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

Abstract

Passion fruit (Passiflora edulis) is an important fruit crop with high economic value. Genetic engineering plays an important role in crop improvement with desired traits and gene functional studies. The lack of a simple, efficient, and stable transformation system for passion fruit has greatly limited gene functional studies. In this study, a simple and efficient Agrobacterium-mediated in planta transformation system for passion fruit was established, using Agrobacterium virulent strain EHA105 harboring the binary vectors pCAMBIA1301 and pCAMBIA1302 with GUS and GFP reporter genes. The system requires less time and labor costs than conventional transformation systems, and no additional phytohormones and sterile conditions are required. Regeneration efficiency of 86% and transformation efficiency of 29% were achieved, when the wounds were wrapped with Parafilm and the plants were kept in darkness for 15 days. Approximately 75% of the regenerated plants had a single shoot and 26% multiple shoots. The transformation was confirmed at the DNA and RNA levels as well as by GUS staining and GFP fluorescent measurements. The developed protocol will contribute to the genetic improvement of passion fruit breeding.

Amenability of an Agrobacterium tumefaciens-mediated shoot apical meristem-targeted in planta transformation strategy in Mango (Mangifera indica L.)

“…Diffused light has positive effect on the shoot regeneration and dark incubation was always found useful in regeneration and transformation experiments. 28 , 29 , 53 , 54 Seedlings took 4–5 weeks for recovery during which, they were regularly irrigated with sterile-double distilled water and Hoagland solution ( Fig. 2g ).…”

“… 58 In our experiment, GFP and GUS were used as screenable marker genes and similar studies have been reported in mango, 6 pummelo, 28 and passion fruit. 29 …”

“…Similar kind of molecular characterization in the chimeric plants of other perennial tree species have been reported in pummelo, 28 and passion fruit. 29 …”

“…Tissue culture-independent in planta transformation has been demonstrated in many crops such as Brassica rapa , 17 B. napus , 18 B. campestris , 19 Arabidopsis thaliana , 20 Medicago truncatula , 21 Raphanus sativus , 22 Solanum lycopersicum , 23 Glycine max , 24 Melilotus alba , 25 Zea mays , 26 Oryza sativa , 27 Citrus maxima 28 and Passiflora edulis . 29 Several in planta transformation strategies have been developed using different tissues, i.e ., seed, epicotyl, shoot apical meristem, flower, fruit etc. 30 …”

Virus-induced gene silencing (VIGS) as a tool for functional genetic analysis in passion fruit plants

“…Potyvirus represents the largest group of plant RNA viruses and comprises a positive-sense single-stranded RNA genome encapsidated by viral coat protein. The potyvirus genome encodes a large polyprotein that is further cleaved into ten mature viral proteins by its own viral proteases (Revers and García, 2015;Cui and Wang, 2019;Dai et al, 2020;Yang et al, 2021). Although potyviruses have been broadly engineered into protein expression vectors, such as tobacco etch virus (TEV), potato virus A (PVA), turnip mosaic virus (TuMV), plum pox virus (PPV), sugarcane mosaic virus (SCMV), wheat streak mosaic virus (WSMV) and zucchini yellow mosaic virus (ZYMV) (Xie et al, 2021), very few potyvirus-based VIGS vector have been reported.…”