Plant Regeneration Protocol for Recalcitrant Passionflower (Passiflora quadrangularis L.)

Abstract

This research was designed to provide the first protocol to establish an efficient solution for direct organogenesis regeneration in Passiflora quadrangularis using nodal explants from young shoots. Passifloraceae tissue culture has been associated with problems such as recalcitrance, sensitivity to ethylene accumulation and browning of explants due to the presence of phenols in the tissues. Due to the high rate of endogenous contamination of the explants, a preliminary experiment was performed. The best results of surface sterilization were obtained using the pretreatment with 70% EtOH, 1 min and 50% NaOCl, 10 min along with the treatment of Rifampicin 15 µg/mL and Benomyl 2 g/L. The effects of plant growth regulators on the induction of direct organogenesis, multiplication of shoots in subcultures and in vitro rooting were evaluated. Additional compounds such as AgNO3 and Pluronic F-68 were added to the culture media in order to reduce the effects of phenols and the sudden browning of the explants. Shoot proliferation increased to the sixth subculture after which it decreased. A maximum of 7.17 shoots were obtained from one shoot on Murashige and Skoog (MS) medium supplemented with 2 mg/L 6-benzylaminopurine and 1 mg/L thidiazuron. Supplementation of ½ MS medium with 1 mg/L 1-naphthaleneacetic acid was conducing to root formation in 61.11% of shoots. After acclimatization, the plants showed vigorous growth, green leaves, and well-developed roots. Although this species has previously shown difficulty in in vitro propagation, this protocol established based on the results proved to be efficient and reproducible.