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The gene TaWOX5 overcomes genotype dependency in wheat genetic transformation

Nature Plants volume 8, pages 110–117 (2022)Cite this article

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An Author Correction to this article was published on 30 May 2022

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Abstract

Although great progress has been achieved regarding wheat genetic transformation technology in the past decade1,2,3, genotype dependency, the most impactful factor in wheat genetic transformation, currently limits the capacity for wheat improvement by transgenic integration and genome-editing approaches. The application of regeneration-related genes during in vitro culture could potentially contribute to enhancement of plant transformation efficiency4,5,6,7,8,9,10,11. In the present study, we found that overexpression of the wheat gene TaWOX5 from the WUSCHEL family dramatically increases transformation efficiency with less genotype dependency than other methods. The expression of TaWOX5 in wheat calli prohibited neither shoot differentiation nor root development. Moreover, successfully transformed transgenic wheat plants can clearly be recognized based on a visible botanic phenotype, relatively wider flag leaves. Application of TaWOX5 improved wheat immature embryo transformation and regeneration. The use of TaWOX5 in improvement of transformation efficiency also showed promising results in Triticum monococcum, triticale, rye, barley and maize.

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Fig. 1: Effects of TaWOX5 and TaWUS on transformation efficiency of selected wheat genotypes.
Fig. 2: Shoot regeneration in different wheat genotypes at increased frequency using TaWOX5.
Fig. 3: Regeneration improvement in immature embryos of poor physiological status by application of TaWOX5 in two wheat genotypes.

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Data availability

Accession numbers and gene names are available from the phylogenetic tree in Extended Data Fig. 1. The accession numbers of genes identified in this study are available in Supplementary Table 1, and their sequences are provided in the Supplementary sequence file. The accession number of pWMB111 is MZ458107. Raw data for experiments are available in Supplementary Tables 2 and 3. Transgenic lines and plasmids generated are available from the corresponding authors on request. Source data are provided with this paper.

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Acknowledgements

We thank W. Xiao at Saint Louis University, USA and H. Li at the Institute of Crop Sciences, Chinese Academy of Agricultural Sciences for critical revisions of this manuscript. This research was financially supported by grants from the National Natural Science Foundation of China (no. 31971946) to K.W., the Science and Technology Department of Ningxia in China (no. 2019BBF02020) to X.Y. and the Agricultural Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences (nos. S2021ZD03 and 2060302-2-19) to K.W. and X.Y..

Author information

Author notes

  1. These authors contributed equally: Lei Shi, Xiaona Liang.

Authors and Affiliations

  1. Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, P. R. China

    Ke Wang, Lei Shi, Xiaona Liang, Pei Zhao, Wanxin Wang, Lipu Du & Xingguo Ye

  2. College of Life Science, Capital Normal University, Beijing, P. R. China

    Junxian Liu & Yanan Chang

  3. Plant Innovation Center, Japan Tobacco Inc., Iwata, Japan

    Yukoh Hiei, Chizu Yanagihara & Yuji Ishida

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  1. Ke Wang
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Contributions

K.W. contributed to funding acquisition, experimental design, vector construction, wheat and barley transformation, data analysis and manuscript writing. L.S. contributed to gene identification, vector construction and transgenic detection. X.L. performed medium modification and wheat transformation. P.Z. was involved in gene identification and sequence analysis. W.W. was involved in barley transformation and manuscript writing. J.L. performed transformation of T. monococcum and rye. Y.C. performed transformation of triticale. Y.H. performed transformation of maize. C.Y. contributed to vector construction. L.D. contributed material management and medium preparation. Y.I. contributed to experimental design, wheat transformation and manuscript editing. X.Y. conceived the study, supervised experiments, conducted formal analysis and contributed to project administration, funding acquisition and manuscript editing.

Corresponding authors

Correspondence to Ke Wang, Yuji Ishida or Xingguo Ye.

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Competing interests

K.W., X.Y. and L.D. (all ICS-CAAS) are co-inventors in Chinese patent application no. ZL201710422896.6. X.Y., K.W., L.S. and L.D. (all ICS-CAAS) and Y.I. and C.Y. (both JT) are co-inventors in international patent application no. PCT/CN2018/090239, in which ICS-CAAS and JT had shared ownership; the share of the latter was assigned to Kaneka Corporation, a Japanese chemical company, on 29 January 2021. The remaining authors declare no competing interests.

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Nature Plants thanks Sadiye Hayta and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Phylogenetic relationships among TaWOX5 and TaWUS proteins from wheat, and WOX proteins from Arabidopsis.

The phylogenetic tree was constructed based on the sequences of TaWOX5 and TaWUS proteins in wheat and WOX proteins in Arabidopsis in MEGA X using the neighbor-joining approach with 1,000 bootstrap replicates. Scale plate and legend in left display tree scale and bootstrap value. AtWUS: NP_565429; AtWOX1: NP_188428; AtWOX2: NP_200742; AtWOX3: NP_180429; AtWOX4: NP_175145; AtWOX5: NP_187735; AtWOX6: NP_565263; AtWOX7: NP_196196; AtWOX8: NP_199410; AtWOX9: NP_180944; AtWOX10: NP_173494; AtWOX11: NP_187016; AtWOX12: NP_197283; AtWOX13: NP_195280; AtWOX14: NP_173493. TaWOX5: MN412513; TaWUS-A: MW452946; TaWUS-B: MW452947; TaWUS-D: MW452945.

Extended Data Fig. 2 Shoot regeneration of the immature embryos of different wheat genotypes promoted by the TaWOX5 gene.

a: Shoot regeneration of the wheat embryos transformed with control vectors. b: Shoot regeneration of the wheat embryos transformed with TaWOX5 gene containing vector. The calli on plates were some overcrowding.

Extended Data Fig. 3 Detection of transgenic wheat plants by QuickStix Kit and PCR.

a: QuickStix Kit assay for the Bar protein; 1-21: transgenic plants; 22: wild-type Fielder. b: PCR detection for Bar gene, this testing experiment being repeated at least three times with similar results; 1: plasmid of TaWOX5 vector; 2: wild-type Fielder; 3-24: transgenic plants.

Source data

Extended Data Fig. 4

Comparison of the transient infection efficiency of different wheat varieties by expressing anthocyanin biosynthesis genes ZmR and ZmC1 as visible markers.

Extended Data Fig. 5 Normal growth of the regeneration shoots and roots derived from a transformed immature embryo of Fielder using the TaWOX5 gene in three experimental replicates.

a: The growth status of regeneration shoots; b: the growth status of the transgenic plants with healthy shoots and roots.

Extended Data Fig. 6

The plasmids map of pWMB111-TaWOX5 and TaWOX5- SpCas9-TaQ.

Supplementary information

Supplementary Information

Supplementary Tables 1–5 and the sequences of TaWOX5 and TaWUS used in this study.

Reporting Summary

Source data

Source Data Fig. 1

Statistical source data.

Source Data Extended Data Fig. 3

Unprocessed gels.

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Wang, K., Shi, L., Liang, X. et al. The gene TaWOX5 overcomes genotype dependency in wheat genetic transformation. Nat. Plants 8, 110–117 (2022). https://doi.org/10.1038/s41477-021-01085-8

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