Abstract
Key message
A detailed, step-by-step protocol for isolation of rice gametes for transcriptional profiling, with a general workflow that includes controls for RNA contamination from surrounding cells and tissues is presented.
Abstract
Characterization of the transcriptome and other -omics studies of flowering plant gametes are challenging as a consequence of the small sizes and relative inaccessibility of these cells. Collecting such poorly represented cells is also complicated by potential contamination from surrounding sporophytic, adjacent gametophytic tissues and difficulties in extracting high-quality intact cells. Here we present detailed, step-by-step procedures for collecting intact, unfixed rice (Oryza sativa) egg cells and sperm cells without enzymatic treatments. In addition, we also present a general workflow for assessing sample purity by RT-PCR, using primers specific for marker genes preferentially expressed in surrounding cells and tissues. These protocols should facilitate future studies of genome-scale characterization of gametes in this important model crop.
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Notes
Care should be taken to identify mature florets that are staged correctly. In mature florets, stamens should occupy most of the floret prior to anthesis.
For RT-PCR, we used MyTaq Red Mix (Bioline BIO-25043). All primers listed in Supplementary Table 1 performed using an annealing temperature of 59 °C.
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Acknowledgements
We thank Imtiyaz Khanday, Jonathan Gent, Sarah Anderson and Daniel Jones for helpful advice for optimizing the experimental methods. We thank Debra Skinner for assistance in artwork. This research was funded by the National Science Foundation (Award No. IOS-1547760) and the USDA Agricultural Experiment Station (Project No. CA-D-XXX-6973-H).
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Communicated by Thomas Dresselhaus.
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A contribution to the special issue ‘Cellular Omics Methods in Plant Reproduction Research’.
Scott D. Russell and Venkatesan Sundaresan: Senior authors.
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Li, C., Xu, H., Russell, S.D. et al. Step-by-step protocols for rice gamete isolation. Plant Reprod 32, 5–13 (2019). https://doi.org/10.1007/s00497-019-00363-y
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DOI: https://doi.org/10.1007/s00497-019-00363-y