 15 minutes, and then sterilize with 10% sodium hypochlorite for 15 minutes. During this period, keep shaking the beaker to make it fully contact; after the disinfection is completed, rinse it with sterile water for 3 times; Cut the orchid fruit, take out the pulp with seeds with sterile tweezers, wash it in a petri dish filled with a small amount of sterile wat...

Embodiment 3

[0238] In this example, the Phalaenopsis protocorm cultivated in Example 2 was dipped by the dipping method of Agrobacterium to realize transgenic operation. The dipping solution was the recombinant Agrobacterium strain GV3101 prepared in Example 1, which contained a plasmid expression vector pCAMBIA1301 -RcOOMT2 , pCAMBIA1303 -VwF3'5'H , the bacterial selection marker gene of the plasmid expression vector is Kan, the plant selection marker gene is Hyg, and the reporter gene is GUS. A brief introduction is as follows.

[0239] The transgenic operation of Agrobacterium infection includes the following steps.

[0240] (1) Pre-cultivation of receptor material

[0241] The excellent protocorm obtained in Example 2 with a size of about 0.5 cm and consistent growth was cut into pieces, transferred to the pre-cultivation medium, and pre-cultivated for 3 days to keep the recipient material vigorous vitality. The state is transformed, and the damage caused by the impregnating...

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Abstract

The invention belongs to the technical field of genetic engineering breeding, and in particular relates to a breeding method using protocorm based on germinated phalaenopsis seeds as a receptor. The method comprises the following steps of obtaining the protocorm, culturing in multiplication, pre-culturing, dip-dyeing agrobacterium, co-culturing, eluting the agrobacterium, screening, culturing, forming shoots and seedlings, and rooting. A set of phalaenopsis transgenosis receptor system is constructed by using the protocorm from the germinated phalaenopsis seeds as a receptor material, and meanwhile, a set of genetic transformation system based on agrobacterium dip-dyeing and a corresponding detection system are established, the receptor material can be fast obtained, the performance is stable, a source is wide, and the breeding method is good to selective antibiotic sensitivity, and the problem that the phalaenopsis is insensitive to antibiotic in the induction type protocorm process, a resistance transgenosis material can be conveniently screened, a good and pure new phalaenopsis system can be obtained, and the breeding method has the good popularization applicable value on prompting the new variety development of the phalaenopsis.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering breeding, in particular to a breeding method based on protocorms germinated from Phalaenopsis seeds as receptors. Background technique [0002] Phalaenopsis( Phalaenopsis aphrodite ) belongs to the genus Phalaenopsis of the family Orchidaceae, and is an important potted flower with high ornamental value and economic value. However, due to the lack of blue or scented varieties of Phalaenopsis in nature, traditional hybrid breeding methods cannot realize Diversified demand for Phalaenopsis flower color and fragrance. Therefore, the new transgenic breeding technology has been applied to a certain extent in the cultivation of new varieties of Phalaenopsis. Existing commonly used transgenic breeding techniques include: Agrobacterium-mediated method for indirect transformation of exogenous genes, biolistic method for direct transformation of exogenous genes, microinjection method and PEG-m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01H4/00
Inventor 张和臣王利民孟月娥董晓宇符真珠张晶李艳敏王慧娟蒋卉
Owner HENAN ACAD OF AGRI SCI
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