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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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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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  1. Albourie J-M, Tourvieille J, De Labrouhe DT (1998) Resistance to metalaxyl in isolates of the sunflower pathogen Plasmopara halstedii. Eur J Plant Pathol 104(3):235–242.

  2. Ambrosini A, Beneduzi A, Stefanski T, Pinheiro FG, Vargas LK, Passaglia LMP (2012) Screening of plant growth promoting Rhizobacteria isolated from sunflower (Helianthus annuus L.). Plant Soil 356(1):245–264.

  3. Arai-Sanoh Y, Takai T, Yoshinaga S, Nakano H, Kojima M, Sakakibara H, Kondo M, Uga Y (2014) Deep rooting conferred by DEEPER ROOTING 1 enhances rice yield in paddy fields. Sci Rep 4(1):5563.

  4. Arias DM, Rieseberg LH (1995) Genetic relationships among domesticated and wild sunflowers (Helianthus annuus, Asteraceae). Econ Bot 49(3):239–248

  5. Badouin H, Gouzy J, Grassa CJ, Murat F, Staton SE, Cottret L, Lelandais-Brière C, Owens GL, Carrère S, Mayjonade B, Legrand L, Gill N, Kane NC, Bowers JE, Hubner S, Bellec A, Bérard A, Bergès H, Blanchet N, Boniface M-C, Brunel D, Catrice O, Chaidir N, Claudel C, Donnadieu C, Faraut T, Fievet G, Helmstetter N, King M, Knapp SJ, Lai Z, Le Paslier M-C, Lippi Y, Lorenzon L, Mandel JR, Marage G, Marchand G, Marquand E, Bret-Mestries E, Morien E, Nambeesan S, Nguyen T, Pegot-Espagnet P, Pouilly N, Raftis F, Sallet E, Schiex T, Thomas J, Vandecasteele C, Varès D, Vear F, Vautrin S, Crespi M, Mangin B, Burke JM, Salse J, Muños S, Vincourt P, Rieseberg LH, Langlade NB (2017) The sunflower genome provides insights into oil metabolism, flowering and Asterid evolution. Nature 546(7656):148–152.

  6. Benzle KA, Finer KR, Marty D, McHale LK, Goodner BW, Taylor CG, Finer JJ (2015) Isolation and characterization of novel Agrobacterium strains for soybean and sunflower transformation. Plant Cell, Tissue and Organ Cult 121(1):71–81.

  7. Boyer JS (1982) Plant productivity and environment. Science 218(4571):443–448.

  8. Chen YR, Yordanov YS, Ma C, Strauss S, Busov VB (2013) DR5 as a reporter system to study auxin response in Populus. Plant Cell Rep 32(3):453–463

  9. Collier R, Fuchs B, Walter N, Kevin Lutke W, Taylor CG (2005) Ex vitro composite plants: an inexpensive, rapid method for root biology. Plant J 43(3):449–457.

  10. Combard A, Brevet J, Borowski D, Cam K, Tempé J (1987) Physical map of the T-DNA region of Agrobacterium rhizogenes strain NCPPB2659. Plasmid 18(1):70–75.

  11. Coutu C, Brandle J, Brown D, Brown K, Miki B, Simmonds J, Hegedus DD (2007) pORE: a modular binary vector series suited for both monocot and dicot plant transformation. Transgenic Res 16(6):771–781.

  12. Curtis MD, Grossniklaus U (2003) A gateway cloning vector set for high-throughput functional analysis of genes in planta. Plant Physiol 133(2):462–469

  13. Davey MR, Jan M (2010) Sunflower (Helianthus annuus L.): genetic improvement using conventional and in vitro technologies. J Crop Improv 24(4):349–391.

  14. Estrada-Navarrete G, Alvarado-Affantranger X, Olivares J-E, Díaz-Camino C, Santana O, Murillo E, Guillén G, Sánchez-Guevara N, Acosta J, Quinto C, Li D, Gresshoff PM, Sánchez F (2006) Agrobacterium rhizogenes transformation of the Phaseolus spp.: A tool for functional genomics. Mol Plant Microbe Interact 19(12):1385–1393.

  15. Everett NP, Robinson KEP, Mascarenhas D (1987) Genetic engineering of sunflower (Helianthus Annuus L.). Bio/Technology 5(11):1201–1204.

  16. Fabijan D, Yeung E, Mukherjee I, Reid DM (1981) Adventitious rooting in hypocotyls of sunflower (Helianthus annuus) seedlings. Physiol Plant 53(4):578–588.

  17. Fan M, Xu C, Xu K, Hu Y (2012) LATERAL ORGAN BOUNDARIES DOMAIN transcription factors direct callus formation in Arabidopsis regeneration. Cell Res 22(7):1169–1180.

  18. Feng Z, Zhu J, Du X, Cui X (2012) Effects of three auxin-inducible LBD members on lateral root formation in Arabidopsis thaliana. Planta 236(4):1227–1237.

  19. Fick GN, Zimmer DE (1974) Fertility restoration in confectionery sunflowers 1. Crop Sci 14(4):603–604.

  20. Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50(1):151–158.

  21. Geldner N, Dénervaud-Tendon V, Hyman DL, Mayer U, Stierhof Y-D, Chory J (2009) Rapid, combinatorial analysis of membrane compartments in intact plants with a multicolor marker set. Plant J 59(1):169–178.

  22. Georgiev MI, Agostini E, Ludwig-Müller J, Xu J (2012) Genetically transformed roots: from plant disease to biotechnological resource. Trends Biotechnol 30(10):528–537.

  23. Goh T, Joi S, Mimura T, Fukaki H (2012) The establishment of asymmetry in Arabidopsis lateral root founder cells is regulated by LBD16/ASL18 and related LBD/ASL proteins. Development 139(5):883–893.

  24. Goh T, Toyokura K, Yamaguchi N, Okamoto Y, Uehara T, Kaneko S, Takebayashi Y, Kasahara H, Ikeyama Y, Okushima Y, Nakajima K, Mimura T, Tasaka M, Fukaki H (2019) Lateral root initiation requires the sequential induction of transcription factors LBD16 and PUCHI in Arabidopsis thaliana. New Phytol 224(2):749–760.

  25. Guivarc'h A, Caissard JC, Brown S, Marie D, Dewitte W, Onckelen HV, Chriqui D (1993) Localization of target cells and improvement of Agrobacterium-mediated transformation efficiency by direct acetosyringone pretreatment of carrot root discs. Protoplasma 174(1–2):10–18.

  26. Han KH, Meilan R, Ma C, Strauss SH (2000) An Agrobacterium tumefaciens transformation protocol effective on a variety of cottonwood hybrids (genus Populus). Plant Cell Rep 19(3):315–320

  27. Hansen J, Jørgensen J-E, Stougaard J, Marcker KA (1989) Hairy roots—a short cut to transgenic root nodules. Plant Cell Rep 8(1):12–15.

  28. Hebbar P, Berge O, Heulin T, Singh SP (1991) Bacterial antagonists of sunflower (Helianthus annuus L.) fungal pathogens. Plant Soil 133(1):131–140.

  29. Hewezi T, Mouzeyar S, Thion L, Rickauer M, Alibert G, Nicolas P, Kallerhoff J (2006) Antisense expression of a NBS-LRR sequence in sunflower (Helianthus annuus L.) and tobacco (Nicotiana tabacum L.): evidence for a dual role in plant development and fungal resistance. Transgenic Res 15(2):165–180.

  30. Hiratsu K, Matsui K, Koyama T, Ohme-Takagi M (2003) Dominant repression of target genes by chimeric repressors that include the EAR motif, a repression domain. Arabidopsis Plant J 34(5):733–739

  31. Hu J, Seiler G, Kole C (2010) Genetics, genomics and breeding of sunflower. Science Publishers Inc, Lebanon

  32. Jefferson RA (1987) Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol Biol Rep 5:387–405

  33. Jefferson RA, Mayer JE (2006) Microbial Beta-glucuronidase genes, gene products and uses thereof. US Patent US 7087420 B1, 8 Aug 2006

  34. Kaya MD, Okçu G, Atak M, Çıkılı Y, Kolsarıcı Ö (2006) Seed treatments to overcome salt and drought stress during germination in sunflower (Helianthus annuus L.). Eur J Agron 24(4):291–295.

  35. Kereszt A, Li D, Indrasumunar A, Nguyen CDT, Nontachaiyapoom S, Kinkema M, Gresshoff PM (2007) Agrobacterium rhizogenes-mediated transformation of soybean to study root biology. Nat Protoc 2:948.

  36. Kochian LV (2012) Plant nutrition: rooting for more phosphorus. Nature 488(7412):466–467.

  37. Kraus D (2014) Consolidated data analysis and presentation using an open-source add-in for the Microsoft Excel-« spreadsheet software. Medical Writing 23(1):25–28

  38. Kuta DD, Tripathi L (2005) Agrobacterium -induced hypersensitive necrotic reaction in plant cells: a resistance response against Agrobacterium-mediated DNA transfer. Afr J Biotechnol 4(8):752–757

  39. Lee HW, Cho C, Pandey SK, Park Y, Kim M-J, Kim J (2019) LBD16 and LBD18 acting downstream of ARF7 and ARF19 are involved in adventitious root formation in Arabidopsis. BMC Plant Biol 19(1):46.

  40. Lee HW, Kang NY, Pandey SK, Cho C, Lee SH, Kim J (2017) Dimerization in LBD16 and LBD18 Transcription Factors Is critical for lateral root formation. Plant Physiol 174(1):301.

  41. Lee HW, Kim NY, Lee DJ, Kim J (2009) LBD18/ASL20 regulates lateral root formation in combination with LBD16/ASL18 downstream of ARF7 and ARF19 in Arabidopsis. Plant Physiol 151(3):1377–1389

  42. Li M, Leung DWM (2003) Root induction in radiata pine using Agrobacterium rhizogenes. Electron J Biotechnol 6(3):254–261.

  43. Lin MJY, Humbert ES, Sosulski FW, Downey RK (1975) Distribution and composition of Pectins in sunflower plants. Can. J. Plant Sci. 55(2):507–513.

  44. Macı́as FA, Torres A, Galindo JLG, Varela RM, Llvarez JA, Molinillo JMG (2002) Bioactive terpenoids from sunflower leaves cv. Peredovick®. Phytochemistry 61(6):687–692.

  45. Mankin SL, Hill DS, Olhoft PM, Toren E, Wenck AR, Nea L, Xing L, Brown JA, Fu H, Ireland L, Jia H, Hillebrand H, Jones T, Song H-S (2007) Disarming and sequencing of Agrobacterium rhizogenes strain K599 (NCPPB2659) plasmid pRi2659. In Vitro Cell Dev Biol Plant 43(6):521–535.

  46. Masalia RR, Temme AA, Ndl T, Burke JM (2018) Multiple genomic regions influence root morphology and seedling growth in cultivated sunflower (Helianthus annuus L.) under well-watered and water-limited conditions. PLoS ONE 13(9):e0204279.

  47. Michalec-Warzecha Ż, Pistelli L, D’Angiolillo F, Libik-Konieczny M (2016) Establishment of Highly Efficient Agrobacterium Rhizogenes-mediated Transformation for Stevia Rebaudiana Bertoni explants. Acta Biol Crac Ser. Bot 58(1):113–118.

  48. Michniewicz M, Frick EM, Strader LC (2015) Gateway-compatible tissue-specific vectors for plant transformation. BMC Res Notes 8(1):63.

  49. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15(3):473–497.

  50. Ogura T, Goeschl C, Filiault D, Mirea M, Slovak R, Wolhrab B, Satbhai SB, Busch W (2019) Root system depth in arabidopsis is shaped by EXOCYST70A3 via the dynamic modulation of auxin transport. Cell 178(2):400–412.e16.

  51. Okushima Y, Fukaki H, Onoda M, Theologis A, Tasaka M (2007) ARF7 and ARF19 regulate lateral root formation via direct activation of LBD/ASL genes in Arabidopsis. Plant Cell 19(1):118–130

  52. Ozyigit II, Dogan I, Artam Tarhan E (2013) Agrobacterium rhizogenes-mediated transformation and its biotechnological applications in crops. In: Hakeem KR, Ahmad P, Ozturk M (eds) Crop improvement: new approaches and modern techniques. Springer, Boston, pp 1–48

  53. Pradhan Mitra P, Loqué D (2014) Histochemical staining of Arabidopsis thaliana secondary cell wall elements. J Vis Exp 87:e51381.

  54. Putnam DH, Oplinger ES, Hicks D, Durgan BR, Noetzel DM, Meronuck RA, Doll JD, Schulte EE (1990) Sunflower. Purdue University Center for New Crops and Plant Products Alternative Field Crops Manual. NewCROP (New Crops Resource Online Program)

  55. Rao IM, Miles JW, Beebe SE, Horst WJ (2016) Root adaptations to soils with low fertility and aluminium toxicity. Ann Bot 118(4):593–605.

  56. Roche J, Hewezi T, Bouniols A, Gentzbittel L (2007) Transcriptional profiles of primary metabolism and signal transduction-related genes in response to water stress in field-grown sunflower genotypes using a thematic cDNA microarray. Planta 226(3):601–617.

  57. Rousselin P, Molinier J, Himber C, Schontz D, Prieto-Dapena P, Jordano J, Martini N, Weber S, Horn R, Ganssmann M, Grison R, Pagniez M, Toppan A, Friedt W, Hahne G (2002) Modification of sunflower oil quality by seed-specific expression of a heterologous Δ9-stearoyl-(acyl carrier protein) desaturase gene. Plant Breed 121(2):108–116.

  58. Schneiter AA, Miller JF (1981) Description of sunflower growth stages 1. Crop Sci 21(6):901–903.

  59. Seiler GJ (1994) Primary and lateral root elongation of sunflower seedlings. Environ Exp Bot 34(4):409–418.

  60. Shahzad Z, Kellermeier F, Armstrong EM, Rogers S, Lobet G, Amtmann A, Hills A (2018) EZ-Root-VIS: a software pipeline for the rapid analysis and visual reconstruction of root system architecture. Plant Physiol 177(4):1368–1381.

  61. Škorić D (2014) Sunflower breeding for resistance to abiotic stresses/mejoramiento de girasol por resistencia a estreses bióticos/sélection du tournesol pour la résistance aux stress abiotiques. Helia 32(50):1–16.

  62. Sujatha M, Vijay S, Vasavi S, Reddy PV, Rao SC (2012) Agrobacterium-mediated transformation of cotyledons of mature seeds of multiple genotypes of sunflower (Helianthus annuus L.). Plant Cell Tiss Organ Cult 110(2):275–287.

  63. Taylor CG, Fuchs B, Collier R, Lutke WK (2006) Generation of composite plants using Agrobacterium rhizogenes. In: Wang K (ed) Agrobacterium protocols. Methods in Molecular Biology Humana Press, New York, pp 155–168

  64. Uga Y, Sugimoto K, Ogawa S, Rane J, Ishitani M, Hara N, Kitomi Y, Inukai Y, Ono K, Kanno N, Inoue H, Takehisa H, Motoyama R, Nagamura Y, Wu J, Matsumoto T, Takai T, Okuno K, Yano M (2013) Control of root system architecture by DEEPER ROOTING 1 increases rice yield under drought conditions. Nat Genet 45(9):1097–1102.

  65. Valdes Franco JA, Collier R, Wang Y, Huo N, Gu Y, Thilmony R, Thomson JG (2016) Draft genome sequence of Agrobacterium rhizogenes strain NCPPB2659. Genome Announc 4(4):e00746–e816.

  66. Veena V, Taylor CG (2007) Agrobacterium rhizogenes: recent developments and promising applications. In Vitro Cell Dev Biol Plant 43(5):383–403.

  67. Vitha S, Benes K, Phillips JP, Gartland KM (1995) Histochemical GUS analysis. Methods Mol Biol 44:185–193.

  68. Yordanov Y, Regan S, Busov V (2010) Members of the lateral organ boundaries domain (LBD) transcription factors family are involved in regulation of secondary growth in Populus. Plant Cell 22:3662–3677

  69. Zhang Z, Finer JJ (2015) Sunflower (Helianthus annuus L.) organogenesis from primary leaves of young seedlings preconditioned by cytokinin. Plant Cell, Tissue Organ Cult 123(3):645–655.

  70. Zhang Z, Finer JJ (2016) Low Agrobacterium tumefaciens inoculum levels and a long co-culture period lead to reduced plant defense responses and increase transgenic shoot production of sunflower (Helianthus annuus L.). In Vitro Cell Dev Biol Plant 52(4):354–366.

  71. Zhong C, Nambiar-Veetil M, Bogusz D, Franche C (2018) Hairy roots as a tool for the functional analysis of plant Genes. In: Srivastava V, Mehrotra S, Mishra S (eds) Hairy roots: an effective Tool of plant biotechnology. Springer, Singapore, pp 275–292

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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).

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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).

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