Agrobacterium rhizogenes-mediated transformation of passionfruit species: Passiflora cincinnata and P. edulis f. flavicarpa

摘要

In vitro-grown passionfruit (Passiflora cincinnata and P. edulis f. flavicarpa) seedlings were decapitated and inoculated with an overnight grown Agrobacterium rhizogenes R1601 suspension culture. The first responses were observed 20-30 d after inoculation. Hairy roots differentiated at the inoculation sites were used to establish individual root clones and used to initiate long term cultures on semi-solid medium. A series of control, non-infected shoots were also set up in order to ensure that any induced responses were not a result of normal rooting or healing responses. Hairy roots showing root tips of 1.5-2.0 cm in length were carefully detached from the shoots and transferred individually for further growth and proliferation to Petri dishes containing semi-solid MS medium supplemented with Timentin (350 mg L-1) and kanamycin (150-200 mg L-1). Elongating hairy roots were subcultured onto fresh medium every 15 days. During this time the clones retained their high growth rates and antibiotic resistance phenotypes. The regenerated roots displayed typical features of hairy roots such as hairiness, plagiotropism, branching and growth habit. The nptII and nos genes were detected by PCR in genomic DNA from root clones of both species, at the 6th passage, whereas nos gene was detected in regenerants derived from somatic embryos of P. cincinnata. Physiological confirmation of the transformed nature was provided by the auxin autotrophic response and resistance to kanamycin. Spontaneous plant regeneration from roots growing on selective semi-solid MS medium devoid of growth regulators was occasionally observed via organogenesis for P. edulis f. flavicarpa. P. cincinnata displayed higher rates of regeneration, and surprisingly regenerants were recovered via both organogenesis and somatic embryogenesis. Histological analysis revealed the direct pathway of shoot regeneration through organogenesis. P. cincinnata regenerated plants were successfully acclimatized under ex vitro conditions..