Optimization of system for Agrobacterium tumefaciens -mediated AtMYB118 genetic transformation into friable embryogenic calli in Hevea brasiliensis
[Objective]: An orthogonal design L25 (56) was used to optimize Agrobacterium -mediated AtMYB118 gene genetic transformation system of friable embryogenic calli in Hevea brasiliensis in order to provide references for genetic improvement of its clones. [Method]: Kanamycin at a concentration of 75.0 mg/L was used to select trans - formed calli. The effects of six factors viz . , preculture time, Agrobacterium growth phase (OD600) , acetosyringone (AS) concentration, infection time, co-culture temperature and co-culture time on genetic transformation were evaluated by GUS transient expression detection. [Result]: Six factors were found to significantly affect transformation frequency of long-term cultural friable embryogenic calli. The highest transformation efficiency was achieved by 0-day preculture, long-term cultural friable embryogenic calli as recipients infected by Agrobacterium cultures corresponding to OD600=0.7 for 7 min, followed by co-culture for 5 days in a co-culture medium containing 200 ?mol/L AS at 25 ?. After 4-6 months, 17 GUS positive callus lines were achieved. Polymerase chain reaction (PCR) and reverse transcription-polymerase chain reaction (RT-PCR) analysis results of transgenic tissues further confirmed that uidA, nptII and AtMYB118 genes had been inserted into genome of calli and expressed. At last , 164 transgenic embryoids were obtained from 1539 cultured embryoids. A transformation efficiency of 10.6% was achieved for longterm cultural friable embryogenic calli using this protocol. Four AtMYB118 transgenic plantlets were obtained. [Conclusion]: The optimized genetic transformation system using friable embryogenic calli of rubber tree as recipients was effective and available,which would provide technological supports on genetic improvement of clones.
| Main Authors: | , , , , , , , , , , , , , , |
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| Format: | article biblioteca |
| Language: | eng |
| Subjects: | F30 - Génétique et amélioration des plantes, F60 - Physiologie et biochimie végétale, Hevea brasiliensis, http://aims.fao.org/aos/agrovoc/c_3589, http://aims.fao.org/aos/agrovoc/c_1556, |
| Online Access: | http://agritrop.cirad.fr/575434/ http://agritrop.cirad.fr/575434/1/document_575434.pdf |
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| Summary: | [Objective]: An orthogonal design L25 (56) was used to optimize Agrobacterium -mediated AtMYB118 gene genetic transformation system of friable embryogenic calli in Hevea brasiliensis in order to provide references for genetic improvement of its clones. [Method]: Kanamycin at a concentration of 75.0 mg/L was used to select trans - formed calli. The effects of six factors viz . , preculture time, Agrobacterium growth phase (OD600) , acetosyringone (AS) concentration, infection time, co-culture temperature and co-culture time on genetic transformation were evaluated by GUS transient expression detection. [Result]: Six factors were found to significantly affect transformation frequency of long-term cultural friable embryogenic calli. The highest transformation efficiency was achieved by 0-day preculture, long-term cultural friable embryogenic calli as recipients infected by Agrobacterium cultures corresponding to OD600=0.7 for 7 min, followed by co-culture for 5 days in a co-culture medium containing 200 ?mol/L AS at 25 ?. After 4-6 months, 17 GUS positive callus lines were achieved. Polymerase chain reaction (PCR) and reverse transcription-polymerase chain reaction (RT-PCR) analysis results of transgenic tissues further confirmed that uidA, nptII and AtMYB118 genes had been inserted into genome of calli and expressed. At last , 164 transgenic embryoids were obtained from 1539 cultured embryoids. A transformation efficiency of 10.6% was achieved for longterm cultural friable embryogenic calli using this protocol. Four AtMYB118 transgenic plantlets were obtained. [Conclusion]: The optimized genetic transformation system using friable embryogenic calli of rubber tree as recipients was effective and available,which would provide technological supports on genetic improvement of clones. |
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