Passion Fruit Woodiness Disease in Coastal Lowlands of Kenya and Genetic Transformation System of Farmer Preferred Passion Fruit (Passiflora Edulis Sims)

Abstract

Passion fruit (Passiflora edulis [Sims]) is an important economic crop grown for both export and domestic market world-wide. In Kenya, passion fruit productivity is low due to both biotic and abiotic constraints. Passion fruit woodiness disease (PWD) complex is the most damaging viral disease causing yield losses of up to 100%. The most effective way to manage PWD is by using resistant cultivars. Conventional breeding of passion fruit for resistance against PWD is hampered by sexual barriers, narrow genetic variability, high ploidy levels and long generation cycles. Thus there is need to incorporate alternative approaches such as genetic transformation in passion fruit improvement. The objectives of this study were to determine the occurrence of passion fruit woodiness disease in selected Counties at the Coastal lowlands of Kenya and develop regeneration and transformation systems for farmer preferred passion fruit genotypes. A survey of PWD was undertaken in major passion fruit growing areas in Kwale and Kilifi Counties. Evaluation of five passion fruit genotypes for resistance to PWD was carried out under greenhouse and field conditions. For shoot induction and multiplication from leaf and nodal explants, respectively, Murashige and skoog (MS) medium supplemented with benzylamino purine (BAP) ranging from 1.0 - 3.0 mg L-1 as well as 2.0 mg L-1 BAP in combination with 0.5 mg L-1 kinetin were tested. The effect of putrescine on root induction was tested. The genetic fidelity of both in vitro regenerated and putrescine treated plants was established using sequence-related amplified polymorphism markers. Callus induction and somatic embryogenesis was carried out using leaf explants and immature seeds on MS supplemented with 0.5 – 16.0 mg L-1 2, 4- dichlorophenoxyacetic acid (2, 4-D) and 1.0 mg L-1 thidiazuron (TDZ). Data on disease severity was used to compute area under disease progress curve (AUDPC). The data was subjected to analysis of variance and means were separated according to Tukey‟s Honest Significant Difference test at 5% confidence level. The highest disease incidence of 59.16% and 51.43% was observed at Kilifi and Kwale Counties, respectively. A significant difference (p < 0.05) in symptom severity was observed within the tested genotypes with purple and banana passion fruits presenting the highest and lowest AUDPC values, respectively, both under greenhouse and field conditions. Efficient induction and multiplication of shoots was achieved on MS supplemented with 2 mg L-1 and 3 mg L-1 BAP, respectively. The homogenous banding pattern based on SRAP analysis confirmed genetic uniformity of in vitro regenerated and macropropagated plants. Exogenous application of putrescine induced root formation on nodal explants. The conditions for optimum gus expression in the histochemical assays were optical bacterial density of 0.5, 30 min infection time, 200 μM acetosyringone and 3 days co-cultivation period. The regenerated putative transgenic lines showed presence of transgene by PCR. The in vitro regeneration system developed can be utilized for mass clonal propagation of passion fruit. The Agrobacterium mediated transformation system can be used to introduce genes which encode beneficial traits such as resistance to PWD and other agronomically important traits into KPF4 variety.