Plant and Soil

, Volume 412, Issue 1–2, pp 61–80 | Cite as

A high-throughput method to quantify root hair area in digital images taken in situ

  • Christopher VincentEmail author
  • Diane Rowland
  • Chaein Na
  • Bruce Schaffer
Regular Article


Background and aims

Root hair growth and development are important features of plant response to varying soil conditions and of nutrient and water uptake. Most current methods of characterizing root hairs in the field are unreliable or inefficient. We describe a method to quantify root hair area in digital images, such as those collected in situ by minirhizotron systems.


This method uses ImageJ and R open source software and is partially automated using code presented here. It requires manual tracing of a subset of root hair images (training data set) to which a multivariate logistic regression is fit with each color channel in the image as an independent variable. Thereafter the model is applied to complete sets of selected root hair sections to estimate total root hair area.


There was good agreement between the training data sets and the predictions of the regression models in castor (Ricinus communis L.), maize (Zea mays L.), and papaya (Carica papaya L.).


This method enables time-efficient and consistent quantification of root hairs using in situ root imaging systems that are already widely in use.


Root hair Rhizotron Digital image analysis 



The authors wish to thank Andrew Schreffler, Alex Tran, Amy Mayedo and Lamar Jahna for their work in making root hair area selections and manually tracing the images, as well as José Clavijo for review of the manuscript and valuable input.


  1. Bates TR, Lynch JP (1996) Stimulation of root hair elongation in Arabidopsis thaliana by low phosphorus availability. Plant Cell Environ 19:529–538CrossRefGoogle Scholar
  2. Bates TR, Lynch JP (2000) The efficiency of Arabidopsis thaliana (Brassicaceae) root hairs in phosphorus acquisition. Amer J Bot 87:964–970CrossRefGoogle Scholar
  3. Box JE, Smucker AJM, Ritchie JT (1989) Minirhizotron installation techniques for investigating root responses to drought and oxygen stresses. Soil Sci Soc Am J 53:115. doi: 10.2136/sssaj1989.03615995005300010021x CrossRefGoogle Scholar
  4. Brown LK, George TS, Thompson JA, Wright G, Lyon J, Dupuy L, Hubbard SF, White PJ (2012) What are the implications of variation in root hair length on tolerance to phosphorus deficiency in combination with water stress in barley (Hordeum vulgare)? Ann Bot 110:319–328CrossRefPubMedPubMedCentralGoogle Scholar
  5. Campbell DN, Rowland DL, Schnell RW et al (2014) Developing a castor (Ricinus communis L.) production system in Florida, U.S.: evaluating crop phenology and response to management. Ind Crops Prod 53:217–227. doi: 10.1016/j.indcrop.2013.12.035 CrossRefGoogle Scholar
  6. Ciss S (2015) randomUniformForest: random Uniform Forests for Classification, Regression and Unsupervised Learning. R package version 1.1.5
  7. Datta S, Prescott H, Dolan L (2015) Intensity of a pulse of RSL4 transcription factor synthesis determines Arabidopsis root hair cell size. Nature Plants 1(10):15138CrossRefPubMedGoogle Scholar
  8. Dittmer HJ (1949) Root hair variations in plant species. Am J Bot 36:152–155. doi: 10.2307/2437782 CrossRefGoogle Scholar
  9. Domingos P (2000) A unified bias-variance decomposition. In: Proceedings of 17th International Conference on Machine Learning. Stanford CA Morgan Kaufmann. pp 231–238Google Scholar
  10. Eshel A, Beeckman T (eds) (2013) Plant roots: the hidden half, fourth edition, 4th edn. CRC Press, Boca RatonGoogle Scholar
  11. Gilroy S, Jones DL (2000) Through form to function: root hair development and nutrient uptake. Trends Plant Sci 5:56–60CrossRefPubMedGoogle Scholar
  12. Haling RE, Brown LK, Bengough AG, Valentine TA, White PJ, Young IM, George TS (2014) Root hair length and rhizosheath mass depend on soil porosity, strength and water content in barley genotypes. Planta 239:643–651CrossRefPubMedGoogle Scholar
  13. He Z, Ma Z, Brown KM, Lynch JP (2005) Assessment of inequality of root hair density in Arabidopsis thaliana using the Gini coefficient: a close look at the effect of phosphorus and its interaction with ethylene. Ann Bot 95:287–293CrossRefPubMedGoogle Scholar
  14. Heeraman DA, Juma NG (1993) A comparison of minirhizotron, core and monolith methods for quantifying barley (Hordeum vulgare. Plant Soil 148:29–41. doi: 10.1007/BF02185382 CrossRefGoogle Scholar
  15. Højsgaard S, Halekoh U (2014) doBy: Groupwise statistics, LSmeans, linear contrasts, utilities. R package version 4.5–13 URL
  16. Hosmer D, Lemeshow S, Sturdivant RX (2013) Applied logistic regression. John Wiley & SonsGoogle Scholar
  17. Intrator N (1993) On the combination of supervised and unsupervised learning. Phys Stat Mech Appl 200:655–661CrossRefGoogle Scholar
  18. Jones VAS, Dolan L (2012) The evolution of root hairs and rhizoids. Ann Bot 110:205–212CrossRefPubMedPubMedCentralGoogle Scholar
  19. Keyes SD, Daly KR, Gostling NJ et al (2013) High resolution synchrotron imaging of wheat root hairs growing in soil and image based modelling of phosphate uptake. New Phytol 198:1023–1029. doi: 10.1111/nph.12294 CrossRefPubMedGoogle Scholar
  20. Lamont B (1983) Root hair dimensions and surface/volume/weight ratios of roots with the aid of scanning electron microscopy. Plant Soil 74:149–152CrossRefGoogle Scholar
  21. Lynch JP (2007) Roots of the second green revolution. Aust J Bot 55:493–512CrossRefGoogle Scholar
  22. Lynch JP, Chimungu JG, Brown KM (2014) Root anatomical phenes associated with water acquisition from drying soil: targets for crop improvement. J Exp Bot 65:6155–6166CrossRefPubMedGoogle Scholar
  23. Mackay AD, Barber SA (1985) Effect of soil moisture and phosphate level on root hair growth of corn roots. Plant Soil 86:321–331CrossRefGoogle Scholar
  24. Meisner CA, Karnok KJ (1991) Root hair occurrence and variation with environment. Agron J 83:814–818CrossRefGoogle Scholar
  25. Mendrinna A, Persson S (2015) Root hair growth: it’s a one-way street. F1000Prime Rep 7:23. doi: 10.12703/P7-23 CrossRefPubMedPubMedCentralGoogle Scholar
  26. Milchunas DG (2012) Biases and errors associated with different root production methods and their effects on field estimates of belowground net primary production. In: Mancuso SS (ed) Measuring roots. Springer-Verlag, Berlin, pp 303–340CrossRefGoogle Scholar
  27. Müller M, Schmidt W (2004) Environmentally induced plasticity of root hair development in Arabidopsis. Plant Phys 134:409–419CrossRefGoogle Scholar
  28. Oldroyd GED (2001) Dissecting symbiosis: development in Nod factor signal transduction. Ann Bot 8:709–718CrossRefGoogle Scholar
  29. Peterson RL, Farquhar ML (1996) Root hairs: specialized tubular cells extending root surfaces. Bot Rev 62:1–40CrossRefGoogle Scholar
  30. Richardson AE, Lynch JP, Ryan PR, Delhaize E, Smith FA, Smith SE, Harvey PR, Ryan MH, Veneklaas EJ, Lambers H, Oberson A, Culvenor RA, Simpson RJ (2011) Plant and microbial strategies to improve the phosphorus efficiency of agriculture. Plant Soil 349:121–156CrossRefGoogle Scholar
  31. Samson BK, Sinclair TR (1994) Soil core and minirhizotron comparison for the determination of root length density. Plant Soil 161:225–232. doi: 10.1007/BF00046393 CrossRefGoogle Scholar
  32. Schnall JA, Quatrano RS (1992) Abscisic acid elicits the water-stress response in root hairs of Arabidopsis thaliana. Plant Physiol 100:216–218CrossRefPubMedPubMedCentralGoogle Scholar
  33. Segal E, Kushnir T, Mualem Y, Shani U (2008) Water uptake and hydraulics of the root hair rhizosphere. Vadose Zone J 7:1027–1034CrossRefGoogle Scholar
  34. Siqueira JO, Saggin-Júnior OJ (2001) Dependency on arbuscular mycorrhizal fungi and responsiveness of some Brazilian native woody species. Mycorrhiza 11:245–255. doi: 10.1007/s005720100129 CrossRefGoogle Scholar
  35. Stetter MG, Schmid K, Ludewig U (2015) Uncovering genes and ploidy involved in the high diversity in root hair density, length and response to local scarce phosphate in Arabidopsis thaliana. PLoS ONE 10:e0120604. doi: 10.1371/journal CrossRefPubMedPubMedCentralGoogle Scholar
  36. Upchurch DR (1987) Conversion of minirhizotron-root intersections to root length density. Minirhizotron Obs Tubes Methods Appl Meas Rhizosphere Dyn asaspecialpubli:51–65. doi:  10.2134/asaspecpub50.c5
  37. Urbanek S (2013) tiff: Read and write TIFF images. R package version 0.1-5 URL
  38. Vadez V (2014) Root hydraulics: the forgotten side of roots in drought adaptation. Field Crops Res 165:15–24CrossRefGoogle Scholar
  39. van Noordwijk M, de Jager A, Floris J (1985) A new dimension to observations in minirhizotrons: a stereoscopic view on root photographs. Plant Soil 86:447–453. doi: 10.1007/BF02145465 CrossRefGoogle Scholar
  40. van Someren M (1986) A bias-variance analysis of a real world learning problem: the CoIL challenge 2000. Mach Learn 57:173Google Scholar
  41. Vandamme E, Renkens M, Pypers P et al (2013) Root hairs explain P uptake efficiency of soybean genotypes grown in a P-deficient Ferralsol. Plant Soil 369:269–282. doi: 10.1007/s11104-012-1571-2 CrossRefGoogle Scholar
  42. Vieira RF, Jochua CN, Lynch JP (2007) Method for evaluation of root hairs of common bean genotypes. Pesq Agropec Bras 42:1365–1368CrossRefGoogle Scholar
  43. Vincent C, Rowland DL, Schaffer B (2015) The potential for primed acclimation in papaya (Carica papaya L.): determination of critical water deficit thresholds and physiological response variables. Sci Hortic 194:344–352CrossRefGoogle Scholar
  44. Wickham H (2007) Reshaping data with the reshape package. J Stat Software, 21(12), 1-20 URL
  45. Wickham H (2009) ggplot2: elegant graphics for data analysis. Springer, New YorkCrossRefGoogle Scholar
  46. Withington JM, Elkin AD, Bułaj B et al (2003) The impact of material used for minirhizotron tubes for root research. New Phytol 160:533–544. doi: 10.1046/j.1469-8137.2003.00903.x CrossRefGoogle Scholar
  47. Yamaguchi J (2002) Measurement of root diameter in field-grown crops under a microscope without washing. Soil Sci Plant Nutr 48:625–629CrossRefGoogle Scholar
  48. Yan X, Liao H, Beebe SE, Blair MW, Lynch JP (2004) QTL mapping of root hair and acid exudation traits and their relationship to phosphorus uptake in common bean. Plant Soil 265:17–29CrossRefGoogle Scholar
  49. Yazdanbakhsh N, Fisahn J (2009) High throughput phenotyping of root growth dynamics, lateral root formation, root architecture and root hair development enabled by PlaRoM. Funct Plant Biol 36:938–946CrossRefGoogle Scholar
  50. Zhu J, Kaeppler SM, Lynch JP (2005) Mapping of QTL controlling root hair length in maize (Zea mays L.) under phosphorus deficiency. Plant Soil 270:299–310CrossRefGoogle Scholar

Copyright information

© Springer International Publishing Switzerland 2016

Authors and Affiliations

  • Christopher Vincent
    • 1
    Email author
  • Diane Rowland
    • 2
  • Chaein Na
    • 3
  • Bruce Schaffer
    • 4
  1. 1.Horticultural Sciences Department, Citrus Research and Education CenterUniversity of FloridaLake AlfredUSA
  2. 2.Agronomy DepartmentUniversity of FloridaGainesvilleUSA
  3. 3.Department of AgronomyGyeongsang National UniversityJinjuKorea
  4. 4.Horticultural Sciences Department, Tropical Research and Education CenterUniversity of FloridaHomesteadUSA

Personalised recommendations