Multiplexed gene editing in citrus by using a multi-intron containing Cas9 gene.

Several expression systems have been developed in clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR/Cas9) framework allowing for gene editing of disease-associated genes across diverse citrus varieties. In this study, we present a new approach employing a multi-intron containing Cas9 gene plus multiple gRNAs separated with tRNA sequences to target the phytoene desaturase gene in both 'Carrizo' citrange and 'Duncan' grapefruit. Notably, using this unified vector significantly boosted editing efficiency in both citrus varieties, showcasing mutations in all three designated targets. The implementation of this multiplex gene editing system with a multi-intron-containing Cas9 plus a gRNA-tRNA array demonstrates a promising avenue for efficient citrus genome editing, equipping us with potent tools in the ongoing battle against several diseases such as canker and huanglongbing.

Development of an Antioxidant-Rich Sugar-Free Plantain Candy and Assessment of Its Shelf Life in a Flexible Laminate.

Research background: Candy is a popular confection worldwide, and it would be beneficial to society if it were converted into a source of antioxidant molecules to eliminate its adverse health effects. The amount of antioxidants available even in fruit candies is questionable due to the high thermal processing losses they undergo and the presence of various food additives. Plantains (Musa paradisiaca) are less known as good sources of biotherapeutic antioxidants, namely l-tryptophan, serotonin and melatonin, and consumption of this highly nutritious fruit is limited to underdeveloped and developing countries. The objectives of this study are: to develop a functional antioxidant-rich sugar-free plantain-based candy with valuable contents of the mentioned biomolecules in synergy; and to ensure its extended shelf life without compromising its physicochemical properties and functionality by wrapping it with a suitable packaging laminate. Experimental approach: To accomplish the first objective, lyophilized plantain powder, sorbitol and mannitol were used as base materials with minimal additives under minimal processing conditions to reduce processing loss. Sensory, proximate, physicochemical and phytochemical properties, including the antioxidant synergy among the mentioned biomolecules of the developed candies were evaluated. For the second objective, the candies were enclosed in two different flexible packaging laminates and the optimal packaging was determined based on the microbiological safety and sensory appeal of the wrapped candies. Subsequently, the above-mentioned properties of the packaged (in the most suitable laminate) candies were evaluated at regular time intervals during storage for assessment of their shelf life. Results and conclusions: The candy had a characteristic flavour of plantain, uniform dark brown colour, rich mouthfeel, pleasant aroma, moderately hard texture and moderate sweetness, along with high antioxidant activity and considerable content of l-tryptophan, serotonin and melatonin (present as a synergistic consortium). During storage of the packaged candy under ambient conditions, it remained microbiologically safe for up to 56 days, and also maintained sensory attributes, antioxidant activity and synergy compared to the control candy. Novelty and scientific contribution: This newly developed semi-hard sugar-free candy with high antioxidant content, containing three important antioxidants, namely l-tryptophan, serotonin and melatonin, could be a good source of biotherapeutic molecules and a substitute for commercial candies consumed globally.