Banana (Musa acuminata) is widely cultivated in the tropical and subtropical regions of the world and produced in over 135 different countries (Drenth and Kema, 2021). Malaysia has a stable growth of banana plantations recording banana as the second-highest fruit crop produced and ranking fourth in overall agricultural production, following oil palm, rubber, and paddy (Tan et al., 2019). There are more than 100 cultivars of banana in Malaysia, with ‘Pisang Berangan’ being the most widely grown local banana cultivar (Mohamad et al., 2012). Banana plantations give emphasis on the fruit for economic returns, while leaves are used for food packaging (Nguyen et al., 2020) or composting (Padam et al., 2014). For each tonne of banana plants the leaf biomass is around 480 kg (Fernandes et al., 2013), representing a large volume of underutilised biomass that has the potential to be used as a platform for the expression of recombinant proteins, which in return can increase the value for farmers and boost the economies of low- and middle-income countries, the primary producers of this crop.

In the past decades, a variety of expression systems, such as bacterial cells, fungal cells, mammalian cell culture, insects, animals, and plants, have been explored to generate recombinant proteins (Schillberg et al., 2019). The commercialisation of recombinant proteins is still dominated by mammalian, yeast, and bacterial cells, but these traditional cell-based expression systems have limitations in terms of high costs due to the demand for a high level of sophisticated infrastructure, and safety concerns related to the risk of contamination with pathogens (Gharelo et al., 2016). Plant-made recombinant systems have been proposed as alternatives for protein production as they can produce proteins rapidly at a large scale and offer lower production costs (McNulty et al., 2020; Ridgley et al., 2023). Plant systems pose a lower risk of environmental contamination and are free from human pathogens (Mohammadinejad et al., 2019). The use of plant systems also enables the reproduction of proteins in batches that comply with current good manufacturing practices (Ma et al., 2015).

The transient expression of recombinant proteins in plants has long been studied by monitoring reporter genes such as beta-glucuronidase (GUS) and Green Fluorescent Protein (GFP) mainly to study the mechanisms of expression of recombinant proteins in these systems (Jefferson et al., 1987, Shimomura et al., 1962). Lately, the most common products in plant expression systems include recombinant antibodies, antibody fragments, and fusion proteins (Schillberg et al., 2019) and recent promising examples include plant-made vaccines against COVID-19 (COVIFENZ® COVID-19 vaccine) produced in Nicotiana benthamiana which has been approved for use in Canada (Su et al., 2023). Together, these indicate that pharmaceutical molecular farming is on the rise and rapidly maturing as a technology. (Lim et al., 2018) demonstrated that the recombinant anti-toxoplasma single-chain variable fragment (scFv) antibody expressed in Nicotiana benthamiana plants was able to inhibit the invasion of human fibroblasts by the parasite Toxoplasma gondii. It is estimated that about one third of the world population has been exposed to Toxoplasma gondii (Djurković-Djaković et al. 2019). Toxoplasma gondii is a parasitic protozoan that causes Toxoplasmosis disease capable of infecting warm blooded animals including humans (Nayeri et al., 2021). Toxoplasmosis, usually asymptomatic or mild in healthy individuals, poses a significant threat to immunocompromised individuals which may lead to encephalitis in patients with HIV, and affect pregnant women resulting in severe neurological and developmental issues for the unborn child (Daher et al., 2021). Given the prevalence of Toxoplasma gondii and the potential risks associated with infection, it is crucial to prepare antibodies against this pathogen for diagnostic and therapeutic purposes. Hence, anti-toxoplasma scFv antibody is a highly valuable protein candidate for efficient recombinant expression systems which could also be the solution for the large-scale production of the antibodies.

Previous works investigated gene expression in banana plants mainly using embryogenic cell suspensions (Schenk et al., 1999), single meristematic bud (Sreeramanan et al., 2006), and apical meristem (Elayabalan et al., 2017). The use of detached leaves for transient gene expression has been reported for cowpea (Juranić et al. 2020), citrus (Acanda et al., 2021), and avocado (Salazar-González et al., 2023). However, to our knowledge, no prior study has reported the exploitation of detached banana leaves, a relatively large and abundant leaf biomass in many tropical and sub-tropical countries, for transient recombinant protein expression.

Hence, in the current study, we established a transient transformation protocol for banana leaves using Agrobacterium-mediated vacuum infiltration to express GUS and GFP, then further investigated the expression of an anti-toxoplasma scFvTG130 antibody. This study is the first report on the use of detached leaves of banana (Musa acuminata cv. Berangan) as a platform for transient protein expression.