Genetic transformation of Paulownia elongata S. Y. Hu., mediated by Agrobacterium tumefaciens and biolistic system

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Carlos R. Castillo-Martínez
Ma. Alejandra Gutiérrez-Espinosa
Jorge Cadena-Iñiguez
Marco T. Buenrostro-Nava
Valeria Martínez-Sias

Keywords

(BA) 6-bencil-adenine, (ANA) naphthalenic acid, micro-propagation.

Resumen

Objective: The most appropriate conditions for genetic transformation through direct (bioballistic) and indirect (Agrobacterium tumefaciens) transformation systems in Paulownia elongata were established.


Design/methodology/approach: Starting from in vitro propagation through both direct and indirect organogenesis, internodal stem segments with 0.5 to 1 cm length were determined as the best explant. The optimum dose for selection media was determined to be 15 mg L-1 of kanamycin. It was possible to obtain transgenic plants under both transformation systems. In the case of Agrobacterium tumefaciens, two hours of incubation, 48 h of co-cultivation, and optical density of 0.9 were used; while for bioballistics, the best conditions were 120 PSI of shot pressure, shot height at level 6 (16 cm), and vacuum pressure of 22 Hg mm, with particle inflow gun system (PIG).


Results: Both systems produced complete transformants, chimeras, as well as those confirmed by histochemical X-GLUC and PCR analysis, producing a total of 14 positive plants by A. tumefaciens transformation from 26 trials and ten positive plants by the bioballistic system from 30 trials; a construction with chitinase and glucanase, NPT II selection gene and the GUS reporter gene were used.


Findings/conclusions: So far, this has been the first report including integration of chitinase and glucanase genes.

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