Whole-Tissue Three-Dimensional Imaging of Rice at Single-Cell Resolution

The three-dimensional (3D) arrangement of cells in tissues provides an anatomical basis for analyzing physiological and biochemical aspects of plant and animal cellular development and function. In this study, we established a protocol for tissue clearing and 3D imaging in rice. Our protocol is based on three improvements: clearing with iTOMEI (clearing solution suitable for plants), developing microscopic conditions in which the Z step is optimized for 3D reconstruction, and optimizing cell-wall staining. Our protocol successfully 3D imaged rice shoot apical meristems, florets, and root apical meristems at cellular resolution throughout whole tissues. Using fluorescent reporters of auxin signaling in rice root tips, we also revealed the 3D distribution of auxin signaling events that are activated in the columella, quiescent center, and multiple rows of cells in the stele of the root apical meristem. Examination of cells with higher levels of auxin signaling revealed that only the central row of cells was connected to the quiescent center. Our method provides opportunities to observe the 3D arrangement of cells in rice tissues.

Augmenting in vitro Shoot Multiplication by Growth Regulators and Photoperiod in Vigna mungo L. (Hepper.)

An efficient in vitro protocol was developed for the mass multiplication of Vigna mungo vars. PU30 and PU31, an important legume crop through apical meristems and cotyledonary nodal explants. Both apical meristems and cotyledonary nodal explants were cultured on Murashige and Skoog (MS, 1962) medium fortified with 0.5 – 1.50 mg/L 6- benzyl aminopurine (BA) or Kinetin and 3 % (w/v) sucrose. The rate of multiplication was higher when the cultures were incubated under continuous light (24h) than the 16h photoperiod. The average number of multiple shoots per culture was enhanced from 2.43 to 5.46 in the case of var. PU30 and 3.12 to 5.82 in the case of var.PU31 within 4 weeks of culture under 24h photoperiod. The multiplication rate was enhanced till 5th subculture declined thereafter. Rooting was readily achieved upon transferring the shoots to half-strength MS basal semisolid medium supplemented with 0.1– 1.0 mg/L indole-3-butyric acid (IBA) and 2% (w/v) sucrose after 2 weeks of culture. The average number of roots per explants ranged from 3.12 to 5.76. About 80% of regenerated plantlets were hardened in the greenhouse and successfully established in the soil. There is no morphological variation among the regenerated plantlets. This protocol can be used for genetic transformation study for crop improvement of black gram.

An Update to the TraVA Database: Time Series of Capsella bursa-pastoris Shoot Apical Meristems during Transition to Flowering

Transition to flowering is a crucial part of plant life directly affecting the fitness of a plant. Time series of transcriptomes is a useful tool for the investigation of process dynamics and can be used for the identification of novel genes and gene networks involved in the process. We present a detailed time series of polyploid Capsella bursa-pastoris shoot apical meristems created with RNA-seq. The time series covers transition to flowering and can be used for thorough analysis of the process. To make the data easy to access, we uploaded them in our database Transcriptome Variation Analysis (TraVA), which provides a convenient depiction of the gene expression profiles, the differential expression analysis between the homeologs and quick data extraction.