Transgene method for peanut

A transgenic, peanut technology, applied in the field of bioengineering, can solve the problems of chimera, escape, low transformation and regeneration efficiency, etc.

Inactive Publication Date: 2008-05-28
INST OF SUBTROPICAL AGRI CHINESE ACAD OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] In summary, no matter the gene transformation method based on tissue culture or non-tissue culture transformation method, there are low transformation and regeneration efficiency, genotype dependence, escape, chimera problems, and the transformation from transformation to transgene (pure One or several of the disadvantages such as long cycle and heavy workload of seeds
At present, there is no peanut transgenic method that achieves high-throughput, high-efficiency transformation and solves the problems of escape and chimera.

Method used

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  • Transgene method for peanut
  • Transgene method for peanut
  • Transgene method for peanut

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Obtaining repeated somatic embryogenic cultures of peanut.

[0036] Peanut varieties Yueyou 116 (cultivated by Guangdong Academy of Agricultural Sciences, continuous flowering subspecies pearl bean type, subsp. Fastigiata Var. vulgaris, spanish type Spanish type), Luhua 9 (cultivated by Shandong Academy of Agricultural Sciences, continuous flowering subspecies intermediate type), 'Georgia Green' (Cultivated by the University of Georgia, USA, alternate flowering subspecies common type, Subsp. hypogaea Var. hypogaea, runner / virginia type Lanna / Virginia type). These varieties are high-quality or disease-resistant peanut varieties, which are one of the main cultivated varieties in various regions. The pods were planted and harvested according to normal cultivation management and fertilizer and water measures (Yueyou 116 and Luhua 9 were kept by myself, and 'Georgia Green' was obtained from the University of Georgia, USA). Ripe fruit pods are stored in sealed pla...

Embodiment 2

[0039] Example 2: The exogenous DNA or gene is constructed in an appropriate plant expression frame and connected to the same vector, extracting and purifying the exogenous gene plant expression vector (plasmid) and determining its concentration.

[0040] The target gene human Bcl-xl gene plant expression cassette in pZP211-Bcl-xL (Dickman MB, ParkYK, Oltersdorf T, Li W, Clemente T, French R. Abrogation of diseasedevelopment in plants expressing animal antiapoptotic genes. Proc NatlAcad Sci USA, 2001 , 98:6957-6962) were digested with PstI and cloned into pUC19 (preserved in our laboratory), and then subcloned into plasmid pRT-66 (Dr.R. Gift) plant expression vector, plasmid pRT-66 with selection marker gene (hygromycin resistance hpt gene) plant expression cassette ( R, Maas C, Horicke-Grandpierre C, Schell J, Steinbiss H-H. Expression vectors for high-level gene expression in dicotyledonous and monocotyledonous plants. Meth Enzymol, 1993, 217:66-78). Plasmid vector constr...

Embodiment 3

[0041] Example 3: Microprojectile Bombardment Transformation of Duplicate Somatic Embryogenic Cultures and Selection of Independently Resistant Cell Lines.

[0042] Get about 10-15 repeated somatic embryogenic embryogenic tissues from the peanut repeated somatic embryogenic culture after 3-10 months of initiation of cultivation in Example 1, and place them in the SEM medium within about a 3 cm diameter area ( about 7cm 2 ). Mix the prepared 50 μl gold microparticles (diameter 0.6 or 1.0 μm, Bio-Rad) suspension (60 mg / ml) (Ozias-Akins et al. 1993) with 5 μl (1 μg / μl) containing the screening gene (hpt) under the control of the CaMV35S promoter Mix with the plasmid of the plant expression cassette of the gene of interest (human Bcl-xl) for non-screening. Add 50 μl of 2.5M calcium chloride and 20 μl of 0.1M spermidine successively while shaking. Shake to suspend for 5-8 minutes, and briefly centrifuge at 10,000rpm. Gold microparticles were rinsed with 100% ethanol and resuspe...

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Abstract

The invention discloses a process for transgene of peanuts, of which the steps include that initially repetitive somatic embryogenesis culture of the peanuts is established, then exogenous DNA or genes are constructed in a plant expression frame or an expression vector and the concentration of the exogenous DNA is extracted, purified and identified, thirdly the repetitive somatic embryogenesis culture is transformed via micro-projectile bombardment and independent resistant cell lines are screened, fourthly, plant regeneration and passage happens, and fifthly transgenic plant and transgenic molecules in the progenies is analyzed. The invention employs the repetitive somatic embryogenesis culture after which is started to be cultured for 1 to 10 months and is induced by mature peanut seeds which are stored for 0 to 6 years as a transgenic acceptor. The transgenic efficiency is high and the transformation flux is high, and the screen efficiency after the bombardment is high without escaping and chimeras. Genotype dependence does not exist in the whole process, the regeneration of the plant is easy and highly effective, transgenic descendant seeds can be abundantly obtained, and the exogenous gene is capable of stably conforming and expressing inheritance.

Description

technical field [0001] The invention belongs to the field of bioengineering and relates to a peanut transgenic method, which is applicable to all peanut varieties, strains or germplasm resource materials. Background technique [0002] Plant transgenic technology transfers the target gene (exogenous gene) isolated from animals, microorganisms, viruses or plants to the genome of the recipient plant through various methods through various transformation systems, making the exogenous gene Stable inheritance in recipient plants, and endow plants with new agronomic traits, such as insect resistance, disease resistance, stress resistance, high yield, high quality, etc. [0003] Peanut is the main oil crop in my country, rich in oil and protein. The use of bioengineering and transgenic means for variety improvement and genetics research is an important content of peanut genetic engineering. Since the first transgenic peanut was obtained in 1993 (Ozias-Akins P, Schrmll JA, Anderson...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09A01H1/00
Inventor 邓向阳
Owner INST OF SUBTROPICAL AGRI CHINESE ACAD OF SCI
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