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Genetic transformation method for stab-vacuum infiltration-assisted agrobacterium tumefaciens-mediated castor seeds

A genetic transformation method and technology of castor seeds, applied in the field of acupuncture-vacuum infiltration assisted Agrobacterium-mediated genetic transformation of castor seeds, can solve problems that cannot meet the requirements of castor molecular level research, difficult regeneration of isolated tissue culture, Castor bean transgene lacks in-depth problems, such as low success rate, long cultivation time and high transformation rate.

Active Publication Date: 2016-07-20
GUANGDONG OCEAN UNIVERSITY
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Problems solved by technology

However, the disadvantages of the above technologies are that it takes a long time, the regeneration of isolated tissue culture is difficult, the transformation efficiency is low, and the reproducibility is poor. The castor regeneration system cannot be better combined with the existing plant transformation methods.
Therefore, the research on castor-bean transgenesis is still not in-depth, and it is not enough to carry out research on the molecular level of castor-bean. It is urgent to establish a relatively mature castor-bean genetic transformation system.

Method used

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  • Genetic transformation method for stab-vacuum infiltration-assisted agrobacterium tumefaciens-mediated castor seeds
  • Genetic transformation method for stab-vacuum infiltration-assisted agrobacterium tumefaciens-mediated castor seeds
  • Genetic transformation method for stab-vacuum infiltration-assisted agrobacterium tumefaciens-mediated castor seeds

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Embodiment 1

[0028] Plant material: Castor Bean Thailand 202, provided by Yunnan Academy of Agricultural Sciences.

[0029] Strains: Agrobacterium EHA105; plant expression vector pCAMBIA1305.1, which carries GUS , Hyg Gene.

[0030]Drugs: LATaq, ExTaq, and pMD18-Tvector were purchased from Bao Biological Engineering (Dalian) Co., Ltd. (TaKaRa); antibiotics, RNaseA, TaqDNAPolymerase, dNTP (10mmol / L), CTAB, β-mercaptoethanol (β-mercaptoethanol), pancreatic Peptone, agar powder, ampicillin (Amp), yeast powder, DEPC, glycerol, calcium chloride, absolute ethanol, boric acid and other reagents were purchased from Sangon Bioengineering (Shanghai) Co., Ltd.

[0031] Using acupuncture-vacuum infiltration assisted Agrobacterium-mediated genetic transformation of castor seeds, the pCAMBIA1305.1 plant expression empty vector was transformed into castor Thailand 202, the steps are as follows:

[0032] 1. Activation and suspension of Agrobacterium: Streak the EHA105 bacterial solution containing the...

Embodiment 2

[0041] Plant material: Castor inbred line HY1, provided by Plant Molecular Breeding Laboratory, College of Agriculture, Guangdong Ocean University. HY1 was a wild material collected in Dongyuan County, Heyuan City, Guangdong Province in 2005. After more than 6 years of self-crossing and stable material, this material has developed root system, darker and thicker leaves than other cultivated materials. After years of planting observation, it shows drought resistance, Waterlogging tolerance, barren tolerance, shade tolerance and high resistance to leafhopper characteristics.

[0042] Strains: Agrobacterium EHA105; plant expression vector pYLRNAi- PAL Overexpression and antisense expression vectors (constructed by Plant Molecular Breeding Laboratory, School of Agriculture, Guangdong Ocean University), which contain Hyg Gene.

[0043] The pYLRNAi- PAL The overexpression and antisense expression vectors were respectively transformed into the castor inbred line HY1, and the st...

Embodiment 3

[0053] Plant material: Castor inbred line YC2, provided by Plant Molecular Breeding Laboratory, College of Agriculture, Guangdong Ocean University.

[0054] Strains: Agrobacterium EHA105; plant expression vector pYLRNAi- PEPC1 Antisense expression vector (constructed by Plant Molecular Breeding Laboratory, School of Agriculture, Guangdong Ocean University), which contains Hyg Gene.

[0055] The pYLRNAi- PEPC1 The antisense expression vector was transformed into castor bean inbred line YC2, the steps are as follows:

[0056] 1. Activation and suspension of Agrobacterium: Infection containing pYLRNAi- PEPC1 The EHA105 bacterial solution of the antisense expression vector was streaked on the YEB plate containing Kan (50mg / L) and Rif (25mg / L) antibiotics, and cultured in the dark at 28°C for 66-72h. Use a spoon to dig all the bacteria on the plate into the 1 / 2MS liquid medium added with 100mg / LAS, shake well to suspend the bacteria.

[0057] 2. Seed disinfection and pre-c...

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Abstract

The invention belongs to the technical field of plant genetic engineering transformation and discloses a genetic transformation method for stab-vacuum infiltration-assisted agrobacterium tumefaciens-mediated castor seeds. The method comprises the steps of pre-culturing castor seeds, stabbing epicotyls of the pre-cultured castor seeds, performing vacuum infiltration for enabling an agrobacterium tumefaciens solution containing target genes to infect the castor seeds, performing co-culture, removing agrobacterium tumefaciens, and performing transplantation, wherein according to the vacuum infiltration, the stabbed seeds are treated in the agrobacterium tumefaciens solution for 5-20 minutes under the vacuum negative pressure of -30kPa to -70kPa, air is discharged for 2-5 minutes, and the stabbed seeds are treated for 2-5 minutes under the negative pressure of -30kPa to -70kPa again. According to the method, the pre-cultured seeds are directly taken as receptors, so that in vitro tissue culture is not required; and a transgenic plant can be obtained in 15 days from castor seed pre-culture to extracted DNA detection, so that the transformation cycle is short, the transformation rate is high, the operation is relatively simple, and the repeatability is high.

Description

technical field [0001] The invention relates to the technical field of plant genetic engineering transformation, in particular to an acupuncture-vacuum infiltration assisted Agrobacterium-mediated genetic transformation method of castor seeds. Background technique [0002] Castor is one of the top ten oil crops in the world, and it is a special industrial oil source crop and a new type of oil crop. Castor oil has unique quality and renewable performance as "green oil". The research on castor oil has been paid more and more attention at home and abroad, and great progress has been made. Now the main problems that restrict the large-scale promotion and development of castor are diseases, insect pests and ricin, so cultivating varieties with resistance to diseases and insect pests and low toxicity proteins is the key to castor breeding, but it is difficult to obtain these new germplasm resources by conventional methods. With the complete genome sequencing of castor in 2010, an...

Claims

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Application Information

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IPC IPC(8): C12N15/82
CPCC12N15/8205
Inventor 陆建农殷学贵施玉珍刘颖李卫锦陈旺东
Owner GUANGDONG OCEAN UNIVERSITY
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