Optimization process for de-novo organogenesis and regeneration of transgenic papaya plants using leaf tissue as explants

Papaya (Carica papaya L.) is widely grown in tropical and subtropical regions. The destructive disease caused by emerging strains of Papaya ringspot virus (PRSV) demands effective transgenic resistance to target atypical virus strains threatening the crop. Papaya transformations are mainly conducted on the explants of immature zygotic or somatic embryos, which are difficult to obtain and proceed, and are largely affected by seasonal factors. Here, we attempted to develop an efficient process for organogenesis using various tissues of ex-vitro or in-vitro grown papaya seedlings. Leaf lamina, hypocotyl and stem sections of seedlings of the papaya variety Sindhi were used for establishment of callus culture through 12 different callus induction treatments (CIT 1–12), with various combinations of plant growth regulators (PGRs). Our results revealed that CIT-11 and CIT-10 enhanced callus induction from ex-vitro leaf discs with midrib, with 86% and 80% efficiency respectively, superior to 53% of CIT-10 using in-vitro leaf discs with midrib. The expression of binary vector construct pSN-PRSV CP in Agrobacterium strain GV3101 was verified in Nicotiana benthamiana and papaya by RT-PCR analysis. Calli derived from leaf tissues (with midrib) of papaya, transformed with the binary vector were successfully regenerated on the shoot induction treatment SIT-13 (Gamborg B5 medium + 0.5 mg/L TDZ + 0.01 mg/L NAA) and were subsequently rooted on root induction treatment RIT-4 (Gamborg B5 medium + 1 mg/L IBA). The transformed explants were regenerated with an efficiency of 26%. The whole process is unique in term of explant selection, source of explant (ex-vitro grown papaya plants) and media formulations as, the leaf tissue from ex-vitro grown papaya showed highest callusing and regeneration efficiency overall.