Optical coherence tomography as a novel tool for non-destructive measurement of the hull thickness of lupin seeds J. C. Clements 1 , A. V. Zvyagin 2 , K. K. M. B. D. Silva 2 , T. Wanner 2 , D. D. Sampson 2 and W. A. Cowling 3 1 Centre for Legumes in Mediterranean Agriculture, E-mail: clem@cyllene.uwa.edu.au; 2 Optical and Biomedical Engineering Laboratory, Department of Electrical and Electronic Engineering and 3 School of Plant Biology, The University of Western Australia, 35 Stirling Hwy, Crawley, WA 6009, Australia With 4 figures and 2 tables Received June 10, 2003/Accepted February 4, 2004 Communicated by W. Swiecicki Abstract Hull thickness is an important component of seed quality, which effects dehulling ability, feed or food nutritional aspects and cooking times. A breeding objective in Lupinus angustifolius crop improve- ment is to reduce hull thickness and a rapid screening method is needed to efficiently screen genotypes. Optical coherence tomography (OCT) imaging using infrared illumination at 980 nm was used to compare hull thickness of genotypes of four lupin species. OCT- derived hull layer thickness correlated highly with actual hull thickness determined by environmental scanning electron microscopy (r ¼ 0.90) and allowed reliable distinction between mutant (thin- hulled) and parent genotypes of L. angustifolius. The imaging could clearly penetrate lupin seed to a depth of approximately 200 lm. The use of OCT to measure hull thickness has the advantage that it is rapid and non-destructive and should be very useful in selecting thin hull lines of lupins and other species on a single seed basis in germplasm or progeny from crosses. Key words: Lupinus — hull — optical coherence tomo- graphy — scanning electron microscopy — seed coat testa — seed quality Crop improvement is becoming increasingly concerned with seedqualityaspectssuchasprotein,oilcontent,anti-nutritional compounds and constituents that influence processing (Basra 1995,MooreandYaklich2000).Forbothcerealsandlegumes, hullthicknessandproportionofseedweightareimportantfor ease of dehulling and food processing, reduced fibre content (andconsequentlyhigheroilandprotein)andreducedcooking times. Lush and Evans (1980) suggested that selection for reduced hull content has occurred over the millennia in the majorpulsecrops,withtheexceptionoflupins. Naturalgeneticvariabilityforhullproportionandthickness occurs in several legume species, e.g. for pea (Ali Khan 1993), common bean (Phaseolus vulgaris, Escribano et al. 1997), chickpea (Waldia et al. 1996) and lupin (Reader and Dracup 1998,ClementsandDracup2001,Clementsetal.2002).Ofthe agriculturally important lupin species, both Lupinus angusti- folius and L. luteus haveahighpercentageofseedweightinthe hull(24and25%,respectively)(LushandEvans1980,Reader and Dracup 1998, Clements et al. 2002) compared with only 7%insoybean(LushandEvans1980)and9%infieldpea(Ali Khan 1993). Lupinus angustifolius has a relatively low metabolizable energycontentforpigandpoultryasaconsequenceofitshigh percentage of hull. The hull consists of cellulose (50%), hemicelluloses and pectins (Brillouet and Riochet 1983). The kernelisalsohighinnon-starchpolysaccharideswithinthecell walls and oligosaccharides that constitute approximately 30% and 10%, respectively, of the dehulled kernel (Brillouet and Riochet 1983, Evans 1994). De-hulling lupins improves nutri- tive value for monogastric animals such as pigs, poultry and fish species (Edwards and Barneveld 1998) but the cost of this process tends to make it uneconomical. Significant genetic variation occurs for hull proportion in L. angustifolius (Clements et al. 2002) and breeding for reduced hull content has the potential to increase metabolizable energy, protein or oil (Stombaugh et al. 2000, Clements et al. 2002), harvest index and yield. A rapid method for screening germplasm would be preferred for selecting new lines with reduced hull content. Methods for estimating hull proportion range from manual removal and weighing (Black et al. 1998a, Clements et al. 2002) to the use of dehulling devices (Black et al. 1998b). Flinn et al. (1998) investigated the use of near-infrared spectroscopy for food processing characteristics of field pea and chickpea, and further development of the method is necessary. Several technologies that allow in vivo imaging of plant tissues such as magnetic resonance imaging (Faust et al. 1997) and confocal microscopy (Running et al. 1995) have been limited by factors such as low resolution, long acqui- sition time and limited penetration, respectively. Optical coherence tomography (OCT) is a relatively new imaging technique (Huang et al. 1991, Masters 1999) that has shown significant promise in the micron-resolution subsurface ima- ging of semitransparent and turbid media. Most research in OCT has focussed on in vivo imaging of tissues such as the eye, the skin, and the superficial layers of internal hollow organs. However, OCT has recently been adapted to imaging of living plant tissues (Hettinger et al. 2000). OCT provides a unique set of capabilities, including: (i) greater penetration than confocal microscopy; (ii) resolution typically in the 10 micron range; (iii) video-rate image acquisition; and (iv) portability. Such capabilities make it a potentially useful tool in seed hull screening. In this paper, the utility of OCT for rapid, non-destructive screening for reduced seed hull thick- ness of lupins is demonstrated. U. S. Copyright Clearance Center Code Statement: 0179–9541/2004/2303–0266 $ 15.00/0 www.blackwell-synergy.com Plant Breeding 123, 266—270 (2004) Ó 2004 Blackwell Verlag, Berlin ISSN 0179-9541