Establishment of a transformation and regeneration protocol for Uganda’s passion fruit using gus reporter gene

Abstract

Declining yields from passion fruit farming due to a number of problems ranging from pests, diseases, environmental stress, low yields, have initiated widespread enterprise abandonment by farmers. Crop improvement efforts by conventional breeding techniques have had little success due to a number of limitations necessitating for research into other alternative approaches, such as plant tissue culture and genetic engineering, for Passiflora improvement. However, currently there is lack of an efficient and reproducible transformation system that is compatible with a regeneration method for the successful transformation of Passiflora varieties in Uganda. In addition, the high heterogeneity of genus Passiflora makes the application of tissue culture technology and transformation very difficult due to a number of physiological and developmental problems owing to the species increased genetic variability. The study thus aimed at optimizing the existing regeneration and transformation protocols to suit Uganda‟s Passiflora cultivars. Regeneration of yellow passion fruit was induced on Murashige and Skoog (MS) media supplemented with 8.9 μM BAP with the best shoot regeneration efficiency of 12.87%; however the purple passion fruit proved to be recalcitrant to tissue culture. Genetic transformation protocol for yellow passion fruit involved heat shocking of one-day pre-cultured leaf disc explants at 45°C for one minute followed by infection with Agrobacterium AGL1 (OD600 of 0.5) transformed with pCAMBIA2301 harboring the Gus gene for 18-22 minutes. Leaf discs were then vacuum infiltrated for 1½ minutes at 750mmHg followed by a three-day co-cultivation period at 26°C ± 1°C in darkness on MS + acetosyringone (100 μmL-1) before culturing on selection media supplemented with Benzylaminopurine (BAP) 8.9 μM, kanamycin (100 mgL-1) and cefotaxime (300 mgL-1). Cultures were incubated in the dark for 8 weeks at 26°C ± 1°C and then changed to a 16-hr photoperiod at 26 ± 1°C. Developed shoots were then transferred to elongation media (MS + 0.44 μM BAP) before rooting them on MS + 5.37 μM Naphthaleneacetic acid (NAA). Genetic transformation was identified through GUS staining and PCR using primers specific to the Gus gene. One transgenic plantlet out of two trials was obtained which was confirmed by PCR using Gus specific primers. The in vitro shoot regeneration and transformation protocol developed though viable had a very low transformation efficiency of 0.456%; there is thus a need for further investigation to optimize the developed protocol in order to increase its transformation efficiency with an ultimate goal of improving the agronomic value of Uganda's passion fruit.