In planta transformation of Polianthes tuberosa for concomitant knockdown of flp-1, flp-12 and flp-18 genes induced root-knot nematode resistance

Abstract

Polianthes tuberosa is a lucrative crop owing to its huge demand in floriculture, perfume and pharmaceutical industry. Root-knot nematodes, especially Meloidogyne incognita poses a major impediment on tuberose yield. Currently nematode management relies on the use of hazardous nematicides and other inefficacious means viz. crop rotation and soil solarization. The ongoing search for an ecologically sustainable and target specific approach, led to the progression of genetic modification based techniques to target genes critical for nematode survival. FMRFamide like peptide (FLPs) genes have drawn interest as prospective targets because of their diverse physiological roles in nematodes. Here, the effect of concomitant silencing of three vital nematode FLP genes (Mi-flp-1, Mi-flp-12 and Mi-flp-18) using host induced RNAi in tuberose was assessed against M. incognita. Firstly, the fusion (flp-1, flp-12 and flp-18) gene cassette was functionally validated using host-delivered RNAi in tobacco model system. Transgenic tobacco expressing dsRNA of fusion gene significantly reduced the nematode multiplication factor (MF) by 82.6%. Since, genetic transformation of tuberose has been neglected so far, and transgenic development is an inevitable step for host-generated RNAi, an Agrobacterium mediated transformation protocol was optimized in tuberose. Tuberose tubers were pricked at the apical meristem of the axis and at the tiny white meristematic buds emerging from them, followed by infection with Agrobacterium hosting pCAMBIA 3301 vector. Maximum transformation efficiency was achieved with 200 μM acetosyringone as observed from the GUS staining. For HIGS, fusion transgenic events were developed in tuberose and molecular analysis confirmed the presence and integration of fusion transgene using PCR, qRT-PCR and Southern blot analysis. Bioefficacy analysis of transgenic plants against M. incognita resulted in 73.75% reduction in nematode fecundity, thereby demonstrating the effectiveness of the three flp genes for nematode management in tuberose. This study established first ever report of in planta Agrobacterium-mediated transformation of tuberose for host delivered RNAi for M. incognita resistance.