Consumer and health-related traits of seed from selected commercial and breeding lines of industrial hemp, Cannabis sativa L.

Journal Pre-proof Consumer and health-related traits of seed from selected commercial and breeding lines of industrial hemp, Cannabis sativa L Carolyn J. Schultz, Wai L. Lim, Shi F. Khor, Kylie A. Neumann, Jakob M. Schulz, Omid Ansari, Mark A. Skewes, Rachel A. Burton PII:

S2666-1543(20)30006-5

DOI:

https://doi.org/10.1016/j.jafr.2020.100025

Reference:

JAFR 100025

To appear in:

Journal of Agriculture and Food Research

Received Date: 8 October 2019 Revised Date:

6 January 2020

Accepted Date: 30 January 2020

Please cite this article as: C.J. Schultz, W.L. Lim, S.F. Khor, K.A. Neumann, J.M. Schulz, O. Ansari, M.A. Skewes, R.A. Burton, Consumer and health-related traits of seed from selected commercial and breeding lines of industrial hemp, Cannabis sativa L, Journal of Agriculture and Food Research, https:// doi.org/10.1016/j.jafr.2020.100025. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier B.V.

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Yunma-1 Yunma-2 ECO_202 ECO_209 Han-NE ECO_16AH ECO_222 USO-31 KC Dora ECO_YMR17 ECO_MR17 ECO_254 Midlands S Midlands X ECO_253 ECO_FR17 ECO_AMB17 Ferimon 12 ECO_50GC Frog-1

1000 seed weight (g)

70

40 65

30 60

20 55

10 50

0

Heart %

50

Lignin (%w/w)

H B C

E testa

radicle

I J

K

L F Neg

Neg

D

G

25 70

20 65

15 60

10 55

5 50

0 0

Heart %

ECO_209 ECO_222 Ferimon 12 ECO_AMB17 ECO_202 ECO_253 ECO_FR17 Midlands X USO-31 ECO_254 Han-NE KC Dora Midlands S ECO_YMR17 ECO_16AH Yunma-2 ECO_50GC Frog-1 ECO_MR17 Yunma-1

0

ECO_209 ECO_222 Ferimon 12 ECO_AMB17 ECO_202 ECO_253 ECO_FR17 Midlands X USO-31 ECO_254 Han-NE KC Dora Midlands S ECO_YMR17 ECO_16AH Yunma-2 ECO_50GC Frog-1 ECO_MR17 Yunma-1

Crystalline cellulose (%w/w)

A

40

30

20

10

CBM3a

Negative

CBM3a

cotyledon

plumular leaf

A

B

C

D

Coomassie

Oil red + calcoflor white

E

K

e F

j

f G

h i

g

H

I

J

A

Glycan class Antibody

Hull

Heart

B

Neg

C

LM10

D

Neg

E

LM11

F

LM11 + arabinofuranosidase

G

Neg

H

LM19

I

LM19 + Na2CO3

J

Neg

K

LM19

L

LM19 + Na2CO3

A

LM15

B

LM15

C

LM15

D

LM19

E

LM19

F

LM19

G

LM19 +Na2C03

H

LM19 +Na2C03

I

LM19 +Na2C03

J

LM20

K

LM20

L

LM20

0 18:3n-6 (GLA)

18:2n-6 (LA)

18:3n-3 (ALA)

18:1n-9 (OA)

18:0 (SA)

16:0 (PA)

% Total Lipids 80

60

40

20

Category Seed traits Cellulose Complex carbohydrates (sugars) Starch Soluble sugars Protein

Lipids

Antinutrients CV <10%

Trait

CV

1000 seed weight heart % crystalline cellulose xylose (hulls) arabinose (hulls) galactose (hulls) resistant starch non-resistant starch sucrose (hull) sucrose (heart) raffinose (heart) protein total lipid total saturated fats total monounsaturated linoleic acid (LA, ω6) α-linolenic acid (ALA, ω3) oleic acid (OA, ω9) palmitic acid (PA) steric acid (SA) γ-linolenic acid (GLA, ω9) phytate lignin CV 10–19.9%

CV 20–29.9%

40.1 5.5 11.5 23.5 21.7 17.0 6.7 22.75 40.4 19.4 43.9 7.4 8.4 7.9 13.6 3.2 16.8 14.7 9.5 9.4 63.9 16.0 5.7 CV ≥30%

1

 

Highlights • • • • •

Hemp cultivars and breeding lines have variation for omega-3 α-linolenic acid Compositional analysis of heart (kernel) and hull (husk) fractions of hemp seed Hemp seed contain non-cellulosic dietary fiber including xylan, xyloglucan & pectin Hemp hearts are low in starch (<2%) and soluble sugars Hemp proteins are found in protein bodies

Consumer and health-related traits of seed from selected commercial and breeding lines of industrial hemp, Cannabis sativa L. Carolyn J. Schultza,b, Wai L. Lima, Shi F. Khora,b, Kylie A. Neumanna,b, Jakob M. Schulza, Omid Ansaric,d,e, Mark A. Skewesf, Rachel A. Burtona,b* a

School of Agriculture, Food, and Wine, University of Adelaide, Waite Campus, Glen Osmond, SA,

Australia b

The Australian Research Council Centre of Excellence in Plant Energy Biology, The University of

Adelaide, Glen Osmond, SA, Australia. c

Ecofibre Limited, Brisbane, Qld, Australia

d

e

Ananda Foods Pty Ltd, NSW, Australia

Ananda Hemp Ltd, Cynthiana, Kentucky, USA

f

South Australian Research and Development Institute, GPO Box 397, Adelaide SA, Australia

* Correspondence: Rachel A. Burton [email protected] Keywords: dietary fiber, cellulose, plant polysaccharides, xylan, immuno-histochemical microscopy, protein bodies. Abstract Global food security and sustainability can be enhanced with increased production and use of hemp seed (Cannabis sativa L.) that not only provides healthy sources of protein and oil, but the whole plant can be used for fiber, especially new applications including high-performance textiles. There is limited knowledge of the variation in hemp seed for health and consumer traits, such as the complex plant polysaccharides comprising dietary fiber. To fill this gap, seed from 20 hemp cultivars and advanced breeding lines were analyzed for a variety of traits including complex carbohydrates, protein, lipids, heart percent, phytate and lignin. The analyses revealed different polysaccharides in

hull and heart fractions, with hulls varying in cellulose (22.0–36.7%) and xylan (5.7–17.1%) whereas heart fractions contained xyloglucan and pectin, and comprised different proportions of heart and hull. Staining revealed that cellulose and lignin are localized in hull not heart tissue. Protein content of hemp seed ranged from 19.5 –26.9% and Coomassie staining showed that cells of hemp hearts contain numerous protein bodies but few starch granules. Analysis of selected lines confirmed that hemp hearts are low in starch (<2%). Lipid content varied from 26.6–37.8%, with the omega-6:3 ratio varying from 2.1 to 4.9 with seven lines having a ‘desirable’ omega-6:3 ratio of less than or equal to three. This research provides breeders with additional traits to consider for maximizing consumer satisfaction and human health. 1

Introduction

Industrial hemp, Cannabis sativa L. is a multifaceted crop that is increasingly being considered by farmers looking to diversify and reduce their ecological footprint. Industrial hemp is grown for both fiber and food, and is also used in cosmetics and for medicinal and nutraceutical/ pharmaceutical purposes [1,2]. A major factor contributing to the sustainability of hemp is that most parts of the plant can be used, and innovation is contributing to additional uses of hemp for biofuels [2,3], highperformance textiles [4] and natural insecticides [5]. Hemp has long been used as a food source and in traditional medicine (reviewed in Callaway [6]), and this has stimulated research into its use as a functional food [6,7]. In addition to its healthy oil and protein profiles, several bioactive peptides have been found in hemp seed [8-10], as well as polyphenols and other effective antioxidants [11]. Hemp seed should be relatively easy to get to food markets as seed processing can use existing production facilities with some adjustments [12]. Hemp seed is actually an achene or nut, with a hard outer shell (pericarp, also called hull), a papery testa, and an inner seed (or heart) [13]. Industrial hemp is classified as Cannabis plants with low levels of ∆9-tetrahydrocannabinol (THC, <0.1–1%), depending on the country and state [2,14]. It was recently estimated that the global production of hemp will double in the four years from 2016 to 2020 [2], and some states in Australia have already more than doubled their production (Omid Ansari, personal communication). This resurgence of interest in hemp has led to an increasing number of states in the USA growing low THC hemp [15], and as of 2017 all states in Australia have legalized cultivation of industrial hemp for food and fiber applications [14]. 2

In comparison to other crops hemp has been described as sustainable because it is relatively resistant to biotic and abiotic stresses thereby reducing agronomic inputs [16]. In temperate Australia industrial hemp is grown as a summer crop and requires supplemental irrigation. Hemp has a relatively high water requirement compared to dryland cereals, although much less (70% less) than cotton [12,17-19]. A major advantage of hemp from an agronomic point of view is the rapid growth of seedlings such that it effectively outcompetes weeds [20]. This and the fact that it has relatively few pests and diseases makes hemp better suited for organic production than many other row crops [12,21,22]. In Australia, some pests and diseases have been observed, but in most cases there has been no significant impact on productivity [14]. Industrial hemp is suited to a wide variety of soil types as long as they are fertile with good drainage and water holding capacity [1,14]. When C. sativa is grown for fiber, tall varieties are used because of their increased biomass. Shorter varieties are being bred with higher harvest index that are specifically targeted to food markets [15,23]. Characterization of FINOLA seed showed that it contains approximately 30% oil and 25% protein [6]. Analysis of lipids of FINOLA seed revealed that over 80% are polyunsaturated including two essential fatty acids, linoleic acid (LA, 18:2 omega-6) and α-linolenic acid (ALA, 18:3 omega-3). Hemp seed contains a ratio of omega-6 to omega-3 in a desirable range between 2:1 and 3:1 [6]. Current western diets are generally deficient in omega-3 fatty acids, with a high (>15:1) ratio of omega-6:omega-3 [24-27], due to the increased intake of vegetable oils including sunflower and corn oils, which have ratios of >50:1 omega-6:omega-3 ([25], http://ndb.nal.usda.gov/ndb/search). Another benefit of hemp seeds is that they contain proteins that are rich in several essential amino acids including arginine and the sulfur-rich amino acids methionine and cysteine [6]. Hemp contains other known beneficial and nutraceutical compounds (e.g. polyphenols) [7,11,28] but also some antinutrients including phytate (phytic acid) and trypsin inhibitor that can impact on nutrient uptake especially of minerals (phytate) and amino acids (both phytate and protease inhibitors) [11,29]. However rat model studies confirm the high bioavailability of hemp proteins [30] suggesting that the antinutrients may not be a major concern. A disadvantage of one of the earliest seed cultivars, FINOLA, is that the seed is about 50% smaller than other seed varieties [2]. Comparisons between different lines are usually based on thousand seed weight (TSW). A recent comparison of 33 lines showed TSW ranged from approximately 7.5 g to 23 g, with FINOLA at around 12 g [2], which is at the lower end of the expected range of 12–15 g for FINOLA [31]. Seed size is not the only important consideration for seed varieties. A higher

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proportion of heart to hull tissue (by weight), here called heart %, is desirable since it is the heart that attracts a higher value margin. A study of five hemp cultivars in Romania showed that heart % varied from 59.0% to 69.5% [32]. However, current data on the heart % trait is limiting with very few published studies. Hemp hearts, also known as dehulled seed have more protein and more “digestible” fiber than whole seed and seed meal (milled seed after oil extraction), based on analysis of a “typical” variety [33]. Interpretation of fiber data in food products can be difficult, due to the use of different terminology and methods [34]. Scientific papers often report both neutral detergent fiber (NDF) and acid detergent fiber (ADF), where NDF includes cellulose, ‘hemicellulose’, and lignin whereas ADF only includes cellulose and lignin, thus providing a measure of non-fermentable fiber [35]. One disadvantage of NDF and ADF is that they underestimate the amount of total fiber, as the methods may not recover all of particular components such as pectins and gums [35], which can be some of the healthier fermentable fibers. However, this information is useful because it provides an indirect way to determine lignin content, and high levels of lignin can negatively affect palatability [36]. The nature of the non-cellulosic polysaccharides that contribute to dietary fiber in hemp has not yet been studied. One review suggests that hemp seed contain 25% starch [2], however the two cited references report soluble (or digestible) fiber and not starch [6,29]. Since “detailed chemical characterization of dietary fiber is crucial to explain its effect on health” [37], there is a clear knowledge gap of the nature of the polysaccharides in hemp lines grown for food. Different cell wall polysaccharides have remarkably different functional properties (e.g. viscosity, water holding capacity, nutrient binding) and these in turn influence fermentability, nutrient bioavailability and composition of gut microbes [37-41]. The role of complex carbohydrates in microbiome diversity and human health is becoming clearer [42], and many positive effects relate to the fermentation products or short chain fatty acids (SCFA) such as butyrate, acetate and lactate [40,41,43,44]. It has long been established that plant polysaccharides can differ in length of backbone, number and distribution of side branches and the form of each monosaccharide (furanosyl (f) and pyranosyl (p)) and the linkages between them (α and β) [37,45-47]. Even within the same general class (e.g. xylans) the structures can be markedly different. For example, heteroxylans from different species of Plantago (source of psyllium husks) have different proportions of unsubstituted xylan and linkages along the xylan backbone [48]. It is now understood that microbes exist in cooperative metabolic networks where selected bacterial species initiate degradation and other species continue fermenting 4

the partially degraded polysaccharides, thus supporting microbial biodiversity and a healthy colon [37,43,49]. To fill the knowledge gap on the nature of hemp polysaccharides, we analyzed the composition of 20 different industrial hemp varieties and breeding lines that are currently available in Australia. We report analysis of 1000 seed weight, heart %, % nitrogen (N), lipid profiles, lignin, phytate, and a detailed analysis of the carbohydrates in hemp heart and hull fractions including cellulose, starch, monosaccharide composition of complex carbohydrates, and soluble sugars. In addition, we use specific antibodies to investigate the distribution of plant polysaccharides in hull and heart fractions, thus providing critical information on the distribution of non-cellulosic polysaccharides in hemp seed. 2

Material and Methods

2.1

Genetic resources

Seed for 20 different varieties and breeding lines of Cannabis sativa L. were obtained from within Australia. The lines included seed and dual purpose (seed and fiber) lines representing international varieties and Australian selections/ breeding lines as outlined in Table 1. 2.2

Determination of 1000 seed weight and heart %

One hundred seed were counted out and weighed, then multiplied by 10 to provide 1000 seed weight (n=2 technical replicates). A single replicate of 100 seed was selected for further analysis. Hull and heart tissues were separated using a fine spatula and the separated fractions weighed. The separated hull and heart fractions were ground prior to carbohydrate analysis. Hull fractions were ground in a retsch mill for 30 s, and the softer heart fractions were ground in liquid nitrogen and air dried prior to analysis. These ground fractions were used for quantification of cellulose and soluble sugars and monosaccharide analysis (both heart and hull fractions), lignin (hull only) and starch (heart only) (see below). 2.3

Quantification of cellulose

Crystalline cellulose was analyzed separately in hull and heart fractions (40 mg each, n=2 technical replicates) using an acetic/nitric acid hydrolysis method [50], determined by weight instead of colorimetric assay. Due to high amounts of oil in each sample, the following washes were done to 5

obtain an alcohol insoluble residue (AIR) 1x 70% ethanol, 2x 100% ethanol, 1x acetone and 1x methanol. Washes (2.5 mL) were incubated at room temperature on a rotary shaker for 20 min. After washing, samples were oven-dried at 70°C for 30 min. 2.4

Lignin quantification and staining

Lignin (%) in ground hemp hulls was determined using the acetyl bromide method based on Barnes and Anderson [51], omitting the destarching (DMSO treatment) step, and with minor modifications to the AIR preparation. Modifications included all washing steps performed in a thermomixer (Eppendorf, Macquarie Park, Australia) at 25°C and 1000 revolutions per minute for 30 min, except the 1:1 chloroform:methanol wash (10 min). A 100% ethanol wash was added after the 70% ethanol wash (and prior to the 1:1 chloroform:methanol wash). Lignin staining was adapted from Liljegren [52], as follows. Seed (Midlands X variety) were cut in half with sharp razor blade and placed into 1.5 mL tube containing 100 µl of 2% phloroglucinol dissolved in 95% ethanol and incubated at room temp for 2 min. Then 100 µl of 50% concentrated HCl in water was added and incubated at room temp for 30 seconds with 2–3 inversions. Seed halves were removed and dried on a kimwipe™ (Kimberly-Clark™ Milsons Point, NSW, Australia). Seed halves were mounted on a slide with blu tack and imaged within 30 min. Midlands X was chosen for microscopy as it had an intermediate phenotype for many of the traits assayed early in the study (e.g. total lipid, heart %, cellulose and xylose (monosaccharide)). 2.5

Soluble sugars and monosaccharide analysis

Two technical replicates were performed for each ground hull and heart sample. Twenty mg of each sample was processed to give an alcohol soluble and alcohol insoluble fraction, using 2 x 70% ethanol washes (pooling the supernatants) [47]. For analysis of soluble sugars, the alcohol soluble fraction was dried and resuspended in ultrapure Milli-Q (MQ) water and quantified by high performance anion exchange chromatography. Monosaccharide profiles of complex carbohydrates were obtained by sulfuric acid hydrolysis of the alcohol insoluble reside and were determined using reverse phase-high performance liquid chromatography of 1-phenyl-3-methyl-5-pyrazoline derivatives as per Comino et al. [53]. Calibration curves for each soluble sugar ranged from 0.125 mg L-1 to 4 mg L-1 and for monosaccharide analysis: glucose (160–4000 µM), arabinose and xylose (20– 500 µM) and all other sugars (12–300 µM).

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2.6

Starch quantification and starch and protein staining

Total starch was determined on hemp hearts (six lines, two technical replicates) as the sum of enzyme resistant and non-resistant starch using a resistant starch assay kit (K-RSTAR, Megazyme, Bray, Ireland), compared to the included resistant starch control. Due to high amounts of oil in each sample, the assay was performed on an AIR preparation, using the same method as for the cellulose assay, except that the ethanol and acetone washes were incubated on ice on a rotary shaker for 20 min, with the exception of the methanol wash which was performed at room temperature for 10 min. After washing, samples were oven-dried at 70°C for 30 min. To stain for starch, whole seeds (Midlands X variety) were dehydrated through an ethanol series and embedded in LR white [54]. Seeds were sectioned (1 µm) using a microtome (Histo diamond knife, DiATOME, Hatfield Pennsylvania, USA) and stained on slides with a 2% potassium iodine (SigmaAldrich, Castle Hill, Australia), 1% iodine (Sigma-Aldrich) solution in MQ water for 2 min, rinsed 3 times with water and covered with 50% glycerol for imaging. To stain for protein, whole seeds were dehydrated, and sectioned as above, then stained on slides with 0.2% Coomassie brilliant blue R250 (ProSciTech, Thuringowa Central, Australia) in a 46.5:7:46.5 ratio solution of methanol:acetic acid:MQ water for 2 min and rinsed 3 times with water. Before imaging, the sections were stained with 1% oil red O (ProSciTech) for 2 min, then after washing 3 times, stained with calcofluor white stain (Sigma-Aldrich #18909-100ml), for 2 min before three washes with MQ water. For Coomassie, both color and black and white images were taken of different sections. The oil red staining was not strong due to the dehydration of the seed which would wash out most of the oil. 2.7

Immuno-histochemical microscopy

Fluorescent immuno-histochemistry microscopy was performed on variety Midlands X as described by Burton et al. [55]. Both heart and hull fractions were sectioned along the longitudinal axis after fixation and embedding in LR white resin. Primary monoclonal antibodies were diluted 1/50. The antibodies used were, LM2 [56] and JIM8 [57] to detect arabinogalactan proteins (AGPs), LM10 to detect xylan [58], LM11 to detect xylan/arabinoxylan [58], LM15 to detect xyloglucan [59], LM19 to detect homogalacturonan (HG) [60], LM20 to detect methyl-esterified homogalacturonan [60], and CBM3a (cellulose binding module) to detect cellulose [61]. Alexa Fluor R555 goat anti-rat IgG (H+L) highly cross-adsorbed secondary antibody was used for JIM8, LM2, LM5, LM6, LM10, 7

LM11, LM15, LM19 and LM20 (all diluted 1:200, ThermoFisher Scientific, Australia). For CBM3a, a two stage secondary antibody phase was employed using a mouse anti-histidine monoclonal antibody (1:100 dilution, Sigma-Aldrich) followed by Alexa Fluor R488 goat anti-mouse IgG (1:100 dilution, Invitrogen) as described by Blake et al. [61]. Fluorescence was observed using a Carl Zeiss M2 AxioImager microscope with an AxioCam Mrm camera, and subsequent image processing was performed with Zen (2012) software (Carl Zeiss, North Ryde, Australia). Selected images were cropped in Adobe Photoshop® but no other adjustments were made to the images. For each antibody, the intensity threshold was adjusted based on the no primary antibody control on hull tissue, and the same setting used for heart tissue, except for LM2 and JIM8 where the intensity was re-adjusted against a heart no primary antibody control. Some sections were pre-incubated with a 1/20 dilution of α-L-arabinofuranosidase (Megazyme, Ireland) to remove arabinose from the xylan backbone for 60 min and washed before LM11 treatment [62]. For detection of homogalacturonan with LM19 antibody, sections were either not treated or treated with 0.1 M sodium carbonate (Na2CO3) for 1 h at room temperature prior to primary antibody incubation [60,63]. Some sections were stained with toluidine blue, 10x images taken on a Nikon Ni-E optical microscope and subsequently “stitched” together using the Nikon software NIS-Elements. 2.8

Protein analysis

Whole seed were ground in liquid nitrogen and 150 mg analyzed for total N by the Cereal Breeding Lab (University of Adelaide, Australia), using the EBC Dumas method [64]. Samples were run using ‘Standard Method’ where the combustion tube is at 960°C and the oxygen is dosed for 80 seconds at 170 mL-1. A factor of 6.25 was used to convert total N to protein [65]. 2.9

Phytate

Whole seed were ground in liquid nitrogen and analyzed using a phytic acid kit (K-PHYT, Megazyme), with minor modifications. Ground tissue (250 mg) was digested overnight in 5 mL HCl (0.66M) in a 10 mL polypropylene tube containing 4 ceramic beads (2.8 mm, #13114-325, Mo Bio/Qiagen (Germantown, MD, USA)). Due to the high fat content of the seed, an additional centrifugation step was added after the overnight acid digestion (10 min, 3220 g), and prior to starting the assay. After this additional spin, a 1 mL sub-sample was removed (as per the protocol) and the oil adhering to the tip was removed with a kimwipe™. The remainder of the protocol was unchanged.

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2.10 Lipid analysis Whole seed were ground in liquid nitrogen and samples submitted to the Waite Lipid Analysis Service (University of Adelaide, Australia) for measurement of total lipid content including determination of the fatty acid composition. Ground hemp (50 mg) was extracted in a mix of saline, methanol and chloroform. The lipid-containing chloroform fraction was removed and blown down under nitrogen to dryness. The lipid was then transmethylated for 3 h at 70°C in a 2% sulfuric acid in methanol solution. After transmethylation water and heptane were added to extract the lipids and the heptane layer was run on an Agilent 7890 GC-FID using a 30 m BPX70 column. 2.11 Statistical Analysis Due to the small sample size (n=1 or n=2 technical replicates) for each assay, analysis of variance could not be performed, nor could the significance between means be compared using a multiple comparison test, such as Tukey’s test. Alternative non-parametric methods such as the KruskalWallis test were evaluated, but analyses are not reported because a significant result only indicates that “at least one” of the 20 varieties was significantly different. Comparison tests, such as Dunn’s test do not have sufficient power for n=2 technical replicates to produce significant results (data not shown). To identify traits with high variation between hemp lines we calculated the coefficient of variation% (CV) = (standard deviation (SD) /mean) x 100 [66]. Where the mean was calculated as the twenty individual means of the two technical replicates, we use the term ‘mean’ rather than the correct term ‘pooled mean’ or ‘grand mean’. Error bars on graphs represent SD. 3

Results

3.1

Variation exists for 1000 seed weight and heart %

Thousand seed weight varied considerably from 11.15 ± 0.33 g (ECO_209) to 46.37 ± 1.13 g (Yunma-2) with mean 23.02 ± 9.24 g and coefficient of variation% (CV) of 40.1% (Figure 1). The seed from all lines were also evaluated for heart %. Heart % values ranged from 54.0% (Yunma-1) to 67.4% (Frog-1) with mean 59.9 ± 3.3% and CV of 5.5% (Figure 1). 3.2

Hemp hearts do not contain cellulose

Polysaccharides or complex carbohydrates can contribute to nutritional composition and sensory properties and therefore the separated heart and hull fractions were subjected to compositional 9

analysis. As expected, cellulose was present in the hulls of all samples, ranging from 22.0 ± 1.9 (ECO_209) to 36.7 ± 0 (Yunma-1) % w/w, with mean of 32.0 ± 3.7 and CV of 11.5% (Figure 2A, Table 2). In contrast, there was minimal cellulose in the hearts of six randomly selected samples (below the limit of detection, data not shown), and therefore the remaining 14 heart samples were not tested. To confirm these results and further investigate the structure of hemp seeds, hull and heart fractions were stained with toluidine blue and the cellulose binding protein CBM3 (Figure 2B–G). Immunolocalization confirmed that cellulose is distributed throughout hemp hulls (Figure 2D), but is absent from hemp hearts (Figure 2G). Negative controls for hull and heart tissue are in Figure 2C,F, respectively. Toluidine blue staining of hemp shows the morphology of hull and heart tissues (Figure 2B,E, respectively). Low lignin varieties of hemp may be more desirable to consumers, especially for whole seed use, therefore lignin levels were quantified. Lignin levels in the defatted hemp hulls ranged from 16.0 ± 1.2 (ECO_YMR17) to 19.5 ± 0.5 (ECO_209) with mean 17.6 ± 1.0 and CV of 5.7% (Figure 2H). The data is plotted with heart % to show there is no apparent correlation between lignin and heart %. Lignin staining confirmed the presence of lignin in hemp hulls and its absence from hemp hearts (Figure 2I-L). 3.3

Monosaccharide analysis of complex carbohydrates from hemp hulls and hearts

The nature of the non-cellulosic carbohydrate fibers in hemp hulls and hearts has not been previously reported. The monosaccharides present in the alcohol insoluble fraction of ground hull and heart tissues were analyzed separately. Acid hydrolysis followed by high performance liquid chromatography of the hull fraction revealed xylose as the predominant monosaccharide in the noncellulosic polysaccharides, varying from 5.7–17.1% w/w (Table 2, Supplementary Figure S1A). Only two other monosaccharides, galactose and arabinose, were present in quantifiable amounts (within the calibration range) in the non-cellulosic polysaccharide fractions (Table 2, Supplementary Figure S1C,E), however these were both below 1% w/w. The overall monosaccharide content from heart non-cellulosic polysaccharides was lower than that of the hulls, and not all of the lines had reliable values (above the lowest concentration used to generate the calibration curve). Arabinose was the most abundant monosaccharide in hearts (reliably quantified in 11/20 lines), whereas xylose and galactose were only accurately measured in 3/20 lines and 2/20 lines, respectively (Table 2,

10

Supplementary Figure S1B,D,F). Overall the levels of monosaccharides in hearts were close to the detection limit, influencing the high level of error (Supplementary Figure S1B,D,F). Soluble sugars were also analyzed in the hull and heart fractions. Sucrose was the most abundant sugar in both hull and heart fractions, however it was less abundant in hulls (0.17%–0.71%) compared to hearts (1.5–3.8% w/w) (Table 2, Supplementary Figure S2A,B, respectively). Raffinose was below the level of the calibration curve in 17 out of 20 hull samples but present at reliable levels in all heart samples ranging from 0.07–0.46% w/w (Table 2, Supplementary Figure S2C,D). Glucose and fructose were analyzed in all heart and hull fractions, however at most three samples had levels within the calibration curve with the highest level observed at 0.11% and 0.10% for glucose and fructose, respectively (Table 2, Supplementary Figure S2E–H). Since only trace amounts of glucose were detected in the monosaccharide analysis of the noncellulosic polysaccharides, it was assumed that starch levels in all 20 lines were low. Six lines were randomly selected for determination of total starch in hemp hearts. Total starch content was generally <2%, ranging from 1.25 ± 0.10 % (ECO_209) to 2.04 ± 0.43% (ECO_254) and approximately 10% of the total starch was resistant starch in these two lines at 0.19 ± 0.01% (ECO_209) and 0.18 ± 0.01 (ECO_254) (Table 3). Seed were also stained for starch, revealing numerous small granules, especially in cotyledons (Supplementary Figure S3A,B). The granules are approximately 1–3 µm (Supplementary Figure S3A,B), which is much smaller than the large A-type granules of barley, which are typically about 20 µm [67] (Supplementary Figure S3C). To reveal the nature of other bodies in the hemp hearts, tissue sections were stained for protein (with Coomassie) and cellulose (with calcofluor white) to delineate cell walls (Figure 3). The staining shows that each cell contains numerous round protein bodies stained blue with Coomassie (Figure 3A,C), that are negatively stained with autofluorescence/oil red (Figure 3B,D). The oil red staining was not successful due to the dehydration of the seed prior to embedding which washes out the oil. Coomassie images were also captured in black and white to allow overlay with calcofluor white images, showing the protein bodies are found in most cell types (Figure 3E–J, Supplementary Figure S4), although there are fewer protein bodies in the shoot apical meristem (Figure 3G). The approximate region of the seed that each image (Figure 3E–J) was taken from is indicated on a toluidine blue stain transverse section (Figure 3K). 3.4

Immunolocalization of non-cellulosic polysaccharides in hemp hull and heart tissues 11

Immunolocalization was used to determine the nature and distribution of non-cellulosic polysaccharides in hemp hull and heart tissues. A summary of the antibodies used, and whether the epitopes were present or absent is provided in Figure 4A. The major polysaccharides in hemp hulls, based on monosaccharide analysis (Table 2), are expected to contain xylose and therefore we tested for xylan (LM10), xylan/arabinoxylan (LM11) and xyloglucan (LM15). Strong labelling was observed for both LM10 (xylan) and LM11 xylan/arabinoxylan, and absent in the no primary antibody controls (Figure 4B–F), however no significant labelling was observed for LM15 (xyloglucan) (data not shown). Epitopes for LM10 and LM11 have a similar distribution and are found throughout the walls of hull tissue (Figure 4C,E), suggesting that xylan and possibly arabinoxylan are present. Enzyme treatment with arabinofuranosidase did not alter the distribution or intensity of the LM11 staining (Figure 4F), establishing that xylan and not arabinoxylan is present in hemp hulls. The LM19 epitope recognizes pectin, specifically homogalacturonan (HG) with low levels of esterification, and is distributed throughout hemp hulls with stronger labelling seen in the outer cell walls and scattered, punctate labelling observed throughout the inner hull tissues (Figure 4H,K). The low level of signal in the no primary antibody control (Figure 4G,J) is a common problem in plant cell wall immunolabelling [60]. Chemical unmasking with Na2CO3 increased the intensity of the labelling revealing a diffuse distribution of labelling within the inner tissues in addition to punctate labelling (Figure 4I,L), suggesting that some of the HG is esterified [60,68]. There are organized clumps of cells between the outer and inner layers of hull tissue likely to be vascular bundles (Figure 4K,L and Supplementary Figure S5). Other polysaccharides that were detected at relatively low levels in hemp hulls, based on intensity above the no primary antibody controls, included highly esterified HG (labeled with LM20, predominantly in the inner wall of the hull) (Supplementary Figure S6A–C), rhamnogalacturonan I (RG-I) (labeled with LM5) (Supplementary Figure S6D,E) and AGPs (labeled with LM2) (Supplementary Figure S6F,G). No signal was detected in hull tissues for mannan, xyloglucan (LM15) and (1→3)-β-D-glucan (callose) (data not shown). Monosaccharide analysis of cell wall polysaccharides from hemp hearts (Table 2) revealed that xylose and arabinose are the major sugars. Epitopes for xyloglucan (XG) were detected using LM15 (Figure 5A–C) and showed strong staining throughout heart tissue, with highest intensity at the apex of the radicle (Figure 5B). Hemp hearts also contain both unesterified and low esterified HG, 12

especially in the testa walls surrounding the cotyledons (Figure 5D,E,G,H), and in the plumular leaf (shoot apex) and cotyledons surrounding the shoot meristem (Figure 5F,I). Low levels of highly esterified HG (detected with LM20) are also found in hemp hearts (Figure 5J–L). Moderate to low levels of other polysaccharides are present in hearts including AGPs (JIM8 and LM2), RG-I (LM5), polysaccharides containing (1→5)-α-L-arabinan (LM6), which could be on AGPs and/or RG-I (Supplementary Figure S7) and callose (autofluorescence, data not shown). 3.5

Characterisation of total seed protein and phytate

Hemp seed are a valuable source of protein and therefore total nitrogen was calculated for whole ground seed samples to determine the variation for protein content. The total protein values for the 20 lines ranged from 19.5% (Yunma-1) to 26.9% (Ferimon 12), with a mean value of 24.2 ± 1.8% and CV of 7.4% (Table 4). It is well established that high levels of phytate can reduce the bioavailability of protein [69] and therefore phytate was measured. Values of seed phytate in the 20 lines ranged from 1.45 ± 0.02 g/100 g (KC Dora) to 3.12 ± 0.04 g/100 g (Frog-1), with a mean value of 2.67 ± 0.43 g/100 g and CV of 16.0% (Table 4). 3.6

Characterisation of lipids in whole hemp seeds

Whole ground seed samples were analyzed for total lipid content and composition. The total lipid content varied from 26.6% (Yunma-1) to 37.8% (Ferimon 12) (Table 5). The two most abundant lipids (as percentage of total lipids) in whole ground seeds are the essential fatty acids linoleic acid (LA, 18:2n-6, mean 56.1 ± 1.8 for the 20 lines, CV of 3.2%) and α-linolenic acid (ALA, 18:3n-3, mean 18.1 ± 3.0, CV of 16.8%) (Figure 6, Supplementary Table S1). The other abundant lipids (>1%), in descending order are: oleic acid (OA, 18:1n-9, mean 11.3 ± 1.7, CV of 14.7%), palmitic acid (PA, 16:0, mean 7.0 ± 0.7, CV of 9.5%), steric acid (SA, 18:0, mean 2.8 ± 0.3, CV of 9.4%), and γ-linolenic acid (GLA, 18:3n-6, mean 2.0 ± 1.3, CV of 63.9%) (Figure 6, Supplementary Table S1). The omega-6 to omega-3 ratios were calculated, as the current health advice is to consume a ratio closer to 3:1 than 16:1, which is typical of many Western diets [24-27]. Seven lines had ‘desirable’ omega-6:3 ratio less than or equal to three (ECO_16AH, ECO_50GC, ECO_209, ECO_253, Yunma1, Yunma-2 and Han-NE), whilst the highest ratio was almost 4.9:1 (ECO_MR17) (Table 5, Supplementary Table S1). The highest % omega-3 as a proportion of total lipid was 25.7% (HanNE), almost two-fold more than the lowest amount (14.1%) in ECO_AMB17, and these values are reflected in ratios of omega-6:omega-3 of 2.1 and 4 respectively. 13

4

Discussion

This research is the first detailed study of complex carbohydrates in hemp seed using a combination of chemical analysis and immunolabelling of tissue sections from a selected set of germplasm (varieties and breeding lines) mainly sourced from Ecofibre’s seed bank. The focus of this study was on end-user/health traits where diversity of dietary complex carbohydrates is positively correlated with microbiome diversity and improved human health outcomes [37,38,40,41,43,44]. The new findings on carbohydrate composition are combined with analysis of heart %, 1000 seed weight, lipid composition, total protein, lignin and phytate, thus providing a foundation for selection of plant lines with improved human health attributes for expanding the hemp industry in Australia and internationally. 4.1

Traits with high coefficient of variation

Coefficient of variation % (CV) is a useful parameter for determining the characteristics that vary most between lines. Since the batches of seed used in this study were sourced from different regions of Australia, it is not possible to tell if the source of variation is from genotype (G) or environment (E) or an interaction of both (G x E). However, CV information is still useful for prioritising hemp lines for future field trials, and traits for subsequent analysis, and for identifying traits with a strong genetic component suitable for targeting in breeding programs. In the following discussion, we make the assumption that traits with a high CV have a strong genetic component, but obviously this will need to be tested in the future. There is some support for this assumption based on a hemp field trial from Canada that evaluated 11 cultivars over two years in seven different environments [70]. Analysis of field trial data revealed that most of the traits evaluated had statistically significant genetic (G), environment (E) and G x E components, including seed protein, seed yield, plant height, biomass yield, biomass cellulose and biomass hemicellulose [70]. However, for the 11 varieties studied there was not a significant genetic component for biomass lignin and seed oil, but both had significant G x E components, and seed oil also had a significant environmental component [70]. In our study, heart %, seed lignin and total lipids had a CV of <10% indicating low variability [66], whereas, many traits had intermediate CV% (10–29.9%), including crystalline cellulose, hull monosaccharides (xylose, arabinose and galactose), non-resistant starch, phytate, the essential omega-3 fatty acid ALA. A few traits had high CV (≥30%), including 1000 seed weight, GLA, soluble sucrose (heart) and soluble raffinose (heart) (Figure 7). This information

14

can be used to select a smaller number of appropriate lines for statistical validation of each desired trait, and used by breeders to select lines for future field trials to determine the extent of the genetic and environmental components of each trait. There is high variation (CV, 40.1%) in 1000 seed weight (Figure 1). Thousand seed weight provides an approximation of seed size, as generally the bigger the seed, the heavier it will be, although variation in the composition and ratio of heart % means that this correlation may not always hold. Furthermore, seed size does not usually correlate with overall seed yield which is key for profitability, since yield is heavily influenced by inflorescence architecture and shattering resistance [23]. Higher heart % provides an opportunity to increase profitability, through increases in higher value hemp heart products. The heart % trait has not been widely reported for hemp, with only one published study to our knowledge, where heart % varied from 59.0% to 69.5% [32]. Twelve of the 20 lines analyzed in this study had heart % greater than 59% (from Kc Dora at 59.2% to Frog-1 at 67.4%, Figure 1). This does not guarantee that these lines are high yielding, profitable lines for current use, but rather they are potential lines for breeding, if the heart % trait has a strong genetic component. For other nuts, such as Macadamia, high heart % is called kernel recovery, and is known to be influenced by the genotype of the pollen donor [71]. Kernel recovery in Macadamia is also sensitive to mild drought stress during nut development [72]. To our knowledge there have not been any studies in C. sativa on what factors affect kernel recovery (heart %), but we hypothesize that pollen donor and / or stress could be important, and should be explored in future to determine genotype by environment effects on this trait. Hemp hulls contain crystalline cellulose as the major polysaccharide (Table 2, Figure 2), with xylan as the next most abundant polysaccharide. The identification of xylan as a major complex carbohydrate in hemp hulls is based on monosaccharide analysis (Table 2) and immunolocalization analysis which demonstrates the polysaccharide is present as an unsubstituted xylan and not arabinoxylan (Figure 4). The variability in hull xylose content is of significant interest to the food industry for adding value to agricultural wastes. Xylan is a source for the production of xylan oligosaccharides for prebiotics, which are non-digestible food ingredients that promote growth of bacteria in the colon and can improve health [38]. Xylan oligosaccharides have been produced from peanut shells [73]. The two hemp lines with the highest levels of xylose in their hulls, Frog-1 and Yunma-1 (Supplementary Figure S1A), are likely to have higher levels of xylan (approximately

15

17%) than peanut shells (14-15.5%) [73], assuming that all the xylose is hemp is found in the xylan as suggested by immunolocalization (Figure 4). Hemp hulls and hearts are low in soluble sugars, with hemp hearts generally containing more soluble sugars than the hull fraction (Table 2). The new data reported here is mostly consistent with a previous study that showed that sucrose was the most abundant soluble sugar [65]. The major difference between the earlier study and this study is that they did not detect any raffinose in whole hemp seeds, whereas here raffinose was found in all 20 heart samples (Supplementary Figure S2D). This difference is puzzling since the 2018 study was able to detect raffinose in other food samples (e.g. flaxseed, lupin and buckwheat) [65], however it is not of great importance given the low levels <0.5% w/w observed in the 20 lines analyzed here (Table 2). An important finding from the current study is that starch is not a major component of hemp seed, based on the trace levels of glucose in the polysaccharide fraction (all 20 lines) and <2% total starch in the six lines chosen for starch quantification (Table 3, Supplementary Figure S3). This contrasts with information in a recent review [2] where the authors state that hemp seeds contain 25% starch. The results presented here show that the dietary fiber is not starch but predominantly xylan (5.7 – 17.1%, based on xylose) and pectin in hulls, with a small amount of xyloglucan and pectin in hemp hearts (<1% each) (Table 2). This is important because it suggests that hemp will have a low glycemic index and contains a range of complex carbohydrates that could be fermented in the gut to varying degrees. This could be readily tested in future by including hemp as a protein source in human diets, because SCFAs, the products of gut fermentation, are readily detected in stools making them convenient biomarkers. Other studies adding whole seeds to human diet plans show that increases in SCFAs can be achieved in relatively short timeframes (weeks) [74]. The fate of the different cell wall polysaccharides is an area of active research, for example, xyloglucans from cranberry promote the growth of SCFA producing strains of Bifidobacterium longum [75]. Furthermore a phenol-free xyloglucan derived oligosaccharide fraction (degree of polymerization 7 to 10) from cranberry hulls reduces biofilm formation by strains of E. coli that cause urinary tract infections [76]. Pectins are also substrates for gut bacteria [49], and although present in small amounts in hemp, they should contribute to microbiome diversity, especially as their fine-structure may be different to commonly consumed pectins such as citrus pectin.

16

The fine-structure of the hemp carbohydrates remains to be determined, and xylan would be the obvious first target as it is the major non-cellulosic polysaccharide in hemp. The hull should not be overlooked as a food source. It can be milled for example and used as a high-fiber ingredient, or better still, more emphasis could be placed on developing food products that use the whole seed. In addition to providing the best human health outcomes, use of whole seeds may have the benefit of reducing manufacturing costs and waste products generated through the dehulling process. Whole seed products would also contain lignin (Figure 2H–L), which can be perceived as a negative component. However, rather than being an inert compound, lignin is now thought to contain beneficial antioxidant activity [34]. The mean value of lignin in hemp hulls observed in this current study was 17.6 ± 1.0 (Figure 2H–L), which is higher than a prior study that reported a mean value of 11.2 [77]. However, the values are comparable given that our study used hemp hulls, not whole seed. The other major components of hemp hearts are proteins and lipids. The study conducted by House et al., [30] used a rat bioassay to calculate protein digestibility-corrected amino acid score (PDCAAS) measurements. Importantly they showed that hemp protein had PDCAAS value equal to or greater than certain grains, nuts, and some pulses. Specifically whole hemp seeds have a higher PDCAAS value than almonds and whole wheat, and hemp hearts have a higher score than lentils, pinto beans and rolled oats [30]. Their study showed that hemp hulls had a significant impact on protein digestibility, with dehulled hemp seed having a PDCAAS score of 61%, whereas whole seeds had a score of 51% [30]. This finding could mean that that whole seed from hemp lines such as Frog-1 and ECO_50GC, with high heart %, could have higher protein digestibility, than other lines with similar protein content, but lower heart %. Other possible factors that affect digestibility need to be considered, and these can be evaluated in the future. Protein digestibility can be affected by the nature and structural arrangement of protein in the seed [78]. We demonstrate that the protein is present as protein bodies throughout the seed (Figure 3D– M). To our knowledge this has not been shown before, but is consistent with seed from other dicotyledonous plants [79]. The structure of protein bodies in monocotyledonous seed such as sorghum are known to be formed due to the interaction of several proteins with a hydrophobic protein (γ-kaffrin) on the surface of the body. γ-kaffrin is responsible for creating the stable structure of sorghum protein bodies and may impede digestibility [80]. There is no data on the arrangement of the seed storage proteins within hemp seed, but a recent paper has revealed that the C. sativa genome contains six 11S edestin genes, two 2S albumin genes and one 7S vicilin-like gene [81]. There is 17

likely to be variation in the levels of hemp seed storage proteins between genotypes and with environmental conditions which may in turn affect both protein digestibility and the amount of the most limiting amino acid, Lys. A subset of the most promising lines could be analyzed in the future to reveal the extent of genetic variability for specific proteins and bioavailability of Lys. Strong environment (E) and G x E effects on protein content have been shown, P<0.0001 and P=0.0019 respectively in a Canadian field trial with 11 lines (including cultivars Midlands X, Midlands S and Ferimon) [70], although a relatively low CV (7.4%) was obtained for protein in the 20 hemp lines grown in Australia (Table 4, Figure 7). In the Canadian field trial, the average protein yield for Midlands X, Midlands S and Ferimon 12 ranked 2nd, 6th and 7th, with the highest average protein yielding line being FINOLA, at 25.7%, compared to Midlands X, Midlands S and Ferimon 12 at 25.2%, 23.8% and 23.0% respectively [70]. In the data reported here, from a single batch of seed, three lines, Ferimon 12, ECO_222 and ECO_208 (Table 4) had highest protein levels at 26.9%, 26.2% and 25.9% respectively. Studies in other crops suggest that stress can reduce seed protein content [82] and also change the relative abundance of different storage proteins [83] and amino acid profiles [84]. The relative susceptibility of hemp protein levels and amino acid profiles to environmental factors including heat will be an important area for future selection of hemp lines for food. The 20 hemp lines analyzed in this study had a greater range of total lipid content, with a higher maximum value of 37.8% (Table 5), compared to 34.8% [29] and 30.6% [77] in previous reports. Two of the lines in each of the other studies were also analyzed in this study, Midlands X and Midlands S had slightly higher total lipids in the current study (at 32.2% and 33.5%) (Table 5) compared to the 2015 report where both lines had 29.5% total lipid [77]. A similar small difference was observed for KC Dora (32.8%) and the 2016 study [29] with 31.5% total lipid, however there was a greater different for Ferimon 12 which had the highest total lipid content in this study at 37.8%, but only 30.2% in Galasso et al. [29]. The mean values of the individual lipids are also similar between this study, and the other two studies, with no notable differences (Supplementary Table S1). The main benefit of analyzing the individual lipids is to calculate the ratio of omega-6 to omega-3 fatty acids (or LA:ALA), which is generally around 3:1 for hemp seed [6,77]. For example, the twenty lines reported by Galasso and colleagues [29] had a mean LA:ALA ratio of 3.32. Only a single line, CAN24 had a dramatically reduced ratio at 1.63, with the next lowest ratio at 2.63 (line CAN40) and another three other lines 18

(CAN51, CAN58 and FINOLA) had a ratio ≤ 3. The data reported here (Supplementary Table S1) revealed that eight lines had LA: ALA ≤ 3, with two lines close to 2 (ECO_16AH (2.3) and Han-NE (2.1)). This ratio provides new plant lines to consider in breeding programs to further improve this trait. Lastly, we discuss phytate levels in the 20 hemp lines. Phytate levels in hemp are similar to other oilseeds, most notably, linseed (Linum usitatissimum) where they range from 2.15 –3.69 g/100 g [85]. We found three prior hemp studies that measured phytate. The first study obtained a mean value almost 2-fold higher [29] than current study (Table 4) even after taking into account they used defatted flour rather than whole seeds, but the second study had more comparable values at 3.5 ± 0.2 g/100g [11]. The values reported in our study (Table 4) likely represent real values, as the kit provided oat flour controls gave the expected values, and recovery of total P was as expected for a mixed oat plus hemp control assay compared to the two samples processed individually. Furthermore the mean value reported here of 2.67 ± 0.43 g/100 g is consistent with the value obtained for whole hemp send with a recently “improved” method of phytate determination, of 2.75 g/100 g [86]. Despite the differences in absolute values between this study and Galasso, the samples showed the same trends, with KC Dora having the lowest levels of phytate (in both studies) and Fermion 12 (Ferimon) having higher, above average levels of phytate (Table 4) [29]. Phytate is often considered an antinutrient because it can bind/chelate other nutrients, especially divalent cations (Fe / Zn), and affects the bioavailability of micronutrients such as Zn and digestibility of proteins [69,87]. Another potential negative of high phytate levels is the positive correlation with trypsin inhibitor (0.679, P=0.01) [29]. This could potentially affect the availability of protein in raw foods, but is not an issue in cooked food [87,88]. Phytate does have some positive attributes for human health. It has antioxidant properties and this may in part account for observed anti-cancer activities in, for example, rat studies where pure phytic acid is more efficient at reducing the incidence and growth of mammary tumours compared to all bran (6% phytate) [89]. In summary, the new research presented fills some previous knowledge gaps, most significantly providing details on the nature of the major dietary fiber components of hemp seed. The use of antibodies revealed a high level of diversity of polysaccharides and some cell-type specificity for the different plant cell wall polymers. Cellulose and xylan were identified as the major components of hemp hulls, and hemp hearts contain low levels of starch. The findings contribute to efforts to grow the global hemp industry, especially for food use and to improve human nutrition. Future research 19

into consumer traits is needed, especially in the area of flavour characteristics and new product development. For example, different hemp lines can have different flavour profiles (e.g. strong/weak, hazelnut, walnut) [2] and new products include microgreens [7,12], which are excellent ways to reduce antinutrients such as phytate [88] and increase antioxidants [7]. With growing worldwide interest in hemp as a more sustainable crop, the breadth of traits incorporated into breeding programs is expected to grow, accelerating the improvement of hemp for consumers and human nutrition. 5

Declarations of interest: none

6

Author Contributions

CS performed the phytate assays, data analysis and wrote the manuscript. WL and KN performed the microscopy, SK and JS performed most of the biochemical analyses. OA and MS provided background information and performed review and revision of the manuscript. RB designed the study, supervised the research project and gave critical suggestions on manuscript preparation. All authors have read and approved the manuscript. 7

Funding

This study was carried out as part of a wider project on Industrial hemp funded by the South Australian government through the South Australian Research and Development Institute (SARDI), the research division of Primary Industries and Regions SA (PIRSA). The work was part-funded by the Australian Research Council Centre of Excellence in Plant Energy Biology (Grant number: CE140100008), the University of Adelaide. 8

Acknowledgements

Seed for these trials was provided free of charge by the following companies: Ecofibre Limited, Midlands Seeds Pty Ltd, AusHemp Pty Ltd, The Hemp Corporation Pty Ltd and Hanidel Pty Ltd. 9

Abbreviations

ADF, acid detergent fiber; AGPs, arabinogalactan proteins; ALA, α-linolenic acid; AIR, alcohol insoluble residue; CV, coefficient of variation%; GLA, γ-linolenic acid; HG, homogalacturonan; LA, linoleic acid; MQ, Milli-Q; NDF, neutral detergent fiber; OA, oleic acid; PA, palmitic acid; PDCAAS, protein digestibility-corrected amino acid score; RG-I, rhamnogalactan I; rpm, revolutions

20

per minute; SA, steric acid; SCFA, short chain fatty acids; SD, standard deviation; THC, ∆9tetrahydrocannabinol; TSW, thousand seed weight. 10

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Appendix A. Supplementary Data Supplementary data associated with this article can be found in the online version, at

26

TABLE 1 | Name, source, origin and use of C. sativa lines evaluated. Hemp line

Sourcea

Region of origin

Primary use

Midlands Xb

Midlands

North America

seed

Midlands Sb

Midlands

North America

seed

USO-31

Midlands

Europe

seed & fibre

Ferimon 12

Midlands

Europe

seed & fibre

KC Dora

AusHemp

Europe

seed & fibre

ECO_YMR17

Ecofibre

Australia

seed

ECO_MR17

Ecofibre

Australia

seed & fibre

ECO_16AH

Ecofibre

Australia

seed & fibre

ECO_50GC

Ecofibre

Australia

seed

ECO_AMB17

Ecofibre

Australia

seed & fibre

ECO_FR17

Ecofibre

Europe

seed & fibre

ECO_202

Ecofibre

Australia

seed

ECO_209

Ecofibre

Australia

seed

ECO_222

Ecofibre

Australia

seed

ECO_253

Ecofibre

East Asia

seed

ECO_254

Ecofibre

Australia

seed

Yunma-1

HempCorp China

seed & fibre

Yunma-2

HempCorp China

seed & fibre

Han-NE

HempCorp China

seed

Frog-1

Hanidel

seed & fibre

a

Australia

Full names of seed sources are Midlands Seeds Pty Ltd (Midlands), AusHemp

Pty Ltd (AusHemp), Ecofibre Limited (Ecofibre), The Hemp Corporation Pty Ltd (HempCorp) and Hanidel Pty Ltd (Hanidel). b

Midlands X = CFX-2 and Midlands S = CRS-1 as reported in Vonapartis et

al. [77].

27

TABLE 2 | Composition of carbohydrates in hulls and hearts of 20 hemp lines. Component

Hull

Heart

(% w/w)

(% w/w)

Crystalline cellulose cellulose

22.0–36.7 (20)a

ndb

Monosaccharide analysis 5.7–17.1 (20)a

0.56 (3)

arabinose

0.64 (18)

0.65 (11)

galactose

0.55 (19)

0.18 (2)

xylose

Soluble sugars sucrose

0.17–0.71 (19)a

1.5–3.8 (20)a

raffinose

0.10 (3)

0.07–0.46 (20)a

glucose

0.11 (2)

0.11 (3)

fructose

0.10 (3)

0.08 (1)

a

Range of values observed for up to 20 lines. All other

values are the highest value observed (see Supplementary Figures S1 and S2). Number in parenthesis is the number of lines with values for both technical replicates falling within the calibration curve. For data on individual lines see Figures 2A and S1 and S2. b

nd, not detected (below the limit of detection in six

randomly selected samples), data not shown.

28

TABLE 3 | Starch content in hearts of the six selected hemp lines. Hemp line

Resistant starch

Non-resistant

Total starch

(% w/w)

starch

(% w/w)

(% w/w) ECO_254

0.18 ± 0.01

1.86 ± 0.42

2.04 ± 0.43

ECO_MR17

0.16 ± 0.01

1.65 ± 0.55

1.81 ± 0.54

ECO_16AH

0.17 ± 0.02

1.36 ± 0.11

1.53 ± 0.13

ECO_YMR17

0.19 ± 0.01

1.18 ± 0.01

1.37 ± 0.01

0.17 ± 0

1.17 ± 0.11

1.34 ± 0.11

0.19 ± 0.01

1.06 ± 0.11

1.25 ± 0.10

ECO_50GC ECO_209

29

TABLE 4 | Total nitrogen, protein and phytate, for whole ground seed samples. Hemp line Midlands X Midlands S USO-31 KC Dora Ferimon 12 ECO_YMR17 ECO_MR17 ECO_16AH ECO_50GC ECO_AMB17 ECO_FR17 ECO_202 ECO_209 ECO_222 ECO_253 ECO_254 Yunma-1 Yunma-2 Han-NE Frog-1

Nitrogen Proteina (%) (%) 4.0 25.1 4.0 25.3 3.7 23.2 4.0 24.8 4.3 26.9 3.4 21.0 3.7 23.2 3.7 23.2 4.0 24.8 4.1 25.7 4.1 25.3 4.1 25.8 4.1 25.9 4.2 26.2 3.8 23.9 3.8 23.9 3.1 19.5 3.6 22.7 3.6 22.7 4.0 25.1

Phytate (g/100 g) 2.41 ± 0.09 2.34 ± 0.00 2.36 ± 0.01 1.45 ± 0.02 3.01 ± 0.05 2.48 ± 0.03 2.84 ± 0.15 2.85 ± 0.02 2.94 ± 0.02 2.41 ± 0.08 2.97 ± 0.07 2.91 ± 0.04 3.09 ± 0.04 3.04 ± 0.11 3.08 ± 0.02 2.89 ± 0.03 2.87 ± 0.39 2.12 ± 0.05 2.31 ± 0.06 3.12 ± 0.04

mean 3.9 24.2 2.67 SD 0.3 1.8 0.43 CV% 7.6 7.4 16.0 a Total protein was calculated from total nitrogen using a conversion factor for plants (Mattila et al., 2018a). The three highest (bold) and lowest (underlined).

30

TABLE 5 | Lipid content (lipid %) and composition of whole ground seed. Hemp line

Lipid composition (% of total lipids)a

Lipid % Saturated

Mono-

Omega

Omega

Omega

Omega

Omega

unsaturated

9

7

6

3

6:3b

Midlands X

32.2

10.4

11.3

10.4

0.9

60.3

17.9

3.4

Midlands S

33.5

10.9

12.9

11.9

1.0

58.8

17.4

3.4

USO-31

37.2

11.3

13.6

12.6

1.0

59.3

15.8

3.8

KC Dora

32.8

12.3

14.1

13.0

1.1

57.7

15.9

3.6

Ferimon 12

37.8

10.7

10.9

9.9

1.0

61.0

17.3

3.5

ECO_YMR17

36.4

13.2

14.5

13.4

1.1

56.7

15.5

3.7

ECO_MR17

31.3

10.5

13.9

13.0

0.9

62.8

12.8

4.9

ECO_16AH

33.6

10.6

11.2

10.3

0.9

54.8

23.4

2.3

ECO_50GC

36.0

10.5

10.8

9.8

1.0

58.8

19.9

3

ECO_AMB17

28.8

13.1

17.1

16.0

1.1

55.7

14.1

4

ECO_FR17

32.8

11.0

12.4

11.4

1.0

58.2

18.5

3.1

ECO_202

36.8

10.8

12.5

11.5

1.0

60.2

16.5

3.6

ECO_209

33.6

10.9

12.0

11.1

1.0

56.3

20.8

2.7

ECO_222

35.3

10.6

11.7

10.8

1.0

59.3

18.4

3.2

ECO_253

33.1

10.8

11.7

10.7

1.0

58.0

19.5

3

ECO_254

30.3

10.7

12.8

11.8

1.0

59.8

16.6

3.6

Yunma-1

26.6

11.0

12.9

12.1

0.7

55.9

20.3

2.8

Yunma-2

31.9

10.8

12.4

11.6

0.8

57.5

19.3

3

Han-NE

33.7

9.8

9.5

8.7

0.8

54.9

25.7

2.1

Frog-1

33.2

11.8

14.9

14.0

0.9

56.9

16.4

3.5

mean

33.3

11.1

12.7

11.7

1.0

58.1

18.1

3.2

SD

2.8

0.9

1.7

1.7

0.1

2.1

3.0

0.6

CV%

8.4

7.9

13.6

14.3

11.2

3.6

16.8

18.9

a

Lipids present at ≥1% are summarized in Figure 6 and the full lipid profiles are reported in Supplementary Table S1.

No trans fats were detected in any lines. The three highest (bold) and lowest (underlined), except for omega-6 to omega-3 ratio, where values ≤ 3 are double underlined. b

LA:ALA is reported in Supplementary Table S1, for direct comparison with prior studies [29,77].

31

11

Figure Legends

Note: Figures 2 to 5 to be printed in color for printed version of journal Figure 1. Thousand seed weight (left axis, n=2) and heart % (right axis, n=1) of 20 hemp lines. Lines are arranged, left to right, from lowest to highest heart %. Figure 2. Crystalline cellulose and lignin content of 20 hemp lines (n=2). (A) Crystalline cellulose content in hulls. (B) Hemp hull (part) stained with toluidine blue, (C) hemp hull, negative (Neg) control (no primary antibody), (D) crystalline cellulose visualized with CBM3a binding protein in hemp hull (part). The staining is representative of the entire hull (data not shown). (E) Transverse section of hemp heart stained with toluidine blue (image is an in silico “stitch” of 15 x 15 images with no other modifications). No significant signal for crystalline cellulose with CBM3a in the control (no primary antibody) (F) or CBM3a labeled heart tissues (G). Images (B−G) are of hemp line ‘Midlands X’. The dotted box in E is the approximate region imaged in F and G, and includes part of the testa, two cotyledons, plumular leaf (or shoot apex) and radicle (or root apex). (H) Lignin (%) in hemp hulls (left axis), and for comparison heart % (right axis). Data are plotted in the same order as cellulose (A, above). Transverse sections of hemp seed, unstained control (I,J) and hull cell walls stained red for lignin with phloroglucinol (K,L). Scale bars are 100 µm, except in E, I and K (1 mm). Figure 3. Hemp seeds contain numerous protein bodies. Hemp heart tissue stained to reveal protein bodies (Coomassie) and plant cell walls (cellulose/calcofluor white) (A−D). Hemp cotyledon stained with Coomassie and imaged in color (A,C) and an overlay image of the same region showing autofluorescence and calcofluor white staining (B,D). The dashed boxed regions in A and B are magnified in C and D respectively and show that protein is packaged in spherical protein bodies. (E– J) Coomassie staining (imaged in black and white) and optically overlayed with calcofluor white staining. (K) Transverse section of hemp heart stained with toluidine blue (same image as Figure 2E). The dashed boxed regions in K (labeled e to j) indicate the approximate region of the seed imaged in E–J. Tissues shown include radicle (E), inner radicle and unknown tissue (F), base of the plumular leaf (G), base of the inner cotyledon (H), inner region between shoot meristem and radicle (I) and junction between two cotyledons (J). Separate images of Coomassie and calcofluor white staining for overlays E and F are shown in Supplementary Figure S4. All images are from hemp line ‘Midlands X’. Scale bars are 20 µm (A–D), 50 µm (E–J) and 1 mm (K). 32

Figure 4. Cell walls of hemp hulls contain xylan and pectin epitopes. (A) Summary of labelling of cell wall polysaccharides in hull and heart tissue, indicating presence (green) or absence (yellow) of epitopes and figure parts where data can be found. Tissue sections labeled with LM10 (xylan) (B,C), LM11 (xylan/arabinoxylan) (D,E), LM11 after arabinofuranosidase treatment to reveal xylan (F), LM19 (low esterified homogalacturonan (HG)) (G,H,J,K), and LM19 after Na2CO3 treatment, to reveal unesterified homogalacturonan (I,L). Negative controls (Neg, no primary antibody, B,D,G,J). Image (J) is an in silico 2x magnification of image G, whereas K and L are images taken at 2X higher magnification and show a different region of hull. All images are from hemp line ‘Midlands X’. Scale bars are 100 µm. Figure 5. Cell walls of hemp hearts contain xyloglucan (LM15) and pectin (LM19 and LM20) epitopes. Tissue sections labeled with LM15 (xyloglucan) (A–C), LM19 (low esterified homogalacturonan (HG) (D–F), LM19 after Na2CO3 treatment to reveal unesterified homogalacturonan (G–I) and LM20 (high methyl-esterified HG) (J–L). Tissues are oriented as per Figure 2E (with cotyledon and root apex at the top) and show cotyledons (A,D,G,J), radicle apex (B,E,H,K) and central region including part of the plumular leaf (C,F,I,L). All images are from hemp line ‘Midlands X’. Scale bars are 50 µm. Figure 6. Lipid content of whole ground seed. Mean value of lipids present at ≥ 1% in 20 hemp lines. Lipids present at <1% are reported in Supplementary Table S1. Palmitic acid (PA), steric acid (SA), oleic acid (OA), α-linolenic acid (ALA), linoleic acid (LA) and γ-linolenic acid (GLA). Figure 7. Heat map summary of coefficient of variation % (CV) for hemp seed traits. Traits with low CV (<10%) exhibit low variation between lines, suggesting these traits are not greatly affected by either genotype (G) or environment (E) or a combination of both (GxE). Traits with ≥10% CV warrant further investigation as to the source of variation (e.g. G or E or GxE). CV was only calculated for monosaccharide and soluble sugar data that was above the lowest value of the calibration curve and where reliable data was obtained for ≥ 18 hemp lines (see Supplementary Figures S1,S2). Individual lipids (≥1% of total lipids) are reported in order of abundance, from most abundant (LA) to least abundant (GLA).

33

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AUTHOR DECLARATION

We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome. We confirm that the manuscript has been read and approved by all named authors and that there are no other persons who satisfied the criteria for authorship but are not listed. We further confirm that the order of authors listed in the manuscript has been approved by all of us. We confirm that we have given due consideration to the protection of intellectual property associated with this work and that there are no impediments to publication, including the timing of publication, with respect to intellectual property. In so doing we confirm that we have followed the regulations of our institutions concerning intellectual property. We understand that the Corresponding Author is the sole contact for the Editorial process (including Editorial Manager and direct communications with the office). He/she is responsible for communicating with the other authors about progress, submissions of revisions and final approval of proofs. We confirm that we have provided a current, correct email address which is accessible by the Corresponding Author.

Signed by all authors as follows: Author Name

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Date

Carolyn J. Schultz

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..........................................

Wai L. Lim

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..........................................

Shi F. Khor

.....................................

..........................................

Kylie A. Neumann

.....................................

..........................................

Jakob M. Schulz

.....................................

..........................................

Omid Ansari

.....................................

...08/10/2019.....................

Mark A. Skewes

.....................................

..........................................

Rachel A. Burton*

.....................................

..........................................

* Corresponding Author ([email protected])

AUTHOR DECLARATION

We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome. We confirm that the manuscript has been read and approved by all named authors and that there are no other persons who satisfied the criteria for authorship but are not listed. We further confirm that the order of authors listed in the manuscript has been approved by all of us. We confirm that we have given due consideration to the protection of intellectual property associated with this work and that there are no impediments to publication, including the timing of publication, with respect to intellectual property. In so doing we confirm that we have followed the regulations of our institutions concerning intellectual property. We understand that the Corresponding Author is the sole contact for the Editorial process (including Editorial Manager and direct communications with the office). He/she is responsible for communicating with the other authors about progress, submissions of revisions and final approval of proofs. We confirm that we have provided a current, correct email address which is accessible by the Corresponding Author.

Signed by all authors as follows: Author Name

Signature

Date

Carolyn J. Schultz

.....................................

..........................................

Wai L. Lim

.....................................

..........................................

Shi F. Khor

.....................................

..........................................

Kylie A. Neumann

.....................................

..........................................

Jakob M. Schulz

.....................................

..........................................

Omid Ansari

.....................................

..........................................

Mark A. Skewes

.....................................

.......9/10/2019..................

Rachel A. Burton*

.....................................

..........................................

* Corresponding Author ([email protected])