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Nonglutinous rice callus culture medium and genetic transformation method mediated by agrobacterium tumfaciens

A culture medium, Agrobacterium technology, applied in horticultural methods, botanical equipment and methods, recombinant DNA technology, etc., can solve the problems of low transformation efficiency, easy death, poor growth status, etc.

Active Publication Date: 2010-06-02
WUHAN BIORUN BIO TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these calli grow poorly on the subculture medium provided by them and are easy to die. Calli in this state cannot be used for Agrobacterium infection and transgenic operations. Many repeated experiments in our laboratory have failed. There was no positive callus capable of achieving successful genetic transformation
At present, there is still a bottleneck in indica rice transgenic technology. Only some genotypes of indica rice can be transformed successfully, and the transformation efficiency is not high; Limiting the role of transgenic technology in rice variety improvement

Method used

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  • Nonglutinous rice callus culture medium and genetic transformation method mediated by agrobacterium tumfaciens
  • Nonglutinous rice callus culture medium and genetic transformation method mediated by agrobacterium tumfaciens
  • Nonglutinous rice callus culture medium and genetic transformation method mediated by agrobacterium tumfaciens

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] A new medium suitable for the growth of indica rice callus (named LY medium, the same below):

[0085] Concentration of substances (basic composition: macroelements)

[0086] Nitrate ion (NO 3 -) ) 40 or 45 or 51 or 56 or 62 or 66 or 70mM

[0087] Ammonium ion (NH 4 + ) 10mM or 20mM or 30mM

[0088] Potassium ion (K + ) 30mM or 40mM or 50mM or 60mM

[0089] Phosphate ion (PO 4 3- ) 2mM or 3mM or 4mM or 5mM

[0090] Magnesium Sulfate Heptahydrate (MgSO 4 *7H2O) 150mg / L or 250mg / L or 400mg / l

[0091] Calcium Chloride Dihydrate (CaCl 2 *2H 2 O) 150mg / L or 250mg / L or 400mg / L

[0092] Manganese sulfate (MnSO 4 ) 20mg / L or 30mg / L or 40mg / L

[0093] Zinc sulfate (ZnSO 4 ) 2mg / L or 4mg / L or 6mg / L or 10mg / L

[0094] Boric acid (HBO 3 ) 5mg / L or 10mg / L or 15mg / L

[0095] Potassium iodide (KI) 1mg / L or 3mg / L or 5mg / L;

[0096] Copper sulfate pentahydrate (CuSO 4 *5H 2 O) 0.01mg / L or 0.05mg / L or 0.1mg / L

[0097] Sodium molybdate dihydrate (Na 2 MoO 4 *2H ...

Embodiment 2

[0107] Plasmid pCAMBIA1301 (purchased from CAMBIA Company, Australia) and Agrobacterium strain EHA105 (preserved in this experiment) were used to transform the mature embryo-induced callus of indica rice material 9311 (preserved in this laboratory). A kind of transgenic method mediated by agrobacterium on rice tissue culture medium, its steps are:

[0108] A. Callus induction:

[0109] Induction medium:

[0110] On the basis of LY medium, add: sucrose: 30g / L, acid hydrolyzed casein: 500-800mg / L, glutamine: 300-500mg / L, proline 300-500mg / L, 2,4- d: 0.5-1.5mg / L; cytokinin (kt or ba, 6-ba, etc.) concentration: 0.0-0.5mg / L; boil the plant gel with 800ml deionized water in a microwave oven, and add the ingredients to the fixed solution to 1000ml. Ph 5.4-5.8 30ml or 50ml / bottle is divided into 50ml or 100ml Erlenmeyer flasks, sealed with assembled sealing film, and sterilized at 115 degrees Celsius for 15 minutes. After the mature rice embryos have been removed from the glumes, ...

Embodiment 3

[0147] Using plasmid pCAMBIA1301 (purchased from pCAMBIA company in Australia), Agrobacterium is EHA105 strain (preserved in this experiment), transforming indica rice material 9311, yta, Minghui 63, Jiang-s, 94d-2, ytb / 9311, 71994, 5-34 / Zhe 733, y58s-1, IR40931, kaslath, IR64, DML105, FR13A, GodaHeenati, E32, Tianyuan 96-2, Xian'ais, MC526, GD-1SD and other 20 varieties of induction, cultivation, Agrobacterium transformation.

[0148] A. Callus induction:

[0149] Induction medium:

[0150] On the basis of LY medium, add: sucrose: 30g / L, acid hydrolyzed casein: 500-800mg / L, glutamine: 300-500mg / L, proline 300-500mg / L, 2,4-d : 0.5-1.5mg / L; cytokinin (kt or ba, 6-ba, etc.) concentration: 0.5-0.5mg / L; boil the plant gel with 800ml deionized water in a microwave oven, add the ingredients to the 1000ml. Ph 5.4-5.8 30ml or 50ml / bottle is divided into 50ml or 100ml Erlenmeyer flasks, sealed with assembled sealing film, and sterilized at 115 degrees Celsius for 15 minutes. After...

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Abstract

The invention discloses a nonglutinous rice callus culture medium and a genetic transformation method mediated by agrobacterium tumfaciens. The culture medium contains major elements, minor elements, organic elements and hormone in different culture stages which are required for the growth of nonglutinous rice calluses. The genetic transformation method of nonglutinous rice based on the culture medium comprises the steps of induction, subculture, pre-culture, co-culture, sieving, aftersieving, differentiation, rooting of the calluses and the like. The method is widely suitable for nonglutinous rice genotypes, all genetic transformation of the tested 35 nonglutinous rice genotypes are successful, and the transformation rate is between 20% and 50%. The genetic transformation method solves the technical problem of low genotype reliance and transformation frequency existing in the nonglutinous rice callus culture and mediation of agrobacterium tumfaciens for a long time, and is widely applied to the research in the molecular biology, the functional genomics of the nonglutinous rice and the genetic improvement of varieties. The planting area of the nonglutinous rice accounts for more than 80% of the total production area of the nonglutinous rice, and the genetic transformation method has an important function on the research of the basic biology, the improvement of new varieties and the safety guarantee of future grain.

Description

technical field [0001] The present invention relates to the field of plant tissue culture and transgenic technology, specifically relates to indica rice tissue culture medium, and also relates to an Agrobacterium-mediated transgenic method of the culture medium. Embryo callus induction and subculture, mature embryo callus induction and subculture. And Agrobacterium-mediated callus genetic transformation operation. technical background [0002] Rice is the main food crop in the world. It is divided into two subspecies, japonica rice and indica rice. The respective representative varieties of the two subspecies have undergone whole genome sequencing (japonica rice Nipponbare, indica rice 9311). Therefore, the operation of genetic engineering on it is a hotspot of current research. Genetic transformation methods are commonly used Agrobacterium infection method and particle gun method. All kinds of transformation methods rely on the excellent receptor material—callus tissue, ...

Claims

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Application Information

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IPC IPC(8): C12N5/04C12N15/82A01H4/00A01H5/00
Inventor 李阳李阳生
Owner WUHAN BIORUN BIO TECH
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