Efficient regeneration and Agrobacterium tumefaciens mediated transformation of recalcitrant sweet potato (Ipomoea batatas L.) cultivars
Rolando García González1,2*, Danalay Somonte Sánchez2, Zurima Zaldúa Guerra1, Jesús Mena Campos2, Alina López Quesada2, Rolando Morán Valdivia2, Ariel D. Arencibia2, Karla Quiroz Bravo³ and Peter DS. Caligari3
1Departamento de Ciencias Agrarias y Forestales, Universidad Católica del Maule, Chile. Avenida San Miguel, No.3605.
2Centro de Ingeniería Genética y Biotecnología de Camagüey. A.P. 387, C.P. 70100. Camagüey, Cuba.
3Instituto de Biología Vegetal y Biotecnología, Universidad de Talca. 2 Norte 685, Talca, Chile.
*Author for Correspondence.
Departamento de Ciencias Agrarias y Forestales,
Universidad Católica del Maule, Chile.
Avenida San Miguel, No.3605.
E-mail: This email address is being protected from spambots. You need JavaScript enabled to view it.
Abstract.
Sweet potato is a major world crop and its behavior under in vitro culture is genotype dependent. We study several factors influencing the regeneration and transformation efficiency of two recalcitrant cultivars: Jewel and CEMSA 78354. Growth regulators, explant preparation and removal of apical dominance were evaluated in order to optimize the regeneration steps. At the same time, the influence of environmental conditions for the interaction between Agrobacterium tumefaciens and leaves were evaluated to obtain higher transformation efficiencies.
For Jewel, the best results were obtained when intact leaf explants were cultivated on MS medium supplemented with 0.5 mgL-1 indol-3-acetic acid for four weeks (Regeneration frequency, RF= 2.02). However, for CEMSA 78354 the best results were obtained by culturing leaf explants on MS medium supplemented with 1.0 mgL-1 paclobutrazol and 1.0 mgL-1 naftalenacetic acid (RF= 0.98).
Optimal transformation conditions were obtained for both cultivars by co-cultivating leaf explants with Agrobacterium tumefaciens in liquid MS medium for 24 hours at 28°C in stationary cultures in the dark. Acetosyringone influence on the transformation efficiency was found to be dependent on the co-cultivation temperature but it did not increase the transformation efficiencies. Molecular evidences by PCR, Southern blot and Dot blot demonstrated the effectiveness of the transformation procedure. The protocol described here is currently in use to produce transgenic sweet potatoes from different cultivars with high efficiency.
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