Detection of vector- and selectable marker-free transgenic maize with a linear GFP cassette transformation via the pollen-tube pathway

The pollen-tube pathway is feasible to transform vector- and selectable marker-free linear gene cassettes into plants to address the biosafety issues. However, its transformation frequency is low and the screening of selectable marker-free transformants by PCR analysis is time-consuming and expensive. In this study, a linear GFP cassette (Ubi-GFP-nos) flanked by 25bp T-DNA borders was transformed into maize via the pollen-tube pathway. The forepart of each maize ear was divided into five segments (segments I-V) at an interval of two rows of kernels. The segments that were most likely to contain transgenic kernels were identified by monitoring GFP expression in the immature embryos. A total of 21 ears were transformed with the linear GFP cassette. Seven out of 19 ears exhibited positive GFP expression in the immature embryos. Transgenic kernels were primarily identified in segments III and IV. A total of 121 plants derived from kernels located within segments III and IV of the remaining two ears were screened by PCR analysis. Six plants (4.96%) showed the presence of the GFP cassette. Southern blot analysis showed that the transgenic plants had simple integration patterns. The identification of transgenic kernels would facilitate PCR screening for marker-free transgenic plants.