1
Microstructural characterization of commercial kiwifruit
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cultivars using X-ray micro computed tomography
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Dennis Cantrea, Andrew Eastb, Pieter Verbovena, Ximenita Trejo Arayab, Els Herremansa,
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Bart M. Nicolaïa, Thamarath Pranamornkithb, Michael Lohc, Alistair Mowatd, Julian Heyesb.
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b
BIOSYST-MeBioS, KU Leuven, Willem de Croylaan 42, Heverlee, Belgium
Centre for Postharvest and Refrigeration Research, Massey University, Palmerston North,
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New Zealand
c
Fonterra Research and Development Centre, Palmerston North, New Zealand
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Zespri™ International Ltd, Mt Maunganui, New Zealand
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Corresponding author:
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Andrew East, Centre for Postharvest and Refrigeration Research, Massey University,
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Palmerston North, New Zealand
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Tel.: +64 350 9099; e-mail: A.R.East@massey.ac.nz
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1
ABSTRACT
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The skin is the physical barrier between the fruit and the environment in which it develops.
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Environmental conditions during fruit development have a large influence on fruit quality,
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both at the time of harvest and during subsequent storage. It is hypothesized that some
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features of the skin and sub-epidermal tissues could provide information about the past
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growing conditions to which the fruit was exposed and therefore be of predictive value for
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storage quality. In this study, five commercial kiwifruit cultivars (‘Hayward’, ‘Hort16A’,
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‘G3’, ‘G9’ and ‘G14’) were studied, and ‘Hayward’ fruit were manipulated during growth
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with different light or cultural practices. After harvest at horticultural maturity, X-ray micro
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computed tomography (µCT) was used to investigate features of the skin and the immediate
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parenchyma tissue. Despite orchard management practices (crop load and girdling) being
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observed to effect macro fruit quality parameters (mass, firmness, SSC, and DM), differences
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in microstructure (e.g. porosity) caused by these practices were not observed. However,
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porosity and pore size were found to be highly variable between cultivars. The thickness of
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dense sub-epidermal tissue could be readily measured and the 3-D distribution of raphide
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bundles was visible as high density particles distributed within the parenchyma. Overall,
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µCT was found to be a powerful technique to explore fruit epidermal and sub-epidermal
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structures in three dimensions at a micro level. However, the length of time required for data
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capture and analysis and the large number of samples required to overcome natural variation
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within horticultural products need to be considered. Future work may define the impact of
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differences in porosity or sub-epidermal anatomy on kiwifruit physiology (e.g. firmness
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change or sensitivity to low oxygen storage atmospheres). With this information, µCT could
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be used as a screening tool during plant breeding, or to determine the response to agronomic
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treatments, without conducting lengthy storage trials.
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Highlights
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 Microstructural differences were found among kiwifruit cultivars
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 Differences in raphide were found among kiwifruit cultivars
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 Orchard manipulation caused macro quality differences but not microstructural
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Keywords: X-ray µCT, microstructure, cultivar differences, orchard manipulation, raphide
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2
Introduction
Kiwifruit quality and storability are known to be affected by environmental factors
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during growth and development.
Sunlight exposure during development can enhance
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pigment development, stimulate fruit maturation, enhance skin coloration and result in firmer
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fruit with less incidence of rot in storage (Tombesi et al., 1993) and better fruit storability
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(Antognozzi et al., 1995). This sunlight exposure is directly influenced by canopy density
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(Snelgar et al., 1998). Similarly, application of trunk girdling has been applied to result in
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both increased fruit mass and dry matter, depending on timing, while fruit crop load is known
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to affect final fruit size (Patterson and Currie, 2011). Further studies in manipulating
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appropriate growth conditions may provide an insight in alleviating LTB in kiwifruit during
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storage. It is hypothesized that membrane deterioration plays a critical role during ripening
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and LTB development (Marangoni et al., 1996). Membrane deterioration affects porosity
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either through filling of pores with cellular fluid as a result of cellular breakdown (Kuroki et
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al., 2004), or creation of larger air spaces as a result of tissue collapse (Herremans et al.,
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2013a). This membrane deterioration leading to changes in porosity is also suggested to play
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a critical role in chilling injury in mangoes. Chilling injury damages cell wall causing cell
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wall disassembly and cell leakage (Han et al., 2006). Thus, cellular fluid may fill the
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intercellular gas-filled spaces, completely separating the pores (Narain et al., 1998). The
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decrease in pore size and pore connectivity in cucumber during ripening also suggests
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leakage of intracellular substance into the gas-filled intercellular space (Kuroki et al., 2004).
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The importance of pore network and void volume fraction for gas exchange in apples
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and pears is extensively studied (Herremans et al., 2013a,b; Ho et al., 2013a, b; Verboven et
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al., 2008). For example, some cultivars (e.g., ‘Braeburn’ and ‘Cripps Pink’) develop internal
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browning disorders in storage (Herremans et al., 2013a; James and Jobling, 2009), while
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others (e.g. ‘Granny Smith’) tend to develop skin related injuries (Fan et al., 1999).
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Contemporary research suggests that these differences in storability and susceptibility to
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disorders are associated with skin and flesh properties that influence gas diffusion within the
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fruit tissue (Ho et al., 2009, 2010, and 2011) including differences in porous microstructure
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(Ho et al., 2013b), with those differences strongly affecting optimal storage conditions (Ho et
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al., 2013c). Whether such relationships of flesh properties to optimal storage conditions exist
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for kiwifruit is currently unknown.
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Techniques to visualise and quantify microstructure have been used to answer valid
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research questions and to provide a better understanding on the relation of microstructure to
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quality characteristics during storage. Unlike light and electron microscopy, non-destructive
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techniques provide a more accurate depiction of the microstructure since cells and tissues
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remains intact and undamaged and no tedious sample preparation is required (Veraverbeke et
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al., 2001; Musse et al., 2010). One such non-destructive technique is X-ray micro computed
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tomography (μCT), a three-dimensional visualization technique that creates an image based
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on X-ray attenuation within the sample. The difference in X-ray attenuation of different
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materials creates contrast to differentiate low density and high density materials
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(Karathanasis and Hajek, 1996). In comparison to other non-destructive techniques, µCT has
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excellent spatial resolution that resolves intercellular space down to submicron range, and
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depth of penetration allowing subsurface features to be studied. This technique can help to
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characterize and understand foods by measuring cell size and shape, void space and spatial
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distribution (Lim and Barigou, 2004). The use of µCT allowed visualisation of the fruit void
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network architecture, showing a volume fraction of 5.1 % and 23 % for ‘Conference’ pear
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and ‘Jonagold’ apple cortex, respectively (Verboven et al., 2008; Mendoza et al., 2007). This
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technique was also successfully used in 3-D analysis of raphides in rose peduncles and Lotus
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miyakojimae seeds (Matsushima et al., 2012; Yamauchi et al., 2013).
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The main objective of this study was to gain the first insights into the potential
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differences in 3-D microstructural properties of commercial kiwifruit genotypes. μCT was
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used as a tool to explore the fruit peel and tissue from five cultivars. Additionally, treatments
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to deliberately manipulate ‘Hayward’ kiwifruit during growth via altering light conditions,
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crop load and application of girdling were applied as a preliminary study to investigate if any
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of the crop manipulation techniques would influence cellular arrangement and density within
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the fruit. Insights from this study are expected to reveal relationship between the potential for
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storage capability of cultivars to the changes in cellular arrangement and density as affected
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by environmental factors during growth and development.
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3
Materials and methods
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3.1
Plant material and treatment manipulation
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Currently there are five commercial cultivars exported from New Zealand, being green
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cultivars ‘Hayward’ (A. deliciosa, Zespri™ Green) and ‘G14’ (A. deliciosa × chinensis,
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Zespri™ Sweet Green) and yellow fleshed cultivars (all A. chinensis) ‘Hort16A’ (Zespri™
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Gold), ‘G3’ (Zespri™ Sun Gold) and ‘G9’ (Zespri™ Charm). Single tray samples of ‘G14’,
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‘Hort16A’, ‘G3’, and ‘G9’ were obtained from commercial orchards in New Zealand and
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delivered via airfreight to KU Leuven, Belgium, in June 2013. Five fruit from each cultivar
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were scanned for X-ray µCT. At the time of the measurements, fruit could be considered as
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eating ripe.
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In a growing condition manipulation experiment conducted in the 2013 harvest season,
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a mature ‘Hayward’ kiwifruit orchard trained on a pergola system and located in Te Puna,
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Bay of Plenty, New Zealand was used. This experiment consisted of a 2 × 2 matrix of plant
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manipulation treatments, being fruit crop load (36 or 43 t/ha) and usage (or not) of trunk
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girdling (Patterson and Currie, 2011). Crop thinning occurred on 4 - 5 Jan 2013 and trunk
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girdling occurred on 10 Dec 2012 and 2 Feb 2013. A single sample of 15 fruit was harvested
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from each of the 4 treatments on 7 May 2013 and delivered by airfreight to KU Leuven. Five
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fruit from each treatment were measured in June at which time the fruit could be considered
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eating ripe. Commercial harvest of this experiment occurred on 15 May 2013 at which time
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90 fruit samples were collected and subsequently analysed for at harvest quality (mass,
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soluble solids content, firmness and % dry matter).
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3.2
Micro X-ray CT imaging
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Kiwifruit samples measuring 5 mm x 5 mm x 10 mm including the skin were excised
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from the equatorial region and wrapped in parafilm to prevent dehydration prior to
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subsequent CT scanning. A Skyscan 1172 high resolution µCT scanner (Bruker, Kontich,
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Belgium) was used for acquiring projection images with a source power of 10 W at 60 kV
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and 167 µA. Each projection image was averaged from three frames with each frame taken
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with an exposure time of 295 ms. The sample was rotated on the stage at an increment of 0.4°
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until a rotation angle of 204.8° (180° + fan beam angle) was completed generating 512
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shadow projections with a pixel size of 4.87 µm. Cross section slices were generated from
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the shadow projections using the Feldkamp reconstruction algorithm (Feldkamp et al., 1984)
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implemented in Nrecon 1.6.5.8 software (http://www.skyscan.be/next/downloads.htm).
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Reconstruction parameters for beam hardening, ring artefact reduction and smoothing were
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set to 35, 8 and 2, respectively. The dynamic range or linear attenuation range was limited to
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0 – 0.0854 to generate an 8-bit bitmap grayscale cross section slice.
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3.3
Image processing
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Undamaged and intact µCT tomographs measuring 2 mm x 2 mm x 2 mm, left after
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virtual cropping, were utilised for 3-D image processing and analysis (Figure 1A). Each
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image was segmented with a manual threshold (Figure 1B) to obtain the pores and cell
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assembly, followed by individual pore labelling (Figure 1C) and 3-D image rendering of the
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pore network (Figure 1D, Figure 2B). A spatial graph representation of the skeleton using
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TEASAR algorithm (Sato et al., 2000) was made to show the essential geometry and local
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thickness of the pore network (Figure 2C). Microstructural parameters (Table 1) as described
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by Herremans et al. (2013a) were analysed. Raphide bundles were revealed as a substantial
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number of high density oblate spheroid particles in the epidermal and sub-epidermal region.
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These particles were segmented with a manual threshold and labelled prior to surface
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rendering and subsequent 3-D analysis. Image processing and 3-D analysis were performed
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using CTAn 1.12 (Bruker microCT, Kontich, Belgium) and Avizo 7.1 (VSG, Bordeaux,
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France). Results of the quality measurements and microstructural analysis were subjected to
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analysis of variance using PROC GLM procedure in SAS 9.3 (SAS Institute, Cary, NC,
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USA).
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3.4
Fruit quality measurement
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At harvest, fruit quality measures from the production method manipulation experiment
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applied to ‘Hayward’ kiwifruit were conducted in order to demonstrate the potential effects
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of these treatments on fruit quality. Individual fruit mass (g) was measured using a balance
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(PG-503S, Mettler-Toledo GmbH, Greifensee, Switzerland) with 0.001 g accuracy. Fruit dry
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matter content was determined using oven drying techniques. An equatorial fruit slice with a
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thickness of 2-3 mm thick and of known mass was dried at 60-65°C for 24 h, subsequently
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weighed and dry matter expressed as a percentage of the wet mass. Firmness was measured
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using a QALink Penetrometer (Willowbank Electronics Ltd., Napier, New Zealand), fitted
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with the standard 7.9 mm round Effegi probe. Prior to measurement, 1 mm of the fruit skin
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was removed at two locations (90° apart) around the equator of the fruit with a fruit slicer
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(setting depth: 1 mm). The average peak force (N) required to puncture the skinned tissue to a
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depth of 8 mm at a speed of 20 mm.s-1 was recorded. Soluble solids were measured as °Brix,
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using a digital pocket refractometer (PAL-1, Atago, Japan). Juice was taken from both the
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stem and blossom ends of the fruit and mixed in approximately equal amounts on the
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refractometer.
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4
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4.1
Results
Imaging and rendering
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The microtomographs of kiwifruit tissue from five commercial cultivars showed
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differences in grey scale values according to the X-ray attenuation by the different elements
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of the tissue (Figure 2A). The shade of the cells is lighter than that of the pores as a
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consequence of the higher density of the cells compared to that of air. 3-D renderings of pore
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structure and pore network model of samples are shown in Figure 2B and 2C respectively.
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Both figures seem to indicate considerable differences in tissue microstructure between fruit
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of different cultivars.
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4.2
Differences between cultivars
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3-D rendering of pore structure and pore network model (Figure 2B and 2C) suggests the
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presence of isolated, short and narrow pores in ‘Hayward’ and ‘G3’ while ‘Hort16A’, ‘G9’
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and ‘G14’ showed highly branched, more connected, and thicker pores. The porosity seems
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to be low in ‘G3’ and high in‘Hort16A’ and ‘G14’ cultivars. The statistical analysis of
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microstructural features (Table 2) emphasized cultivar specific difference in porosity of
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kiwifruit. ‘G14’ and ‘Hort16A’ had a significantly higher porosity compared to ‘Hayward’
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and‘G3’ cultivars (Table 2). In addition, fruit of 'G3' had porosity value significantly lower
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than those of the other cultivars. Pores observed below the skin (Figure 2) also differ among
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cultivars and they may significantly affect the gas exchange. ‘G3’ cultivar, for example,
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showed larger pores beneath the skin and smaller pores in the parenchyma tissue. All the
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other cultivars have small isolated pores under the skin but have large and more connected
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pores located in the parenchyma tissue.
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The size range of pores varied by an order of magnitude of 4 from the detection limit of
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2.99 × 10-6 mm3 (Herremans et al., 2013a) to pore volumes as high as 1.35 × 10-2 mm3. The
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pore size distribution (Figure 3) showed further differences between kiwifruit cultivars. ‘G14’
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and ‘Hort16A’ showed the broadest pore size distribution with a pore equivalent diameter up
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to 295 µm. 3-D microstructural analysis data (Table 2) also showed that ‘G14’ had the
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highest maximal individual pore volume. The prevalence of large pores (> 100 µm) in ‘G14’,
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contributing to 40% of its total pore volume, also separates it from other cultivars. In contrast,
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‘G3’ had a narrow pore size range with only 196 µm as the largest pore equivalent diameter
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detected, and with only 0.0024 mm3 maximal individual pore volume, the lowest of the five
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cultivars. It is also differentiated by the prevalence of small pores (< 50 µm) which constitute
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58% of its total pore volume, significantly higher across all cultivars analysed. Pore sizes of
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‘G9’, ‘Hort16A’, and ‘Hayward’ appeared to fall in a similar and intermediate range. Small
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pores (< 50 µm) represent a substantial portion (more than 90%) of the total number of pores
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in all cultivars.
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Pore structure thickness results concur with observations on pore size. The large pores
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in ‘G14’ are accompanied by a thick pore structure which is significantly higher than all the
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other cultivars (Table 2). In comparison, the pores found in ‘G3’ cultivar have a significantly
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lower pore structure thickness compared to other cultivars due to its narrow pores. Pore
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structure thickness of ‘G9’, ‘Hort16A’, and ‘Hayward’ appeared to fall in an intermediate
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range. The pore specific surface area was lowest in ‘G14’ and highest in ‘G3’ (Table 2). The
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smaller pore size fraction in ‘G3’ provided higher surface area per unit volume of tissue
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giving a higher value of pore specific surface area.
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Pore connectivity and pore fragmentation results (Table 2) confirmed the observation
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on the pore network model suggesting highly fragmented and less connected pores in ‘G3’.
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Furthermore, the pore fragmentation and pore connectivity of ‘G3’is significantly different
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across all cultivars studied. The observed highly connected pores in ‘G14’ from the pore
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network model correspond to its low pore fragmentation value. In addition, the ‘G14’ pore
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fragmentation is significantly lower than that of all other cultivars. Pore connectivity results,
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on the other hand, showed no significant difference between ‘G14’, ‘G9’, ‘Hort16A’, and
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‘Hayward’, suggesting the same number of connected structures per unit volume of tissue in
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these cultivars.
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The peel thickness, defined as the distance between the fruit surface and the first pore,
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was not significantly different amongst the 5 cultivars (Table 2). The peel thickness was on
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average between 70 and 85 µm.
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4.3
Effect of growing conditions
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As expected, manipulation of growing conditions resulted in differences in ‘Hayward’
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fruit quality attributes at commercial harvest (Table 3 and 4). High crop load resulted in
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reduced fruit size (mass) and soluble solids content. High crop load also reduced dry matter
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when the vines were girdled, and increased fruit firmness when the vines were not girdled.
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Trunk girdling caused increased fruit size (mass), soluble solids content and decreased
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firmness. However, X-ray CT image analysis failed to identify any significant difference in
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equivalent porosity (Table 4), pore diameter, porosity or peel thickness effects (data not
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shown) caused by either pre-harvest crop load or trunk girdling treatments.
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.
4.4
Presence of raphides
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A substantial number of oblate spheroid shaped high density particles were observed
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within the kiwifruit tissue. These were observed distinctly on the X-ray images (Figure 4).
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These particles were approximately 80-130 µm in length (Figure 5) and in most occasions
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orientated with the long axis parallel with the kiwifruit skin surface. The particles were
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widely spread across the whole pericarp and beneath the surface of the skin. All five
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kiwifruit cultivars showed the presence of these particles, identified as druses (bundles of
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raphides). High counts of these particles were found in ‘G14’ and ‘G3’ (Figure 5) but fruit to
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fruit variability rendered no significant difference in count of these bundles across all
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cultivars. ‘G3’ had the highest mean raphide bundle volume and length. It also contained
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significantly larger raphide bundles compared to ‘Hort16A’ and ‘G14’. More spherical
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bundles were found in ‘G14’ and ‘Hort16A’ cultivars than in ‘Hayward’.
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5
Discussion
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The porosity and pore sizes of kiwifruit are generally small compared to other fruit. For
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example, for ‘Braeburn’ apple, Herremans et al. (2013a) found pore sizes up to 600 µm with
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50% of the pores smaller than 180 µm, thus considerably larger than those of kiwifruit. This
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is not unexpected because apple consists also of larger cells, and has a much larger porosity
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with values up to 25.4% compared to kiwifruit with values up to 6.5%. The porosity and
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small pore size of kiwifruit are more similar to pear and cucumber fruit with values up to 4.1
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and 3.4% respectively (Verboven et al., 2008; Kuroki et al., 2004). We earlier attributed these
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differences to differences in the mechanisms of pore formation where programmed cell death
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could be responsible for larger (lysogenous) pores in apple and cell wall separation for
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smaller (schizogenous) pores in pears. More likely the latter mechanisms is responsible for
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pores in kiwifruit as well. In terms of the number of pores, kiwifruit is also similar to
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cucumber: most (79%) of the pores were smaller than 26 µm equivalent diameter, with 20%
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of the pores were between 27 and 58 µm and fewer than 1% were larger. Kiwifruit had a
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somewhat wider size range distribution with approximately 65% of the pores smaller than 20
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µm, 30% for pores between 20 and 60 µm and 5% larger than 60 µm. Concerning the range
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of porosity values found for kiwifruit cultivars, the least porous cultivar ‘G3’ has a porosity
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which is only 25% of that of the most porous fruit (‘G14’). For comparison, analysis of 3
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apple cultivars and ‘Conference’ pear showed that ‘Kanzi’ had a porosity that was
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approximately 50% that of ‘Jonagold’, while conference pear had a porosity that was less
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than 20% of that of ‘Jonagold’ (Herremans et al. 2013b). The porosity of kiwifruit ranging
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from 1.55 % in ‘G3’ up to 6.5 % in ‘G14’ is comparable to that of ‘Conference’ pear
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(Herremans et al., 2013b) and that of the radial pericarp of tomato (Musse et al., 2010) with
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porosity values of 4.1 and 3.0 % respectively.
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Comparing the pore network of kiwifruit and ‘Jonagold’ apples, kiwifruit has pore
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structure thickness of that is only 28-40 % with that of ‘Jonagold’. Pore fragmentation on the
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other hand, is more than twice that of ‘Jonagold’ (Herremans et al., 2013a). Physically, fruit
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with larger, but especially more connected, air voids enable more efficient gas exchange (Ho
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et al., 2013b).
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diffusivity and, hence, sensitivity to controlled atmosphere conditions. Physically, more air
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voids may affect the cell structure and mechanical properties of the tissue and how this
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weakens with pectin degradation during ripening and storage (Waldron et al., 1997).
These differences in pore structure may account for differences in gas
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Future scans at a higher resolution may reveal pores that are smaller than the detection
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limit. These small pores may further contribute to the pore connectivity and pore analysis
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result based on the current pixel resolution. Further, the hypothetical presence of small pores
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would be more important in less porous kiwifruit cultivars where they would significantly
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contribute to gas exchange
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Kiwifruit peel thickness falls within the range that was measured for apple
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(Veraverbeke et al., 2001; Verboven et al., 2013). Apple has a peel thickness between 60 and
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120 µm, depending on the cultivar, with significant differences between ‘Idared’, on the one
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hand, and ‘Arlet’, ‘Braeburn’ and ‘Royal Gala’, on the other hand. The thickness of the apple
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wax layer also varies depending on the cultivar, with ‘Elstar’, ‘Jonagored’ and ‘Jonagold’
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having a wax layer thickness significantly different from each other (Veraverbeke et al.,
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2001). These large cultivar differences in porosity and sub-epidermal anatomy emphasise the
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value of a technique such as µCT as a screening tool in a breeding programme, once a link
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between these features and fruit storage performance is established.
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This work used commercially-produced fruit which were measured at a table-ripe
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stage. Micro X-ray CT studies suggest that apple browning is associated with cellular
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breakdown which was detected as a disappearance of air spaces in affected tissue followed by
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the creation of large pores after water has diffused out of the affected zone (Herremans et al.,
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2013a). In the case of kiwifruit, it may be possible that as kiwifruit develop LTB or ripen,
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they may also go through porosity changes throughout storage. In investigating kiwifruit
307
ripening with MRI, Taglienti et al. (2009) suggested that as kiwifruit softened, water was “re-
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organised” due to a “release” of water from the cellular matrix. Changes in porosity were
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also observed during ripening of mango. In addition, microstructural changes observed
310
during mango ripening also suggest a release of water from the cellular matrix (Cantre et al.,
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2013). Use of X-ray µCT to determine porosity changes (if any) during ripening and/or LTB
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development may assist in developing understanding of these processes.
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A number of plant species including kiwifruit are known to contain druses. These are
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bundles of raphides or calcium oxalate crystals which promote the burning or ‘catching’
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sensation in the mouth when eaten due to their needle-like nature (Perera et al., 1990).
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Normally, druses are enclosed inside an idioblast cell in the locular gel around the seeds
317
(Perera et al., 1990). Several roles are proposed for raphides: protection against animals,
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detoxifying oxalates, contributing to tissue firmness and regulating free calcium
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concentration in the fruit (Faheed et al., 2012; Nakata, 2012). Calcium uptake and deposition
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during development depends on environmental conditions (Buxton, 2005). The distribution,
321
number and morphological characteristics of these particles were not known prior to this
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study.
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Raphides in rose peduncles and Lotus miyakojimae seeds were successfully quantified
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in 3-D using synchrotron X-ray µCT (Matsushima et al., 2012; Yamauchi et al., 2013), but
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the ease of detection of raphides by conventional X-ray µCT has not been previously
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reported. Furthermore, the presence of raphides this close to the skin is not normally reported
327
or visible with conventional staining systems (e.g., Hallett et al., 2005). This holds promise
328
for studies of their morphological characteristics and 3-D arrangement in space utilizing only
329
a laboratory scale X-ray µCT system. Threshold-binarisation of the µCT images facilitated
330
isolation and labelling of these particles before subsequent 3-D analysis and quantification.
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X-ray µCT further allowed determination of the amount and morphological characteristics of
332
raphides for each kiwifruit cultivar. The non-invasive nature of X-ray µCT provides an ideal
333
tool for in-situ investigation of raphides and will facilitate studies of changes in calcium
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oxalate deposition. If these raphides act as a source or competes for calcium available for
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cross-linking pectins in the apoplast of soft fruit (Perera et al., 1990), the observed cultivar
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differences in size and shape may become important in influencing firmness.
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6
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Conclusions
This work demonstrates X-ray µCT as a research technique to investigate kiwifruit
339
tissue in detail, revealing its pore structure, porosity and raphide features.
X-ray µCT
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revealed the microstructure and showed differences in the cell/void networks near the skin of
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5 different cultivars. Yellow fleshed ‘G3’ was found to have a lower porosity due to a
16
342
majority of small pores, whereas ‘G14’ had the greatest porosity as a result of a high number
343
of larger voids. Other cultivars were intermediary. Orchard management practices (e.g.
344
girdling and crop loading) used to effect fruit quality parameters (mass, firmness, SSC, and
345
DM) did not show any observable differences in microstructure. However, other growth
346
conditions and phenotypic variation may have an effect on porosity and microstructure and
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this remains to be investigated. Raphides were also identified and classified very close to the
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fruit surface with cultivar differences in size and shape characterised, which may influence
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calcium relations (and firmness) in these cultivars.
350
Acknowledgements
351
The work is an output of the Food Structure Design Theme of the Fonterra / Zespri™
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Primary Growth Partnership research project funded by the Ministry of Primary Industries,
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New Zealand. The authors would like to acknowledge the wider staff and student group of
354
the Centre for Postharvest and Refrigeration Research, Massey University, and PlusGroup™
355
Horticulture who donated their time to enable experimental set-up and fruit harvest. Dennis
356
Cantre is an IRO scholar of KU Leuven. Els Herremans is a doctoral fellow of the IWT
357
(Flemish agency for Innovation by Science and Technology).
358
7
359
Antognozzi, E., Boco, M., Famiani, F., Palliotti, A., Tombesi, A., 1995. Effect of different
360
light intensity on quality and storage life of kiwifruit. Acta Horticulturae 379, 483-490.
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8
Tables
507
Table 1. Morphometric parameters and description used to quantify microstructure.
Microstructural
Unit
Description
Volume
mm3
Volume of the sample
Area
mm2
Area of the sample boundary
Specific surface area
mm-1
The surface area of all the solid objects in the sample
parameter
divided by the volume of the object
Porosity
%
Pore volume divided by total volume of the analysed
sample (pores and cells)
Connectivity
(-)
Defined as a measure of the degree to which a structure
is multiply connected. For a network, it represents the
maximal number of branches that can be broken before
the structure is separated into two parts (Odgaard and
Gunderson, 1993).
Pore equivalent diameter µm
The diameter of a sphere of equivalent volume as the
irregularly shaped object (Jennings and Parslow, 1988).
Pore structure thickness
mm
Average of the local thickness of the pores. Calculated
by skeletonisation of the binarised tissue, followed by a
sphere fitting algorithm for each voxel of the skeleton
(Hildebrand and Ruegsegger, 1997).
Fragmentation index
mm-1
Inverse index of connectivity, calculated by comparing
24
volume and surface of the binarised object before and
after an image dilation (Hahn et al., 1992). A lower
fragmentation signifies better connected structures.
508
Herremans et al. (2013a)
509
Table 2. Microstructural parameters of pores and tissue, statistics of the pore size distribution, and peel thickness at eating ripe condition. Mean
510
values are presented with their 95% confidence interval. Values were averaged from 5 fruits per cultivar except for porosity and peel thickness
511
of ‘Hayward’ where values were averaged from 20 fruits. Means followed by different letters are significantly different at 0.05 level of
512
significance.
Cultivar
G9
Hort16A
G3
G14
Hayward
4.6 ± 1.2 (AB)
5.6 ± 1.2 (A)
1.55 ± 0.43 (C)
6.49 ± 0.75 (A)
3.94 ± 0.40 (B)
0.0062 ± 0.0055 (AB)
0.0024 ± 0.0014 (B)
0.0089 ± 0.0040 (A)
0.00256 ± 0.00058 (B)
0.0261 ± 0.0016 (B)
0.0211 ± 0.0013 (C)
0.02933 ± 0.00088 (A)
0.0244 ± 0.0012 (B)
177.1 ± 5.5 (B)
170.8 ± 9.3 (B)
215.2 ± 7.4 (A)
146.8 ± 4.23 (C)
182.7 ± 7.8 (B)
88.1 ± 4.9 (B)
87.6 ± 5.5 (B)
111.5 ± 1.7 (A)
70.8 ± 3.0 (C)
87.0 ± 3.3 (B)
65 × 10 ± 44 × 10 (A)
79 × 10 ± 31 × 10 (A)
145 ± 28 (B)
67 × 10 ± 19 × 10 (A)
87 × 10 ± 56 × 10 (A)
77 ± 20
78 ± 15
73 ± 20
68 ± 15
83.9 ± 9.7
Porosity (%)
Maximal individual pore volume (mm3)
0.0035 ± 0.0014 (B)
Pore structure thickness (mm)
0.0249 ± 0.0010 (B)
Pore specific surface area (mm-1)
Pore fragmentation (mm-1)
Pore connectivity
Peel thickness (µm)
513
514
Table 3. At harvest ‘Hayward’ kiwifruit quality difference as a result of crop load and girdling. Values represent mean and standard deviation (in
515
brackets).
Orchard Treatment
Mass (g)
DM (%)
SSC (°Brix)
Firmness (N)
Porosity (%)
43 t/ha, with girdling
112.8 (11.9)
18.56 (0.01)
7.8 (0.9)
74.1 (6.7)
3.52 (0.36)
43 t/ha, without girdling
104.3 (11.5)
18.84 (0.01)
7.4 (0.8)
81.0 (6.6)
3.97 (0.52)
36 t/ha, with girdling
119.4 (13.3)
19.14 (0.01)
8.4 (0.7)
74.8 (6.5)
3.82 (0.64)
36 t/ha, without girdling
111.9 (11.5)
18.84 (0.01)
7.9 (0.9)
78.2 (6.8)
4.45 (1.47)
90
90
90
90
5
n
516
517
518
519
520
Table 4. Significance table showing the p-value for crop load and girdling effects on at-harvest ‘Hayward’ kiwifruit attributes.
521
p - value
Orchard treatment
Mass
DM
SSC
Firmness
Porosity
(g)
(%)
(°Brix)
(N)
(%)
Crop Loading
< 0.001
0.002
< 0.001
0.138
0.323
Girdling
< 0.001
0.931
< 0.001
< 0.001
0.180
0.680
0.002
0.791
0.014
0.828
Crop Loading * Girdling
28
522
9
Figure captions
523
Fig. 1. Image processing steps of X-ray micro-CT data of kiwifruit tissue: A. grey scale
524
reconstructed X-ray slice (4.4 µm pixel size, scale bar: 1 mm). B. Segmentation of pores by
525
manual threshold. C. Labeling of individual pores. D. 3-D rendering of pores.
526
527
Fig. 2. Typical X-ray microtomograph of commercial kiwifruit cultivars at eating ripeness
528
(A, 4.87 µm pixel size, scale bar: 1 mm), 3-D rendering of the pore structure (B, 400 × 400 ×
529
460 voxel volume), and pore network model (C, 100 × 100 × 100 voxel volume) showing the
530
essential geometry and branching, as well as local thickness (in color bar) of pores.
531
532
Fig. 3. Pore size distribution expressed as volume fraction of individual pores as a function of
533
pore equivalent diameter. Sample volumes analysed (460 × 400 × 400 voxels, 4.87 µm pixel
534
resolution) were extracted in the region immediately adjacent to the skin of commercial
535
kiwifruit cultivars. Values were averaged from 5 fruits per cultivar.
536
537
Fig. 4. Druses within outer kiwifruit tissues visualized by micro X-ray CT. Maximum
538
intensity projection image presents the surface of a ‘G3’ kiwifruit sample showing widely-
539
scattered druses (A, bounding box: 2 mm × 2mm × 2.2 mm). Tomographic CT image shows
540
a transverse reconstructed image of ‘G3’ kiwifruit tissue with druses (B, 2 mm × 2 mm).
541
Surface rendering of typical druses from different kiwifruit cultivars (C, scale: 100 µm).
542
543
Fig. 5. Druse count per volume for each cultivar (A) and its morphometric characteristics
544
showing the mean bundle volume (B), length (C), and sphericity (D) obtained from X-ray CT
545
images (4.87 µm pixel resolution, 460 × 400 × 400 voxel volume). Values were averaged
546
from 5 fruits from each cultivar. Error bar represent 95 % confidence of the mean.
547
30
548
10 Figures
A
B
C
D
549
550
Fig. 1. Image processing steps of X-ray micro-CT data of kiwifruit tissue: A. grey scale
551
reconstructed X-ray slice (4.4 µm pixel size, scale bar: 1 mm). B. Segmentation of pores by
552
manual threshold. C. Labeling of individual pores. D. 3-D rendering of pores.
553
Cultivar
A
B
C
Hayward
Hort16A
G3
G9
G14
554
Fig. 2. Typical X-ray microtomograph of commercial kiwifruit cultivars at eating ripeness
555
(A, 4.87 µm pixel size, scale bar: 1 mm), 3-D rendering of the pore structure (B, 400 × 400 ×
556
460 voxel volume), and pore network model (C, 100 × 100 × 100 voxel volume) showing the
557
essential geometry and branching, as well as local thickness (in color bar) of pores.
32
558
25
G9
Volume fraction (%)
20
Hort16A
G3
15
G14
Hayward
10
5
0
0
559
20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320
Pore equivalent diameter (µm)
560
Fig. 3. Pore size distribution expressed as volume fraction of individual pores as a function of
561
pore equivalent diameter. Sample volumes analysed (460 × 400 × 400 voxels, 4.87 µm pixel
562
resolution) were extracted in the region immediately adjacent to the skin of commercial
563
kiwifruit cultivars. Values were averaged from 5 fruits per cultivar.
564
565
566
A
(C)
Hayward
Hort16A
B
G3
G9
G14
567
568
Fig. 4. Druses within outer kiwifruit tissues visualized by micro X-ray CT. Maximum
569
intensity projection image presents the surface of a ‘G3’ kiwifruit sample showing widely-
570
scattered druses (A, bounding box: 2 mm × 2mm × 2.2 mm). Tomographic CT image shows
571
a transverse reconstructed image of ‘G3’ kiwifruit tissue with druses (B, 2 mm × 2 mm).
572
Surface rendering of typical druses from different kiwifruit cultivars (C, scale: 100 µm).
573
34
7
B
250000
5
Volume (µm3)
Druse count (mm-3)
6
300000
A
4
3
2
200000
150000
100000
1
50000
0
180
0
0.95
160
C
D
0.9
Sphericity
Length (µm)
140
120
100
0.85
80
60
40
0.8
G9
574
Hort16A
G3
G14
Kiwifruit cultivar
Hayward
G9
Hort16A
G3
G14
Hayward
Kiwifruit cultivar
575
Fig. 5. Druse count per volume for each cultivar (A) and its morphometric characteristics
576
showing the mean bundle volume (B), length (C), and sphericity (D) obtained from X-ray CT
577
images (4.87 µm pixel resolution, 460 × 400 × 400 voxel volume). Values were averaged
578
from 5 fruits from each cultivar. Error bar represent 95 % confidence of the mean.