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Method for building cell culture system for chimera obtained through grafting vinca rosea to vinca minor

A technology of cell culture and establishment method, which is applied in the field of establishment of chimera cell culture system obtained by grafting periwinkle vinca minor, can solve the problem of low anti-cancer content, and achieve the effect of convenient and efficient extraction

Active Publication Date: 2016-07-20
上海秋皇医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the content of anti-cancer and anti-stress substances in these two plants is too low, and the active ingredients extracted from the plants are far from meeting the needs of the market

Method used

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  • Method for building cell culture system for chimera obtained through grafting vinca rosea to vinca minor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] The periwinkle and periwinkle of the present embodiment all come from this laboratory.

[0022] This embodiment includes the following steps:

[0023] Step 1, cut the test-tube seedling of Vinca minor at the root, and keep 2 leaves as stock;

[0024] Step 2, peeling off the growth point of a margin base of periwinkle aseptic seedlings under a dissection microscope;

[0025] Step 3, inoculating the Vinca vinca growth point on the vinca minor rootstock under aseptic conditions;

[0026] Step 4, inoculate the graft material obtained in Step 3 into a growth medium, wherein the growth medium is a solid medium of MS+6-BA3.0mg / L+NAA0.4mg / L+GA30.9mg / L. and cultivated at 25°C for 25 days;

[0027] In step 5, a group of relatively complete callus will appear at the place where the scion and rootstock are combined, and the callus is subjected to liquid suspension culture to screen excellent cell lines and further screen the ratio of hormones, wherein the culture medium used in ...

Embodiment 2

[0030] The periwinkle and vinca minor of the present embodiment all come from this laboratory, wherein the test-tube seedling of vinca minor in the step 1 is cut at the root, and 3 leaves are reserved as stock; the concrete steps 1 to 3 of the present embodiment are the same Embodiment 1, step 4 and step 5 are shown in Table 1:

[0031] In this embodiment, 20 vinca vincas and 20 vinca vincas were aseptic as materials respectively, and 12 seedlings were grafted successfully, and chimera calluses appeared at the junction of 9 grafted materials, in which cascristine, The contents of vinblastine and Wenduolin were increased to varying degrees. But the presence of vincamine was not detected from the chimeric callus. Using this chimera as a material, an excellent cell line was screened out, and its content of mandolin was twice that of the parent plant. In subsequent experiments, the chimeric callus was differentiated and cultured, but no regenerated plants were obtained. In addi...

Embodiment 3

[0033] The difference from Example 2 is that the medium used in step 4 and step 5 of this example is, wherein the growth medium in step 4 is MS+6-BA2.0mg / L+NAA0.2mg / L+GA30. 5mg / L solid medium, and cultivated at 25°C for 26 days; Step 5, a group of relatively complete callus will appear at the place where the scion and rootstock are combined, and the callus will be cultured in liquid suspension to screen for excellent cells Line, and further screening hormone ratio, wherein the medium used in liquid suspension culture: MS+6-BA0.4mg / L+NAA1.8mg / L+hydrolyzed casein 19mg / L liquid medium.

[0034] In this embodiment, 20 vinca vincas and 20 vinca vincas were used as materials respectively, and 13 seedlings were successfully grafted, and chimera calluses appeared at the junction of 12 grafted materials. The contents of vinblastine and Wenduolin were increased to varying degrees. But the presence of vincamine was not detected from the chimeric callus. Using this chimera as a material...

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Abstract

The invention discloses a method for building a cell culture system for a chimera obtained through grafting vinca rosea to vinca minor. The method comprises the steps of cutting roots of the vinca minor, then, reserving 2 to 3 leaves for each vinca minor plant so as to prepare rootstock, inoculating growth points of the vinca rosea to the vinca minor rootstock under aseptic conditions, then, enabling each place of combination of a graft and the corresponding rootstock to emerge a callus, carrying out fluid suspension culture on the calli, and carrying out screening so as to obtain an excellent cell line. According to the method, after the growth points of the vinca rosea is grafted to the vinca minor, the calli are induced by using the obtained chimera as a material, and finally, an in-vitro cell suspension culture system for the chimera is built; proven by tests, the content of anticancer substances (such as vinblastine, vincristine and vindoline) in a cell derived from the chimera is increased.

Description

technical field [0001] The invention relates to the technical field of biological cell culture, in particular to a method for establishing a chimera cell culture system obtained by grafting periwinkle vinca minor. Background technique [0002] Micrografting is a technique of grafting rootstock and scion in test tube, which is a combination of tissue culture rapid propagation and grafting technology. Studies have shown that there are a series of complex material and energy transfers and exchanges between the scion and the rootstock after plant grafting, including changes in hormones at the joint site at the initial stage of grafting, alkaloids, small RNAs, flowering stimulating substances, viruses, etc. after symplasmic connection. In addition, the chimera formed after grafting often has some intermediate characters between the rootstock and the scion, and some specific proteins, polysaccharides and small molecular substances appear in the chimera, and these mutated character...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01H4/00A01G1/06
CPCA01G2/30A01H4/005
Inventor 张边江陈全战唐宁
Owner 上海秋皇医药科技有限公司
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