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Composite plants for a composite plant: an efficient protocol for root studies in the sunflower using composite plants approach

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Abstract

Sunflower (Helianthus annuus L.) is an important oilseed crop in the world and the sunflower oil is prized for its exceptional quality and flavor. The recent availability of the sunflower genome can allow genome-wide characterization of genes and gene families. With plant transformation usually being the rate-limiting step for gene functional studies of sunflower, composite plants can alleviate this bottleneck. Composite plants, produced using Agrobacterium rhizogenes, are plants with transgenic roots and wild type shoots. Composite plants offer benefits over creating fully transgenic plants, namely time and cost. Here we outlined the critical steps and parameters for a protocol for the production of sunflower composite plants. We tested more than a dozen genotypes and three constitutive promoters to validate the utility and efficiency of this protocol. Moreover, functional gene characterization by overexpression and RNAi silencing of a root-related transcription factor, HaLBD16, further emphasize the value of the system in the sunflower studies. With the protocol developed here an experiment can be carried efficiently and in only 2 months. This procedure adds to the arsenal of approaches for the functional genetics/genomics in sunflower for characterization candidate genes involved in root development and stress adaptation.

Key message

Composite plants technique described here is fast and efficient approach for roots functional studies in sunflower.

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Acknowledgements

We would like to thank Prof. Gary Stacey (University of Missouri) for providing the K599 strain, and the Department of Biological Sciences for the funding: startup found (YSY), Redden grants (YSY), and Lewis Hanford Tiffany Botany Graduate Research Fund (TP).

Author information

TP and YSY designed the experiment, conducted the research, collected and analyzed data, and drafted the manuscript.

Correspondence to Yordan S. Yordanov.

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Communicated by Qiao-Chun Wang.

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Supplementary file1 (TIF 2996 kb)—Supplemental Fig. 1. Timeline for composite sunflower production. Pictures are the same as in the Fig.1 and are used to envision the main outcome for the stages in the procedure.

Supplementary file2 (TIF 4053 kb)—Supplemental Figure 2. β-Glucoronidase detection in roots. Unstained roots and stained (transgenic) roots of the same explant. Note, more developed roots are non-transgenic.

Supplementary file3 (TIF 3338 kb)—Supplemental Figure 3. Removal of putative non-transgenic roots after removal from the rockwool. Note: More developed adventitious roots are removed from composite plants. Only small putative transgenic roots originating from the teratoma remained.

Supplementary file4 (TIF 10457 kb)—Supplemental Figure 4. Morphology of transgenic roots. Transgenic roots was produced with wild type strain K599/pORE-E4-GusPlus, and histochemical GUS detected produces blue color roots. All roots shown are from plants grown in vermiculate for 10 days. Note, transgenic roots have similar morphology to non-transgenic roots (the three roots on the right).

Supplementary file5 (TIFF 8713 kb)—Supplemental Figure 5. Proportion of transgenic roots (blue) of the composite plant roots. Note, more than half of the roots are transgenic and have blue stanning, an indication of the GUS activity. Shown the same root as in the Fig. 1h.

Supplementary file6—Supplemental data 1 (XLSX 168 kb). Unprocessed data extracted form scanned roots via EZ-Rhizo program.

Supplementary file7 (DOCX 32 kb)—Supplemental Table 1. Characterization of transformation and rooting efficiencies of the sunflower varieties.

Supplementary file8 (DOCX 23 kb)—Supplemental Table 2. Correlation between rooting and transformation traits.

Supplementary file9 (DOCX 32 kb)—Supplemental text 1. Step-by-step protocol for the composite plants production in sunflower.

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Parks, T., Yordanov, Y.S. Composite plants for a composite plant: an efficient protocol for root studies in the sunflower using composite plants approach. Plant Cell Tiss Organ Cult 140, 647–659 (2020). https://doi.org/10.1007/s11240-019-01760-x

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Keywords

  • Agrobacterium rhizogenes
  • Helianthus annuus L.
  • Composite plants
  • Functional genetics
  • Roots transformation
  • Root architecture