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A simple improved-throughput xylem protoplast system for studying wood formation

Nature Protocols volume 9, pages 2194–2205 (2014)Cite this article

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Abstract

Isolated protoplasts serve as a transient expression system that is highly representative of stable transgenics in terms of transcriptome responses. They can also be used as a cellular system to study gene transactivation and nucleocytoplasmic protein trafficking. They are particularly useful for systems studies in which stable transgenics and mutants are unavailable. We present a protocol for the isolation and transfection of protoplasts from wood-forming tissue, the stem-differentiating xylem (SDX), in the model woody plant Populus trichocarpa. The method involves tissue preparation, digestion of SDX cell walls, protoplast isolation and DNA transfection. Our approach is markedly faster and provides better yields than previous protocols; small (milligrams)- to large (20 g)-scale SDX preparations can be achieved in ∼60 s, with isolation of protoplasts and their subsequent transfection taking ∼50 min. Up to ten different samples can be processed simultaneously in this time scale. Our protocol gives a high yield (∼2.5 × 107 protoplasts per g of SDX) of protoplasts sharing 96% transcriptome identity with intact SDX.

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Figure 1: Cell types in the stem of P. trichocarpa and the stem dipping approach for releasing protoplasts from wood-forming cells.
Figure 2: Workflows highlighting the difference between this protocol and previous protocols.
Figure 3: A healthy 3-month-old greenhouse-grown P. trichocarpa plant.
Figure 4: Ex vitro propagation of P. trichocarpa.
Figure 5: Transfection efficiency of SDX protoplasts.
Figure 6: RNA quality of SDX protoplasts.

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Acknowledgements

This work was supported by the Office of Science (Biological and Environmental Research), US Department of Energy grant DE-SC000691 (to V.L.C.). We also thank the North Carolina State University Jordan Family Distinguished Professor Endowment for support.

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Authors and Affiliations

  1. State Key Laboratory of Tree Genetics and Breeding, Northeast Forestry University, Harbin, China

    Ying-Chung Lin, Wei Li, Quanzi Li, Jack P Wang, Guan-Zheng Qu & Vincent L Chiang

  2. Department of Forestry and Environmental Resources, Forest Biotechnology Group, North Carolina State University, Raleigh, North Carolina, USA

    Ying-Chung Lin, Wei Li, Hao Chen, Quanzi Li, Rui Shi, Chien-Yuan Lin, Jack P Wang, Hsi-Chuan Chen, Ling Chuang, Ronald R Sederoff & Vincent L Chiang

  3. College of Forestry, Shandong Agricultural University, Taian, Shandong, China

    Quanzi Li

  4. Department of Forestry, National Chung Hsing University, Taichung, Taiwan

    Ying-Hsuan Sun

  5. Department of Forest Biomaterials, North Carolina State University, Raleigh, North Carolina, USA

    Vincent L Chiang

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  1. Ying-Chung Lin
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Contributions

Y.-C.L., W.L., Q.L., Y.-H.S. and V.L.C. designed the experiment. Y.-C.L., W.L., H.C., Q.L., R.S., C.-Y.L., J.P.W., H.-C.C. and L.C. performed the experiments. Y.-C.L., W.L., Q.L., Y.-H.S. and V.L.C. analyzed the data. R.R.S., L.C. and G.-Z.Q. edited the manuscript. Y.-C.L., W.L. and V.L.C. wrote the paper. All authors discussed and commented on the manuscript.

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Correspondence to Vincent L Chiang.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Table 1

Optimization of PEG-Mediated P. trichocarpa SDX Protoplast DNA Transfection (PDF 108 kb)

Footnotes: an represents the number of biological replicates of transfection efficiency test (see PROCEDURE) for each set of conditions. bThe optimized SDX protoplast transfection condition used for all PtrSND1-B1 overexpression experiments. Supplementary Table 1 was adapted from ref. 7 with permission from http://www.plantcell.org, Copyright American Society of Plant Biologists.

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Lin, YC., Li, W., Chen, H. et al. A simple improved-throughput xylem protoplast system for studying wood formation. Nat Protoc 9, 2194–2205 (2014). https://doi.org/10.1038/nprot.2014.147

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