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Agrobacterium local invasion method applied in soybean hypocotyl explant transformation system, and device thereof

A technology of explants and Agrobacterium, applied in the field of genetic engineering, can solve problems such as adverse effects on the regeneration of adventitious buds of explants

Inactive Publication Date: 2012-12-19
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, while antibiotics inhibit the proliferation of Agrobacterium, they also adversely affect the regeneration of adventitious shoots in explants

Method used

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  • Agrobacterium local invasion method applied in soybean hypocotyl explant transformation system, and device thereof
  • Agrobacterium local invasion method applied in soybean hypocotyl explant transformation system, and device thereof
  • Agrobacterium local invasion method applied in soybean hypocotyl explant transformation system, and device thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] This embodiment provides a device for local infection of Agrobacterium applied to soybean hypocotyl explant transformation recipient system, see attached figure 1 As shown, including a culture dish 1 and a culture dish cover 2, a base plate 3 is fixed in the culture dish, and a through post hole 4 is arranged on the base plate. The bottom distance is 1 mm to 2 mm; the inner diameter of the through column hole is suitable for the size of the stem diameter of the explant. The device can be placed in an autoclave for routine sterilization. There may be several through-pillar holes.

[0054] As preferably, the substrate adopts a PCR plate 3 that is usually used for nucleotide fragment amplification, neatly cut off the small tubes at the bottom of the PCR plate to form through column holes 4, and keep the small tubes on the four corners as supports, and the supports will PCR Plate 3 is supported in a petri dish. According to the size of the petri dish and the size of the ...

Embodiment 2

[0083] Example 2 The effect of pre-culture time on the selection survival rate of hypocotyl explants

[0084] In this example, soybean seedlings with a seedling age of 5 days were cut to obtain hypocotyl explants, which were treated with short-term BA solution (same as in Example 1), and then pre-cultivated for 0, 1, and 2 days. The morphological upper end of the explants was locally infected with an Agrobacterium solution with an OD value of 0.5-1.0 for 20 minutes. After 3 days of co-cultivation, the explants were inoculated vertically into the recovery medium without antibiotics. The explants were cultured for 20 days, and finally the explants were inoculated horizontally on the selection medium and cultured for 30 days. The experimental results are shown in Table 1.

[0085] Table 1 Effect of pre-culture time on explant survival rate

[0086] time / d Survival rate / % 0 20.83b 1 45.83a 2 40.28a

[0087] Note: The data were analyzed using SP...

Embodiment 3

[0089] Example 3 Effect of co-cultivation time on survival rate of hypocotyl explant selection

[0090] In this example, soybean seedlings with a seedling age of 5 days were cut to obtain hypocotyl explants, which were treated with BA solution for a short period of time, and then pre-cultured for 1 day, using Agrobacterium with an OD value of 0.5-1.0 The morphological upper end of the explants was partially infected with the bacterial solution for 20 minutes, and after co-cultivation for 0, 3, and 6 days, the explants were inoculated vertically on the recovery medium without antibiotics and cultured for 20 days , and finally the explants were inoculated horizontally on the selection medium and cultured for 30 days. The experimental results are shown in Table 2.

[0091] Table 2 Effect of co-culture time on explant survival rate

[0092] time / d Survival rate / % 0 20.00b 3 55.83a 6 62.50a

[0093] Note: The data were analyzed using SPSS Statis...

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Abstract

The invention discloses an Agrobacterium local invasion device applied in a soybean hypocotyl explant transformation acceptor system, and an invasion method thereof. The invention provides the simply-designed device successfully applied in the Agrobacterium invasion of the soybean hypocotyl explant conversion acceptor system to inhibit the excess propagation of the Agrobacterium on the surface of the explant in a medium in order to prevent the decay of a plant material, and the device enables the whole explant to be immersed in the Agrobacterium solution, so only the upper end notches of the hypocotyl having an adventive bud regeneration potential undergo the Agrobacterium invasion. Invasion method conditions are further optimized in the invention. The medicine cost of the soybean hypocotyl explant genetic transformation acceptor system can be reduced and the transformation working efficiency can be improved through applying the device and the method.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a local Agrobacterium infection method applied to a soybean hypocotyl explant transformation receptor system. Background technique [0002] Plant genetic transformation is an important and advanced breeding technology. At present, the technology of plant genetic transformation has been basically mature, and the most common transformation method is to introduce the exogenous gene into the cells of the recipient plant using the Agrobacterium-mediated method and integrate it into the chromosomal genome to achieve stable inheritance of the exogenous gene. [0003] In the process of using Agrobacterium to transform soybean hypocotyl explants, in the past, the entire explant was soaked in the Agrobacterium bacterium liquid, so that the whole explant was stained with the bacterium liquid. In order to suppress the overgrowth of Agrobacterium on the surface of th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12M1/00C12N15/84
CPCC12M23/38C12M23/10
Inventor 杨跃生刘颖张奇于磊郭振飞年海
Owner SOUTH CHINA AGRI UNIV
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