Efficient separation, transformation and regeneration system for tobacco protoplast

A protoplast and tobacco technology, applied in the field of genetic engineering, can solve problems such as irregular operation, high pollution rate, complicated steps, etc.

Inactive Publication Date: 2015-07-22
BEIJING GENOVO BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Aiming at the defects of complicated steps, irregular operation, high pollution rate and low transformation efficiency in the separation and transform

Method used

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  • Efficient separation, transformation and regeneration system for tobacco protoplast
  • Efficient separation, transformation and regeneration system for tobacco protoplast
  • Efficient separation, transformation and regeneration system for tobacco protoplast

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] 1. Preparation of mother liquor and culture medium for protoplast isolation, transformation and regeneration

[0053] Table 1 MS trace 100× formula

[0054] ingredients

Final concentrationmg / L

500mL mother liquor (g)

CoCl 2 ·6H 2 o

2.5

0.00125

KI

83

0.0415

MnSO 4 ·H 2 o

1690

0.845

h 3 BO 3

620

0.31

CuSO 4 ·5H 2 o

2.5

0.00125

Na 2 MoO 4 2H 2 o

25

0.0125

ZnSO 4 ·7H 2 o

860

0.43

[0055] Table 2 Iron salt 1000× formula

[0056] ingredients

Final concentrationmg / L

500mL mother liquor (g)

FeNaEDTA

36700

18.35

[0057] Table 3 Medium formula

[0058]

[0059]

[0060]

[0061] Enzyme hydrolysis solution: add 1.2g of cellulase R10 to every 100mL of C medium, isolate enzyme R10 0. 6g, adjust the pH to 5.6 with 1M KOH, filter sterilize, store in -20℃ after aliquoting.

[0062] ...

Embodiment 2

[0092] Use Bensheng tobacco (Nicotiana Benthamiana) and cultivated tobacco (Nicotiana tabacum) such as Honghua Dajinyuan, K326, Yun 87, CB-1 and other varieties, and cultivate them according to the method in Example 1, and obtain tobacco transgenic plants. .

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PUM

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Abstract

The invention relates to an efficient separation, transformation and regeneration system for tobacco protoplast. The regeneration system comprises special separation, transformation and regeneration reagent combination and method for the tobacco protoplast. The reagent combination comprises a 1/2 MS (Murashige and Skoog) medium, an MS medium, enzymatic hydrolysate, a medium C, a W5 washing liquid, the Evans Blue dye, a transformation liquid, a polyethylene glycol solution, a C:B medium, a medium A and an MS regeneration medium. The transformation method comprises steps of culture of aseptic seedlings, separation and purification of the protoplast, transformation of the protoplast, culture and regeneration of the protoplast and the like. The method has the advantages of simple steps, normative operation, low pollution rate, high transformation efficiency and the like.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a high-efficiency separation, transformation and regeneration system of tobacco protoplasts. Background technique [0002] Tobacco (Nicotiana tabacum L.) is an important economic crop in my country. In 2014, the industrial and commercial tax profit of the tobacco industry reached 1.05176 trillion yuan, accounting for more than 10% of the total tax profit. In recent years, the public's demand for tobacco leaf quality and safety has been increasing, so accelerating variety selection is one of the important ways to meet this demand. At the same time, tobacco is an excellent recipient of plant transgenes and a model plant for cytogenetics, and is often used to carry out expression and functional verification of exogenous genes. In addition, in the field of bioengineering, it is often used as a plant bioreactor to effectively express some proteins with practical value. [0003] P...

Claims

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Application Information

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IPC IPC(8): C12N5/04C12N15/82A01H4/00
Inventor 高崑张康
Owner BEIJING GENOVO BIOTECH CO LTD
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