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Optimization of system for Agrobacterium tumefaciens -mediated AtMYB118 genetic transformation into friable embryogenic calli in Hevea brasiliensis

Bi Z.H., Li Z., Wang Z.Q., Dai X.M., Fang J.L., Huang H., Lin W.F., Lardet L., Montoro P., Carron M.P., Leclercq J., Zuo J.R., Mu J.Y., Yang X.H., Li X.. 2014. Journal of Southern Agriculture, 45 (7) : p. 1137-1146.

[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. (Résumé d'auteur)

Mots-clés : hevea brasiliensis; chine

Thématique : Génétique et amélioration des plantes; Physiologie et biochimie végétales

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