Protoplast isolation and genetic transformation method of Brassica napus without restriction of genotype and regeneration system thereof

A technology of protoplast transformation and Brassica napus, which is applied in the field of genetic engineering, can solve the problems of few successful examples, poor reproducibility of experimental operations, and large genotype dependence

Pending Publication Date: 2019-06-14
TIANJIN GENOVO BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Previous reports mainly used the hypocotyls of three Brassica napus cultivars to obtain protoplast regenerated plants (Glimelius, 1984; Kirti, 1988; Kirti and Chopra, 1988; Pua, 1990; Pauk, 1991; Olin-Fatih, 1996) , genotype dependence, high and low transformation efficiency, poor reproducibility of experimental operations, and few successful examples of mesophyll protoplast regeneration plants (Glimelius, 1984; Müller and Sonntag, 1998, Sahab et al, 2018)

Method used

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  • Protoplast isolation and genetic transformation method of Brassica napus without restriction of genotype and regeneration system thereof
  • Protoplast isolation and genetic transformation method of Brassica napus without restriction of genotype and regeneration system thereof
  • Protoplast isolation and genetic transformation method of Brassica napus without restriction of genotype and regeneration system thereof

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0073] The mother solution and the substratum used in the preparation of protoplast isolation, transformation and regeneration used in the following examples are as follows:

[0074] Table 1 MS trace 100× mother solution formula

[0075] 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

[0076] Table 2 Iron salt 1000 × mother liquor formula (solvent is water, solute is FeNaEDTA)

[0077] ingredients

Final concentrationmg / L

500mL mother liquor (g)

FeNaEDTA

36700

18.35

[0078] Table 3 medium formula

[0079]

[0080]

[0081]

[0082] The difference between the liquid form and the solid form of each medium in Table 3 is only w...

Embodiment 1

[0090] Embodiment 1, the separation, purification and transformation of rape protoplast

[0091]1. Separation and purification of rapeseed protoplasts

[0092] 1. Cultivation of sterile vaccines

[0093] 1) Weigh about 1g of rapeseed (Yangyou No. 7) into a 15mL centrifuge tube.

[0094] 2) Add 5 mL of 75% ethanol aqueous solution and shake lightly for 5 min, discard the waste liquid, and rinse once with sterile water.

[0095] 3) Disinfect the surface with a mixed solution of 0.05% Tween20 and 10% hydrogen peroxide by volume percentage for 15 minutes, and then rinse with sterile water for 5 times.

[0096] 4) Sow on demand in a 90mm×15mm petri dish filled with 1 / 2 MS B5 medium (30 grains per dish), place at 24°C, the photoperiod is 16 hours of light / 8 hours of darkness, and the light intensity is 84 μmol / m 2 / s conditions in the growth chamber.

[0097] 5) After 1.5-2 weeks, when the first true leaf grows from the seeds, the leaves are cut and transplanted into a square cu...

Embodiment 2

[0143] Embodiment 2, the separation, purification and transformation of rape protoplast

[0144] 1. Separation and purification of rapeseed protoplasts

[0145] Same as the method of Example 1, only Yangyou No. 7 was randomly replaced with Brassicanapus L to obtain Zhongshuang No. 10 protoplasts, Nanyang 41 protoplasts, Yunyou No. 49 protoplasts, and Ningza No. 11 protoplast, 9603 protoplast, Qinyou No. 11 protoplast.

[0146] 2. Transformation of protoplasts

[0147] According to the second method of Example 1, the transgenic plants Zhongshuang No. 10, Nanyang No. 41 transgenic plants, Yunyou No. 49 transgenic plants, Ningza No. 11 transgenic plants, 9603 transgenic plants and Qinyou No. 11 transgenic plants were obtained .

[0148] The results of the statistical transformation process are shown in Table 4.

[0149] Table 4

[0150]

[0151] The calculation formulas for each indicator in the above table are as follows:

[0152] Protoplast output=X / 9×5000×protoplast sol...

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Abstract

The invention discloses a protoplast isolation and genetic transformation method of Brassica napus without restriction of genotype and a regeneration system thereof, and provides a method for transforming exogenous nucleic acid molecules by using Brassica napus protoplasts. The method comprises the following steps of: 1) preparing protoplasts by enzymolysis of Brassica napus leaves; 2) transferring the exogenous nucleic acid molecules into the protoplasts to obtain the protoplasts with the transferred exogenous nucleic acid molecules; and dividing and culturing the protoplasts with the transferred exogenous nucleic acid molecules in a C: B culture medium to obtain cell clusters formed by division of the protoplasts. According to the invention, a sterile seedling of the Brassica napus is used as a raw material, the mesophyll tissue of the Brassica napus is digested by cellulase R-10 and macerozyme R-10, the protoplasts are isolated by using a density gradient sedimentation method to obtain the high-purity protoplasts. The target gene is introduced into the genome of the Brassica napus protoplasts through the mediation of polyethylene glycol, and a large number of Brassica napus transgenic plants are cultured through shallow liquid culture and subsequent differentiation and regeneration.

Description

technical field [0001] The invention relates to the field of transgenic engineering, in particular to a method for isolating and genetically transforming protoplasts of Brassica napus and the used regeneration system which are not restricted by genotype. Background technique [0002] Rapeseed rape (Brassica napus L) is an important oil crop, not only for human consumption, but also for food industry and industrial processing such as animal feed. Today, traditional breeding methods are increasingly unable to meet the ever-changing demand for varieties. The further improvement of rapeseed varieties largely depends on the expansion breadth and depth of functional genes. Protoplast manipulation methods such as in vitro induction, cell fusion and genetic transformation provide new sources of genetic variation (Vamling and Glimelius, 1990), and somatic cell hybridization also provides new possibilities for genetic material exchange and recombination between distant species ( Jou...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/04C12N15/82A01H4/00A01H5/00A01H6/20
Inventor 冉毅东高崑张康
Owner TIANJIN GENOVO BIOTECH CO LTD
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