A simple and fast Agrobacterium-mediated transformation system for passion fruit KPF4 (Passiflora edulis f. edulis × Passiflora edulis f. flavicarpa)

Abstract

Background Passion fruit (Passiflora edulis Sims) is an important horticultural crop in the tropics and subtropics, where it has great commercial potential due to high demand for fresh edible fruits and processed juice as well as source of raw materials in cosmetic industries. Genetic engineering shows great potential in passion fruit improvement and can compensate for the limitations of conventional breeding. Despite the success achieved in genetic modification of few passion fruit varieties, transgenic passion fruit production is still difficult for farmer-preferred cultivars. Therefore, it is important to establish a simple and fast Agrobacterium-mediated cell transformation of commercial hybrid passion fruit KPF4 (Passiflora edulis f. edulis × Passiflora edulis f. flavicarpa). Results In the present study, we have developed a simple and fast Agrobacterium-mediated transformation system for hybrid passion fruit KPF4 using leaf disc explants. Factors affecting the rate of transient beta (β)-glucuronidase (gusA) expression and consequently transformation efficiency were optimized as follows: Agrobacterium cell density with an OD600 of 0.5, 30 min infection time, 3 days of co-cultivation duration and the incorporation of 200 µM acetosyringone into Agrobacterium infection suspension medium. Using the optimized conditions, transgenic plants of KPF4 were produced within 2 months with an average transformation efficiency of 0.67%. The β-glucuronidase (GUS) histochemical staining confirmed the expression and integration of an intron-containing gusA gene into transformed leaf discs and transgenic plant lines of KPF4. The presence of gusA gene in the transgenic plants was confirmed by polymerase chain reaction (PCR). The results confirmed that the gusA gene was efficiently integrated into the passion fruit genome. Conclusions The developed transformation protocol is simple and rapid and could be useful for functional genomic studies and transferring agronomically important traits into passion fruit hybrid KPF4. This study developed a method that can be used to transfer traits such as resistance to viral diseases, low fruit quality and short storage life. To the best of our knowledge, this is the first report on genetic transformation system for commercial passion fruit hybrid KPF4.

Establishment of a Novel and Efficient Agrobacterium-Mediated in Planta Transformation System for Passion Fruit (Passiflora edulis)

“…Additionally, Correa, Pinto [13] achieved a low transformation efficiency of 0.89% using hypocotyl explants of P. alata. Similar results were obtained by Tuhaise, Nakavuma [15] and Asande, Omwoyo [9] between 0.456% and 0.67% with the KPF4 variety of P. edulis f. flavicarpa using leaf disk as explant for in vitro regeneration and transformation technique. Using an in vitro somatic embryogenesis sonication-assisted transformation system, da Silva, Pinto [16] achieved a transformation efficiency of 28.3% in Passiflora cincinnata anthers.…”

“…The plant transformation system requires a suitable marker gene for the recovery of transgenic plants [48]. We used hygromycin and achieved an effective and reliable transformation efficiency, similar to that reported by Asande et al [9] for passion fruit and Zhang, Zhang [35] for Citrus maxima. Moreover, the bacterial noise for the selection of clean transgenic plants and to avoid concealed infections of Agrobacterium spp.…”

“…Genetic engineering made it possible Plants 2021, 10, 2459 2 of 14 to develop new crop varieties with desired traits [5], such as better pathogen resistance, higher productivity, nutritional value, and water use efficiency [6][7][8]. However, due to high ploidy, self-incompatibility, low genetic variability, and lack of disease resistant genes of the existing passion fruit varieties, the development of new and improved varieties is slow and costly [9]. The first transgenic passion fruit plants were generated in 1994 using Agrobacterium-mediated transformation [10].…”

“…Additionally, Bunnag and Chamnanpon [14] reported low in vitro regeneration and transformation rates for purple passion fruit with A. tumefaciens. Tuhaise, Nakavuma [15] established an in vitro transformation protocol for Uganda's yellow passion fruit and Asande, Omwoyo [9] generated transgenic hybrid passion fruit KPF4 with efficiencies below 1%. Only recently, da Silva, Pinto [16] established a sonication-assisted A.-mediated transformation protocol for anthers of wild passion fruit (P. cincinnata Mast.)…”

Genome-wide identification and expression analysis of the SWEET gene family in daylily (Hemerocallis fulva) and functional analysis of HfSWEET17 in response to cold stress

“…The constructed binary vector was transformed into GV3101. The generation of transgenic tobacco was performed following the leaf disc transformation method [ 67 ].…”