A simple method for transient transformation of pumpkin (Cucurbita maxima) seedlings

Currently there are no efficient stable transformation methods for pumpkin. In this work we report an efficient genetic transient transformation method of whole pumpkin (Cucurbita maxima cv. Big Max, a dicot) plants in the cotyledonary stage via direct inoculation of Agrobacterium tumefaciens and A. rhizogenes. A vector harboring a uidA-GFP translational fusion and the Cauliflower mosaic virus (CaMV) 35S promoter was employed. Transformation with A. rhizogenes resulted in the generation of root tumor phenotype within the lower portion of the stem, two months after inoculation. On the other hand, A. tumefaciens induces callus tissue also in the lower section of the stem one month after inoculation. Sections from these tissues were confirmed to harbor the transgene through histochemical GUS analysis and quantitative (q) PCR and RT-qPCR. Transgene RNA was also detected in phloem sap exudates, suggesting a via for delivery of foreign-expressed proteins in pumpkin. Plants expressing the maize KNOTTED1 (KN1) genes showed distinct phenotype, consisting in leaf deformations with its known role in determining leaf shape. Transformation efficiency, based on transgene presence and appearance of a phenotype, ranged between 17.6 and 56%. The method described in this work can be used to transiently transform adult plants, and may be applicable to other species recalcitrant to Agrobacterium-mediated stable transformation; also, novel traits may be introduced to pumpkin and other plants using this technique.