Characterization of Mesocarp and Kernel Lipids from Elaeis

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Characterization of Mesocarp and Kernel Lipids from Elaeis guineensis Jacq., Elaeis oleifera [Kunth] Cortés, and their Interspecific Hybrids Veronika Maria Lieb, Margarete Rosa Kerfers, Amrei Kronmüller, Patricia Esquivel, Amancio Alvarado, Víctor M. Jiménez, Hans-Georg Schmarr, Reinhold Carle, Ralf M. Schweiggert, and Christof Björn Steingass J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 22 Apr 2017 Downloaded from http://pubs.acs.org on April 27, 2017

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Journal of Agricultural and Food Chemistry

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Characterization of Mesocarp and Kernel Lipids from Elaeis guineensis Jacq., Elaeis

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oleifera [Kunth] Cortés, and their Interspecific Hybrids

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Veronika M. Lieb†,*, Margarete R. Kerfers†, Amrei Kronmüller†, Patricia Esquivel‡, Amancio

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○ Alvarado§, Víctor M. Jiménez#,┴, Hans-Georg Schmarr║,▽, Reinhold Carle†, , Ralf M.

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Schweiggert†, Christof B. Steingass†

10 †

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Institute of Food Science and Biotechnology, Chair Plant Foodstuff Technology and Analysis, University of Hohenheim, Garbenstraße 25, 70599 Stuttgart, Germany

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School of Food Technology, Universidad de Costa Rica, 2060 San Pedro, Costa Rica §

14 #

15 ┴

16 ║

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CIGRAS, Universidad de Costa Rica, 2060 San Pedro, Costa Rica

Food Security Center, University of Hohenheim, 70599 Stuttgart, Germany

Dienstleistungszentrum Ländlicher Raum (DLR) Rheinpfalz, Institute for Viticulture and Oenology, Breitenweg 71, 67435 Neustadt an der Weinstraße, Germany

18 ▽

19

Faculty of Chemistry, University Duisburg-Essen, Universitätsstraße 5, 45141 Essen, Germany

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ASD Costa Rica, P.O. Box 30-1000, San José, Costa Rica

○

Biological Science Department, King Abdulaziz University, P.O. Box 80257, Jeddah 21589, Saudi Arabia

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*

Corresponding author. Tel.: +49 711 459 23041. Fax: +49 711 459 4110. E-mail: veronika.lieb@uni-

hohenheim.de ACS Paragon Plus Environment


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Abstract

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Morphological traits, total lipid contents, and fatty acid profiles were assessed in fruits of

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several accessions of Elaeis oleifera [Kunth] Cortés, Elaeis guineensis Jacq., and their

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interspecific hybrids. The latter featured the highest mesocarp-to-fruit ratios (77.9–78.2%).

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The total lipid contents of both E. guineensis mesocarp and kernel were significantly higher

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than for E. oleifera accessions. Main fatty acids comprised C16:0, C18:1n9, and C18:2n6 in

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mesocarp, and C12:0, C14:0, and C18:1n9 in kernels. E. oleifera samples were characterized

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by higher proportions of unsaturated long-chain fatty acids. Saturated medium-chain fatty

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acids supported the clustering of E. guineensis kernels in multivariate statistics. Hybrid

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mesocarp lipids had an intermediate fatty acid composition, while their kernel lipids

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resembled those of E. oleifera genotypes. Principal component analysis based on lipid

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contents and proportions of individual fatty acids permitted clear-cut distinction of E. oleifera,

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E. guineensis, and their hybrids.

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Keywords: Elaeis guineensis; Elaeis oleifera; interspecific hybrids; mesocarp lipids; kernel lipids

ACS Paragon Plus Environment

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Introduction

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The African oil palm (Elaeis guineensis Jacq.) represents one of the most important oil crops.

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A total of 73 million tons of palm oil was produced in 2016, equaling ~40% of the global

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vegetable oil demand. The most important producers, Indonesia and Malaysia, supply 85% of

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palm oil used worldwide. Approximately 70% of the world production is used in numerous

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applications in the food industry,1 owing to its low costs and distinctive technological

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properties such as its high slip melting point at 33–39 °C.2 E. guineensis fruits are classified

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into thick-shelled (f. dura), shell-less (f. pisifera), and a thin-shelled dura × pisifera cross (f.

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tenera).3 The immense demand of palm oil resulted in extensive E. guineensis monocultures,

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being publicly criticized due to a number of severe environmental issues like deforestation

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and degradation of habitats of endangered animal species.4 In particular, progenies of

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E. guineensis dura palms are prone to bud rot disease.5 Diversification of the currently used

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Elaeis species might mitigate the incidence or might even prevent such a disease.

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Native to Central and South America, the American oil palm (Elaeis oleifera [Kunth] Cortés)

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has been found to be genetically highly concordant with E. guineensis.6 Its oil has several

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desired traits such as a fatty acid profile and melting points similar to those of E. guineensis.

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Furthermore, the oil was reported to contain high concentrations of provitamin A carotenoids

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and vitamin E.7 Nevertheless, E. oleifera is so far not being cultivated on a commercial scale,

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but is exploited for domestic consumption by traditional communities.3,

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E.oleifera is grown at experimental stations as ex-situ germplasm collection and represents a

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highly interesting genetic resource to breed interspecific hybrids with E. guineensis.9 Such

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hybrids have been reported to be less susceptible to bud rot disease.5,

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preliminary analyses have suggested that the oil contains elevated proportions of nutritionally

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relevant micronutrients, particularly oleic acid as well as the aforementioned (pro-)vitamins.

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Consumption of crude palm oil from interspecific hybrids has been shown to induce effects


8

10

Additionally,

In particular,


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on human plasma lipids similar to those of olive oil,11 which is well-known for its numerous

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putative health benefits.12

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This present study provides an in-depth insight into the diversity of Elaeis mesocarp and

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kernel lipids. Morphological fruit traits, total lipid contents, and fatty acid profiles of six

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E. oleifera, two E. guineensis, and two of their interspecific hybrid accessions were assessed.

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We sought to present a methodology for the future authentication of oils obtained from

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different oil palm species using GC-based fatty acid profiling and a subsequent principal

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component analysis (PCA). Moreover, the broad diversity of Elaeis-fatty acid profiles

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reported herein may support the production of tailored palm oils as well as the future targeted

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research into palm oil substitutes.

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Materials and methods Plant material

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All assessed fruit samples were gathered in a genetic collection established in Costa Rica. The

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sampling comprised four accessions of E. oleifera originating from Suriname ('Surinam'),

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Brazil ('Manaos 03', 'Manaos 79'), and Ecuador ('Taisha') as well as two intraspecific

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E. oleifera hybrids, namely 'CA/Col' and 'Manaos/Taisha' ('Manaos 03' × 'Taisha'). Two

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E. guineensis accessions with origin from Papua New Guinea ('Deli dura Dami') and

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Tanzania ('Tanzania dura') as well as the two interspecific E. guineensis × E. oleifera hybrids

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'Compact' and 'Amazon' were included. The hybrid 'Compact' has been back-crossed thrice

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with its E. guineensis parent to foster properties typical of E. guineensis genotypes.13

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'Amazon' represents a hybrid of the E. oleifera accession 'Manaos 03' and the aforementioned

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hybrid 'Compact' (Table 1 and Figure 1). All palms were grown in Coto (Puntarenas, Costa

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Rica) and harvested from October to December 2014. One palm from each genotype was

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randomly selected for collecting the fruits.


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In addition to fresh oil palm fruits, approximately 50 g fruit were sterilized at 140 °C for

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70 min using a laboratory autoclave steam sterilization system to mimic industrial practice.

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Subsequently, mesocarp and kernels were dissected. Samples were lyophilized in a laboratory

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freeze dryer, milled under liquid nitrogen with a laboratory blender, and stored in vacuum-

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sealed aluminum pouches at -80 °C until further analyses.

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Chemicals

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All reagents and solvents were purchased from Merck (Darmstadt, Germany) and VWR

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International (Darmstadt, Germany). Boron trifluoride-methanol solution (14% BF3 in

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methanol), cis-11-vaccenic acid methyl ester, Supelco 37 component FAME mix, and C7–

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C30 saturated alkanes standards were purchased from Sigma-Aldrich Chemie (Steinheim,

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Germany).

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Morphological traits

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Total fresh weights of ten single, manually washed fruits of each genotype were determined

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gravimetrically. Proportions of mesocarp, shell, and kernel were determined from a pooled

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sample. Total mesocarp ratio was expressed as the sum of mesocarp and peel weights relative

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to the total fruit weight.

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Total lipid and fatty acid analysis Sample preparation

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Total free lipids of freeze-dried samples were extracted for 2 h using boiling n-hexane and a

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Soxhlet apparatus. Palm kernel samples were subjected to hydrolysis with concentrated

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hydrochloric acid prior to the hexane extraction according to the Weibull-Stoldt method.14

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After complete evaporation of the organic solvent, total lipids in dry matter (% DM, w/w)



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were quantitated gravimetrically. The obtained extracts were placed in glass tubes, headspace-

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flushed with nitrogen, and stored at -20 °C until further analysis.

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Alkaline hydrolysis and derivatization of extracted oils were performed according to

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previously published protocols15, 16 with some modifications. In brief, a 10–20 mg aliquot of

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the extract was combined with 1 mL of 10% (w/v) methanolic KOH and heated to 80 °C for

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10 min. After adding 2 and 1 mL methanolic BF3 to mesocarp and kernel samples,

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respectively, heating was continued for further 5 min. Subsequently, 2 mL of 10% (w/v)

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NaClaq were added, and fatty acid methyl esters (FAMEs) were extracted using 2 mL n-

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hexane prior to GC analyses.

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GC analysis

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FAMEs were analyzed using a CP 9001 gas chromatograph (Chrompack, Middelburg, The

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Netherlands) equipped with a 30 m × 0.25 mm i.d., df = 0.25 µm, fused silica capillary

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column coated with polyethylene glycol (Supelcowax-10, Supelco, Bellafonte, PA), a CP

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9010 autosampler and a flame ionization detector. Maestro Chromatography Data System

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software, ver. 2.4 (Chrompack) was applied for system control and data acquisition. Helium

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was used as a carrier gas at a constant inlet pressure of 120 kPa and an initial flow rate of

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1.2 mL/min. The injection volume was 1 µL at a split ratio of 1:55. Both injector and detector

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temperatures were set to 250 °C. The temperature program was as follows: isothermal hold at

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120 °C for 1 min, constant raise (4 °C/min) to a final temperature of 240 °C held for further

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5 min (total run time 36 min). The relative fatty acid composition was calculated as area % of

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the total peak area.

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FAMEs were identified using a 6890N gas chromatograph equipped with a split/splitless

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injector coupled to a 5975 Mass Selective Detector (both Agilent Technologies, Santa Clara,

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CA). Chromatographic separation was achieved using a 30 m × 0.25 mm i.d., df = 0.25 µm,

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fused silica capillary coated with a comparable polyethylene glycol stationary phase (DBACS Paragon Plus Environment

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Wax, Agilent J&W Columns, Santa Clara, CA). Helium was used as carrier gas at a constant

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flow of 1.2 mL/min and the aforementioned oven temperature program. Mass spectra were

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recorded in the electron impact positive (EI+) mode at a scan range of m/z 40–350 (scan

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frequency 4.51 Hz) between 2 to 14 min, and m/z 40–450 (3.5 Hz) for the final segment.

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Source and transfer line temperatures were set to 230 °C and 240 °C, respectively. Individual

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FAMEs were assigned by comparing their mass spectra to those of the Wiley 6 N library

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(Wiley and Sons, New York, NY). In addition to the molecular ion [M]+, indicative FAME

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mass fragments were [M - 32]+ and [M - 74]+. Moreover, linear retention indices (LRIs)

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according to Van den Dool and Kratz17 were calculated relative to C7–C30 alkanes.

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Experimental LRIs were compared to those of retention standards (Supelco 37-component

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FAME mix, cis-11-vaccenic acid methyl ester).

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Statistics

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Univariate statistical analysis was performed with SAS 3.5 software (SAS Institute Inc., Cary,

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NC). Significant differences of means (p < 0.05) were determined applying analysis of

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variance (ANOVA) and Tukey’s multiple-range test.

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Multivariate data analysis was conducted for pattern recognition. Unsupervised hierarchical

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cluster analysis (HCA) and principal component analysis (PCA) were calculated based on

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total lipid contents and the relative composition of fatty acids with a % peak area of ≥1.0%

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using Solo software version 8.1.1 (Eigenvector Research, Wenatchee, WA). Data was pre-

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processed using the “autoscale” function, i.e., being mean centered and weighing all variables

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by their standard deviation to ensure homogeneity of variances.18 Contiguous block cross-

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validation with 12 PCs and 6 data splits was used for PCA. HCA was performed using

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Ward’s method of agglomeration and Euclidean distances.

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Results and discussion Morphological traits

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Total fruit weights varied significantly between the analyzed Elaeis accessions (Table 1).

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Most E. oleifera fruits (3.3–12.4 g) were lighter than those of E. guineensis (10.3–19.2 g). In

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particular, the E. guineensis accession 'Deli dura Dami' had significantly higher fruit weights

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(19.2 ± 2.9 g per fruit) compared to all other accessions. In accordance with our findings, the

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fruit weights of Colombian 'Deli' cultivars have been earlier reported to reach comparably

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high fruit weights of up to 21.4 g, despite some considerable variability.19 In contrast, fruits of

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the E. guineensis accession 'Tanzania dura' were significantly lighter compared to the

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assessed 'Deli' cultivar. Dura samples from Tanzania featured high fruit weights of

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16.9 ± 4.1 g as summarized by Corley and Tinker.3

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In comparison to our E. guineensis accessions, the E. oleifera accessions 'CA/Col' (3.3 ± 0.5 g

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per fruit) and 'Surinam' (3.4 ± 0.7 g per fruit) were characterized by small fruit weights. These

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results are in agreement with previous morphological studies.8,

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weights of 'Taisha' (E. oleifera) and intraspecific E. oleifera hybrid 'Manaos/Taisha' reached

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on average 10.4 and 12.4 g per fruit, respectively, thus being in the range of the E. guineensis

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fruits (10.3–19.2 g per fruit). Fruits of three E. oleifera accessions with Brazilian origin, i.e.

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'Manaos 79', 'Manaos 03', and 'Manaos/Taisha', had total fruit weights between 4.7–12.4 g per

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fruit being in accordance with previous reports summarized by Ooi et al.20

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The fruit weights of the interspecific back-cross 'Compact' (94% E. guineensis) were

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significantly lower (4.7 ± 0.6 g per fruit) than those of E. guineensis accessions. The hybrid

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'Amazon' was characterized by a higher fruit weight of 7.8 ± 2.1 g per fruit in comparison to

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'Compact' (Table 1).

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Compared to literature data,8, 20 our assessed E. oleifera samples were characterized by higher

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mesocarp (38.7–86.0%) and reduced kernel proportions (2.8–7.5%). In agreement with our

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results, a broad diversity of morphological characteristics has been reported among Brazilian ACS Paragon Plus Environment

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Nevertheless, the fruit

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oil palm fruits.9,

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parthenocarpic fruits.21 In our study, the parthenocarpic E. oleifera accession 'Manaos 03' was

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characterized by small seeds without kernel resulting in a low shell ratio of 14.0% and a high

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mesocarp proportion of 86.0%. Such parthenocarpic fruits might be of high commercial

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interest for the production of mesocarp oils. Interestingly, the second Manaos accession

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'Manaos 79' produced non-parthenocarpic fruits, which showed lower mesocarp (38.7%) and

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higher shell (53.8%) fractions. Mean mesocarp proportions among other non-parthenocarpic

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E. oleifera accessions were highest in 'Taisha' (68.1%). Similarly, Arias et al.9 have described

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an Ecuadorian 'Taisha' accession with a mesocarp-to-fruit ratio of 63.7%. The E. oleifera

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accession 'Surinam' investigated herein featured a lower kernel-to-fruit ratio (7.5%) compared

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to palm fruits from Suriname (17%) as reviewed earlier.20

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Overall, E. guineensis mesocarp proportions varied between 46.4 and 62.2% in 'Tanzania

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dura' and 'Deli dura Dami', respectively. In agreement, dura fruits were earlier reported to

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feature mesocarp portions between 60–65.2%.21-23 A mesocarp proportion of 46.7 ± 10.1%

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has been reported in Tanzanian dura cultivars as reviewed by Corley and Tinker.3 The kernel

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content of 'Deli dura Dami' was lower (3.5%) compared to 'Tanzania dura' and literature.21-23

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The assessed E. guineensis accessions were thick-shelled, resulting in higher shell proportion

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(34.3–43.9%) than dura cultivars described in literature (28.9–32%).21-23

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In agreement with mesocarp ratios of hybrids (67.2–75.0%) reported in literature,13, 20 our

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interspecific hybrids 'Compact' (77.9%) and 'Amazon' (78.2%) displayed highest mesocarp

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ratios of all non-parthenocarpic palm fruits. Remarkably, these hybrids were further

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characterized by the lowest shell (13.8%, 17.1%) and high kernel proportions (5.0%, 7.9%).

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In accordance with earlier findings summarized by Ooi et al.,20 hybridizing the F1 progeny

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'Compact' (77.9%) with 'Manaos 03' (86.0%) of our study apparently reduced the mesocarp-

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to-fruit ratio of the resulting interspecific hybrid 'Amazon' (78.2%). Simultaneously, the

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kernel proportion increased after hybridizing (Table 1). In general, the morphological

Several E. oleifera genotypes have been described previously to bear



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characteristics of interspecific hybrids were described to depend on the fruit morphology of

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their E. guineensis parent. The inheritance of their mesocarp-to-fruit ratios has been shown to

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be incompletely dominant, but still determined by the E. guineensis parent.20 Hence,

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mesocarp proportion of the 'Amazon' hybrid was expectedly similar to that of its E. guineensis

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parent 'Compact'. The interspecific hybrid 'Amazon' featured lowest shell proportions (13.8%)

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of all fruits being in agreement with previous data3 describing such E. oleifera × E. guineensis

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pisifera hybrids as being thin-shelled.

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Total lipids content Mesocarp total lipids

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Total lipid contents in dry matter (DM) of fruit mesocarp varied from 16.6% to 83.6% when

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considering all accessions (Table 2). Earlier reports have indicated total lipids of E. oleifera,

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E. guineensis, and hybrid mesocarp to range from 13.1–86.5%.24 In the present study, the

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mean total lipid contents of E. guineensis and hybrids were significantly higher than those of

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all E. oleifera accessions. These findings are in agreement with previously published

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studies.21,

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between those of E. guineensis and E. oleifera.21, 24 In our study, total lipid contents of hybrids

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were significantly higher than E. oleifera while being similar to those of the two E. guineensis

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samples assessed. Besides their significantly lower total lipid contents, E. oleifera fruits

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exhibited a wide range of total lipid contents from 17.4 ± 0.8% in 'Taisha' to 59.0 ± 0.5% in

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'Manaos 03' fruits. Our findings are in agreement with literature, reporting total lipids of

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E. oleifera fruits vary from 21.4–9.4%.8, 24, 25 The differences among E. oleifera accessions

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might possibly be related to their elevated genetic diversity compared to the E. guineensis

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species as reported by Barcelos et al.6 In particular, parthenocarpic fruits have high oil

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concentrations in the dry mesocarp21 such as 'Manaos 03' (59.0%).

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The lipid contents of hybrids have been described as being intermediate

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Kernel total lipids

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Total lipid contents in E. guineensis kernels were significantly higher compared to those of

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E. oleifera samples. In contrast to the broad variation found in E. oleifera mesocarp (17.4–

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59.0%), E. oleifera kernel lipids ranged only from 37.2–43.9%, as has also been observed in

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Colombian E. oleifera kernels.26 E. guineensis kernels featured significantly higher total lipid

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contents (51.3%, 44.7%). These findings are in agreement with earlier reported total lipid

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contents of E. guineensis kernels.27, 28 Interspecific hybrids have been reported to come close

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to the kernel oil content of their parental E. guineensis genotype.20 In our study, 'Amazon'

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bread from 'Manaos 03' and 'Compact' had total lipid contents (38.2 ± 0.3%) similar to those

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of the E. oleifera kernels. However, triple back-crossing with E. guineensis apparently

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resulted in comparable high total lipid contents in 'Compact' kernels (52.1 ± 1.1%) than those

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of assessed E. guineensis samples.

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Fatty acid profiles Mesocarp fatty acids

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The principal fatty acids found in palm mesocarp oil were C14:0, C16:0, 16:1n7, C18:0,

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C18:1n9, C18:1n7, C18:2n6, and C18:3n3 (Table 2 and Figure 2A). The minor fatty acids

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C12:0, C15:0, C17:1n7, C20:0, and C20:1n9 were found in trace amounts (each

Characterization of Mesocarp and Kernel Lipids from Elaeis

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