Abstract
Agrobacterium-mediated in-planta genetic transformation of rough lemon (Citrus jambhiri Lush.) circumventing the need of tissue culture is reported. Mature seeds of rough lemon with removed integument were germinated on moist filter paper and the seeds with emerged plumule (1–2 cm) were pricked at the stem part using a sterile needle followed by immersing in Agrobacterium suspension carrying antifungal β-1,3-glucanase gene under the control of CaMV 35S promoter and NOS terminator. The Agrobacterium-infected seedlings were blot dried and transferred to sowing trays containing field soil + farm-yard manure (3:1) and moved to transgenic glass house for growth of the seedlings. The genomic DNA of T0 seedlings (142) was PCR analysed using gene specific PCR primers leading to identification of eight seedlings (5.63%) carrying β-1,3-glucanase transgene. Transcription of the transgene in eight putative transformants was determined by RT-PCR and quantification of relative gene expression by qRT-PCR revealed upto 4.5-fold increase in seedling number 41, as compared to non-transgenic control.
Published Version
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