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Citrus protoplast transformation and purification method

A protoplast transformation and protoplast technology, applied in the biological field, can solve the problems of high transformation efficiency and the lag of protoplast transient expression technology, and achieve the effects of improving transformation efficiency, shortening enzymatic hydrolysis time, and improving uniformity.

Active Publication Date: 2016-06-08
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] After the first isolation of citrus protoplasts was completed by Vardietal. Electric shock induces the transformation of protoplasts of different varieties of citrus, but the highest transformation efficiency is only 10.9%

Method used

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  • Citrus protoplast transformation and purification method
  • Citrus protoplast transformation and purification method
  • Citrus protoplast transformation and purification method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] This example relates to the purification and transformation of citrus juice spore protoplasts. The citrus variety is Newhall Navel Orange Citrussinensis (L.) Osbeck, with yellow juice spores, picked from the National Indoor Preservation Center for Virus-free Germplasm Resources of Fruit Trees. Specifically include the following steps:

[0071] Prepare protoplasts:

[0072] A. After cleaning the peel of the picked Newhall navel orange, get its equatorial pulp, and place the stripped granulated juice cells in 20ml of 0.7M mannitol aqueous solution at normal temperature for pretreatment for 20 minutes;

[0073] B. Replace the mannitol aqueous solution in step 1 with 10ml of enzymatic solution, vacuum infiltrate for 30 minutes, and then treat it on a horizontal shaker at 40rpm for 5h. The preparation method of 10ml of enzyme solution is to add 7.5ml of 0.93M mannitol, 1ml of 0.1MMES, and 0.24g of CelluloseRS in sequence. And 0.12g Macerozyme, 55 ℃ water bath for 10 minutes...

Embodiment 2

[0087] Adopt different citrus materials, prepare protoplast according to the step in embodiment 1, the result is as follows figure 2 shown, where: figure 2 A is the protoplast that mesophyll extracts; figure 2 B is the protoplast that callus extracts; figure 2 C is the protoplast extracted from sap; figure 2 D is the protoplast extracted from the pericarp.

[0088] The protoplast that mesophyll is extracted is transformed according to the scheme in Example 1, and the results are as shown in Fig. image 3 shown, where image 3 A is the bright field of mesophyll protoplasts co-cultured after transformation; image 3 B is the fluorescence of co-cultured mesophyll protoplasts after transformation.

Embodiment 3

[0090] The method is the same as in Example 1, except that the amount of plasmid used in step (1) is 5 μg, 10 μg, 15 μg, and 20 μg, respectively. The results show that when the amount of plasmid used is 15 μg, the transient transformation efficiency of citrus protoplasts is the highest. For specific transient transformation results, see Figure 4 .

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Abstract

The invention discloses a citrus protoplast transformation method. The method comprises steps as follows: (1) plasmids and a polyethylene glycol-calcium solution with the concentration being 40%-60% are added to citrus protoplast, 10-15 mu g of the plasmids and 110 mu l of the polyethylene glycol-calcium solution are added to every 100 mu l of the citrus protoplast, and the mixture is uniformly mixed and left to stand; (2) the mixture is treated through heat shock for 5-9 min at the temperature of 45-47 DEG C; (3) a W5 solution is added to the mixture treated through heat shock, 440 mu l of the W5 solution is added to every 100 mu l of the citrus protoplast, the mixture is centrifuged after being uniformly mixed, and precipitates are taken; (4) a WI solution is added to the obtained precipitates, 600-800 mu l of the WI solution is added to every 100 mu l of the citrus protoplast, and the mixture is cultured for 24-48 h. According to the citrus protoplast transformation method, the transformation rate is substantially increased.

Description

technical field [0001] The invention belongs to the field of biotechnology, and more specifically relates to a method for transforming and purifying citrus protoplasts. Background technique [0002] my country is one of the important origins of citrus in the world, with abundant germplasm resources and various types. With the continuous improvement of people's quality of life, fruit quality has become the core of the competitiveness of the fruit market. Citrus is favored by consumers for its convenient eating, unique flavor and rich nutritional value. Time is difficult to meet the excellent varieties of market demand. With the rapid development of genetic engineering, transgenic technology provides a new way for the genetic improvement of citrus varieties. [0003] Transient expression technology refers to the technology of transferring the target gene into recipient cells in a short period of time, establishing an efficient expression system in the recipient cells, and o...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N5/04
CPCC12N5/04C12N15/8206C12N2509/00
Inventor 邓秀新杨威徐强郭文武
Owner HUAZHONG AGRI UNIV