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bioRxiv2025

Tagging and harvesting of developing rice spikelets to demarcate grain developmental stages

PremiseIn rice, grain is the major sink for nutrient storage and is a rich source of carbohydrates, seed storage proteins, lipids, minerals, and vitamins, making it a principal food source for over half of the worlds population. Grain development in rice generally spans over a month and has been divided into five stages, S1-S5, depending upon various morphological and physiological changes occurring in the developing seed. To completely understand the process, rice grain development needs to be examined at each stage. However, there are no detailed protocols available to demarcate grain developmental stages. Methods and ResultsThis protocol describes how to tag and harvest the developing rice spikelets. The process starts with anthesis and continues till seed maturation, demarcating the five stages of rice grain development. Seeds are collected, labelled, and stored for use in molecular experiments. Using this protocol, we were able to identify and distinguish different seed developmental stages as observed by histochemical screening. ConclusionThis is an easy, efficient, and cost-effective method. This protocol ensures the collection of the entire panel of rice seed developmental stages. The stored tissue can later be used for various molecular biology based experiments.

plant biology↗

bioRxiv2025

Hyphal induction in Candida albicans: Optimizing medium parameters to accelerate hyphal growth enhanced by GlcNAc

Hyphal formation is a critical virulence trait in Candida species, contributing significantly to host tissue invasion, immune evasion, and disease progression. The morphological transition from yeast to hyphal form is therefore a key target for antifungal intervention. However, conventional hyphal induction media are often complex, expensive, and poorly standardized. In this study, we developed a simplified and cost-effective medium (MF8) containing peptone (0.16%), dextrose (0.4%), and bovine serum albumin (BSA; 0.25%), which supported moderate hyphal growth under nutrient-limited conditions. The addition of N-acetylglucosamine (GlcNAc) markedly enhanced filamentation. Using a Design of Experiments (DOE) approach via JMP software, we evaluated the effects of GlcNAc and magnesium sulfate (MgSO) on hyphal induction. GlcNAc was identified as a significant enhancer (p < 0.05; R{superscript 2} = 0.26), while MgSO had no significant impact (p > 0.05). Under optimized conditions (30 mM GlcNAc, 1 mM MgSO), RT-qPCR analysis revealed strong upregulation of HWP1 (100-fold at 4 h; 85-fold at 6 h) and HGC1 (>6-fold; p < 0.05). A concurrent pH shift from alkaline to acidic during 1-6 hours correlated with hyphal induction and gene activation, suggesting that acidification may serve as an additional cue regulating morphogenesis. IMPORTANCECandida species are highly adaptable opportunistic fungi that persist in diverse host niches due to numerous virulence factors. While typically commensal, they can transition to pathogenic forms, with hyphal formation being a key virulence trait. This yeast-to-hypha switch facilitates tissue invasion, immune evasion, and disease progression, making it a major target in antifungal research. Conventional hyphal induction media are often complex and costly. Developing a simplified, cost-effective, and reproducible medium would enhance studies on morphogenesis and pathogenesis, support drug screening, and potentially reveal novel virulence mechanisms under alternative environmental cues.

microbiology↗