Comparative expression profiling of candidate genes in the premeiotic, meiotic, and post -meiotic rice floral tissues developed in Vivo and in vitro

Abstract

Rice (Oryza sativa L.), a vital staple crop, sustains the food security of over half the global population. Yet, the rising global demand for rice and shrinking arable land necessitate innovative strategies to boost productivity and resilience. Among these, investigating the genetic mechanisms underpinning reproductive processes, specifically meiosis, holds great promise for improving breeding techniques. This study focuses on analyzing the gene expression dynamics of critical candidate genes involved in meiosis across premeiotic, meiotic, and post-meiotic stages in rice florets, developed under both in vivo and in vitro conditions. Using the japonica rice cultivar Nipponbare as a model, the research aims to compare meiosis in vivo and in vitro, while assessing the feasibility of inducing meiosis in somatic cells under controlled laboratory conditions. This research employs advanced tissue culture techniques combined with molecular biology approaches to evaluate gene expression patterns and rates. Many genes like OsMEL1, OsSPO11-1, SPO11-2, OsMIL1, OsAUX1, OsMSP1, OsGAMYB1, OsARF2, OsDMC1, and OsMADS1 have been reported to play a significant role in meiosis. Key candidate genes, such as OsDMC1, OsSPO11-1, OsMADS1, OsAUX1, OsMSP1, and OsARF2, were selected for their known roles in meiotic progression and gamete formation. Media compositions were designed and optimized to mimic the microenvironment required for meiosis in pre-meiosis florets, utilizing plant hormones like auxins, cytokinins, and gibberellins. Fourteen hormonal combinations were tested to identify conditions that support meiosis induction in rice callus and pre-meiosis florets. High-quality RNA was extracted from tissues cultured under these conditions, and quantitative real-time PCR was used to profile the expression of the selected genes. Results revealed significant similarities in gene expression patterns between in vivo and in vitro systems, demonstrating the potential of in vitro models to replicate natural meiosis. Fourteen growth regulator combinations were tested using ½ MS media to support meiotic progression in rice pre-meiosis florets and callus cultures. Of these, eight combinations consistently supported growth. These included media with the following hormone combinations: 2, 4-D (2 mg/L), IBA + 6-BAP (2+1 mg/L), 6-BAP (4 mg/L), IBA (1 mg/L), 2, 4-D + IBA (2+1 mg/L), 2, 4-D (4 mg/L), 2, 4-D + 6-BAP (2+1 mg/L), and 2, 4-D + GA3(2+1 mg/L). The study aimed to support meiotic progression in vitro and evaluate the expression of six candidate genes in pre-meiosis florets. In vitro gene expression analysis revealed expected patterns for most genes in seven of the eight media, although OsMSP1 did not follow the expected trend. While expression levels varied across media, OsDMC1, OsSPO11-1, OsMADS1, OsAUX1, and OsARF2 showed significant upregulation as anticipated. For OsDMC1, the media IBA + 6-BAP (2+1 mg/L & ½ MS) showed the expected progressive fold change, with upregulation observed in both treated florets and callus compared to pre-meiotic and meiotic controls. For OsSPO11-1, the same media combination (IBA + 6-BAP) exhibited progressive fold change in treated callus, while 2, 4-D + 6-BAP (2+1 mg/L & ½ MS) also demonstrated expected results in both treated florets and callus. For OsARF2, IBA (1 mg/L & ½ MS) and 2, 4-D (4 mg/L & ½ MS) achieved the expected progressive fold change in treated florets, although callus samples did not align with expectations. For OsMADS1, 2, 4-D (2 mg/L & ½ MS) and IBA + 6-BAP (2+1 mg/L & ½ MS) showed progressive fold change in treated florets, while callus results varied. For OsAUX1, 6-BAP (4 mg/L & ½ MS) displayed a fold change similar to pre-meiotic and meiotic controls in florets, with upregulation observed in treated samples but not as expected in callus. For OsMSP1, no media demonstrated the expected progressive fold change in either treated florets or callus. Overall, the media combination IBA + 6-BAP (2+1 mg/L & ½ MS) consistently showed potential, delivering expected progressive fold change across multiple genes in both florets and callus, making it the most promising for gene expression regulation and reprogramming. The findings suggest that specific culture conditions can elicit meiosis-like gene expression patterns and potentially induce meiotic progression in somatic cells. However, further research is necessary to confirm meiotic induction and rule out aberrant cell behaviour under these conditions.