Diversity of Kale (Brassica oleracea var. sabellica): Glucosinolate

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The diversity of kale (Brassica oleracea var. sabellica): Glucosinolate content and phylogenetic relationships Christoph Hahn, Anja Müller, Nikolai Kuhnert, and Dirk Albach J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b01000 • Publication Date (Web): 30 Mar 2016 Downloaded from http://pubs.acs.org on April 5, 2016

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Journal of Agricultural and Food Chemistry

The diversity of kale (Brassica oleracea var. sabellica): Glucosinolate content and phylogenetic relationships

Christoph Hahn * 1 , Anja Müller 2 , Nikolai Kuhnert 2 , Dirk Albach 1

1

Carl von Ossietzky University Oldenburg, Institute for Biology and Environmental

Sciences, Carl von Ossietzky Str. 9-11, 26111 Oldenburg, Germany

2

Jacobs University Bremen, School of Engineering and Science, Campus Ring 8,

Research III, 28759 Bremen, Germany

* corresponding author: E-mail: [email protected] Phone: 0049-441-798-3343

1 ACS Paragon Plus Environment


1

Abstract

2

Recently, kale has become popular due to nutritive components beneficial for human

3

health. It is an important source of phytochemicals such as glucosinolates that trigger

4

associated cancer-preventive activity. However, nutritional value varies among

5

glucosinolates and among cultivars. Here, we start a systematic determination of the

6

content of five glucosinolates in 25 kale varieties and 11 non-kale Brassica oleracea

7

cultivars by HPLC-DAD-ESI-MSn and compare the profiles with results from the

8

analysis of SNPs derived from a KASP genotyping assay. Our results demonstrate that

9

the glucosinolate levels differ markedly among varieties of different origin. The

10

comparison of the phytochemical data with phylogenetic relationships revealed that the

11

common name kale refers to at least three different groups. German, American and

12

Italian kales differ morphologically and phytochemically. Landraces do not show

13

outstanding glucosinolate levels. Our results demonstrate the diversity of kale and the

14

importance of preserving a broad genepool for future breeding purposes.

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Keywords: kale, cabbage, glucosinolates, HPLC-ESI-MS, phylogenetic relationships,

18

KASP assay, SNPs


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Introduction

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Brassica oleracea L. is economically one of the most important species of the

21

Brassicaceae family because cabbage is a widely consumed vegetable all over the

22

world. Worldwide production is 70 million tons.1 The major reason for this success is

23

large variation in morphology and use by humans of this vegetable. The morphological

24

variation ranges from white and red cabbage over broccoli, cauliflower, kale, kohlrabi,

25

Brussels sprout to lesser known varieties. All domesticated cabbage varieties are

26

believed to have originated from wild cabbage (Brassica oleracea var. oleracea) native

27

to different coastal habitats at the Mediterranean and Atlantic.2 The presently known

28

cabbage cultivars are likely to have originated independently in different regions of

29

Europe from various wild forms. Romans already cultivated several different kinds of

30

cabbage.3 Evidence for this hypothesis is given by the study of glucosinolates, for

31

example.4 Song et al. suggested that ancient forms of Brassica oleracea may be

32

thousand head kale and Chinese kale / kai-lan.5 The most recent study on the phylogeny

33

of Brassica reported B. oleracea to be phylogenetically close to B. juncea (L.) Czern.

34

(Indian mustard), B. incana Ten. (Mediterranean mustard), B. rapa L. (turnip rape) and

35

B. napus L. (rapeseed, hybrid of wild cabbage and turnip rape), as well as B. cretica

36

Lam. and B. montana DC. (other wild cabbage types).6 Surprisingly little is, however,

37

known about relationships of varieties within B. oleracea. Louarn et al. demonstrated

38

polyphyly of white cabbage using AFLP (Amplified Fragment-length Polymorphism)

39

but did not resolve relationships among varieties.7 In contrast, Mei et al. resolved

40

broccoli as early branching in B. oleracea and sister-group relationships of kohlrabi and

41

savoy cabbage, as well as white cabbage and Chinese kale but sampling with B.

42

oleracea was too small to allow further inferences.8 Including all varieties is a huge task

43

given the enormous diversity in the species.



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Here, we focus on kale (B. oleracea var. sabellica L.), which is a valuable fresh

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vegetable during wintertime given its tolerance against frost. It is mostly assumed to

46

have originated from wild cabbage occurring on cliffs and rocky coasts along the

47

Atlantic but this is still under dispute.2 Typical German kale differs from other cabbages

48

in their curly leaves. However, the term kale is rather widely used for different

49

morphological types of large-leafed B. oleracea varying in leaf morphology and

50

sometimes described as different varieties, for example. In general, kale is characterized

51

by high levels of various nutrients and further constitutional metabolites like

52

glucosinolates, flavonoids and carotenoids.9 In this study we investigate different kale

53

varieties from various origins and countries (like Northern Germany, Italy, USA) that

54

differ in traits like growth height, shape, leaf color and curling. However, what is not

55

known is whether these geographical origins also form cohesive phytochemical and

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phylogenetic groups. The main question is, therefore, whether kale constitutes a

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homogenous group or whether regional groups of kale can be distinguished

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phytochemically and genetically.

59 60

The phytochemical analysis focusses on five frequently occurring glucosinolates in the

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leaves in late autumn. Glucosinolates are a large group of anionic, hydrophilic, sulphur-

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containing secondary plant metabolites. They make a significant contribution to the

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specific spicy cabbage flavor and bitter taste in Brassica crops.10 More than 100

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different glucosinolates are known11 and about 20 are predominant in Brassica sp.12

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They are derived from amino acids and can be grouped into aliphatic, aromatic and

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indole glucosinolates. The endogenous enzyme myrosinase (thioglucoside

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glucohydrolase) catalyzes the hydrolysis of glucosinolates to yield glucose, sulfate and

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aglycones. The aglycones then decompose to breakdown products like isothiocyanates,


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thiocyanates, indoles and organic cyanides (nitriles) – depending on the substrate, pH

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and availability of certain ions.13-15 Several isothiocyanates are found to exhibit

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anticancerogenic effects against various types of cancer, such as breast, lung and colon

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cancer.16-17 Compounds are bioavailable and show interference in early stages of cancer

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development by inducing detoxification enzymes (phase II enzymes), inhibiting

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activation enzymes (phase I enzymes) and triggering of cell cycle arrest or apoptosis.18-

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21

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cultivars is therefore an important aspect of Brassica breeding. We, here, investigate

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quantitatively the occurrence of five glucosinolates, i.e., the aliphatics gluconapin,

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progoitrin and glucoraphanin, the aromatic gluconasturtiin and the indole glucobrassicin

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(Table 1, Fig. 1). Most of these glucosinolates lead to metabolites that are associated

80

with beneficial anticancerogenic properties (especially glucoraphanin22). Additionally,

81

progoitrin and the aromatic gluconasturtiin contribute to the bitterness of cabbage.13, 23

82

Progoitrin in particular is associated with the detrimental effect of potentially causing

83

goiter (through the hydrolytic product oxazolidine-2-thione)24. It is generally assumed

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that bitter compounds are eliminated by breeding from commercially cultured plants.

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Therefore, we hypothesized that landraces have increased levels of these compounds

86

compared with commercial cultivars. Glucosinolate profiles are known to differ among

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Brassica oleracea cultivars.25 Information on a broad sample of kale cultivars and

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determining specific glucosinolate profiles for certain groups of cultivars may therefore

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be beneficial for future breeding and choice of kale types for improved nutrition.

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Whereas glucosinolate levels are known to be influenced by various factors such as life

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cycle, temperature and fertilization, common origin is still considered a crucial aspect of

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phytochemical profiles.12

The occurrence and differences in specific glucosinolates among various Brassica

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For investigating relationships between different kales and their relation to other

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cabbage cultivars we made use of the single-plex SNP genotyping platform KASPTM

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(Kompetitive Allele Specific Polymerase Chain Reaction (PCR)). Genetic variation is

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measured on the basis of Single Nucleotide Polymorphisms (SNPs). They are useful for

98

investigating genetic diversity and discriminating between cultivars and have been

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commonly applied in research on Brassica crops such as B. napus (e.g. Dalton-Morgan

100

et al 26). SNPs are one of the most common type of genetic variation in eukaryotic

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genomes and most commonly found in the DNA between genes. The advantages of this

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assay are low average genotyping errors, cheap genotyping costs, as well as scalable

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flexibility in applications.27

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Here, we use SNP and phytochemical data with the aim to identify glucosinolate

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profiles specific to certain kale varieties. We hypothesize that different groups of kale

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have evolved in different parts of the world differing in their ability to synthesize

107

different glucosinolates. Further, similarities between phytochemical profiles and

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genetic data allow to infer the relationships of kale varieties, especially the origin of the

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American kale varieties.

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Material and Methods

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Plant Material

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In March 2014, seeds of 25 varieties of kale (Table 2) as well as 11 non-kale Brassica

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oleracea cultivars were seeded in a greenhouse under natural daylight. When plants

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reached the 5-leaf stage, plants were transplanted in a randomized scheme into a field at

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the University of Oldenburg botanical garden. Water and fertilizer were applied

117

according to standard cultural practices. Leaves were harvested in August/September

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2014.


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Quantification of the Glucosinolate Content

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Extraction from Plant Material. For each kale variety, 10 g fresh material of the

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youngest fully developed leaves were weighed (excluding the thick midnerves), treated

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at 120 °C for 2h15 (to inactivate the enzyme myrosinase), ground to a fine powder using

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a mixer mill, transferred to a screwtop vial and mixed with 8 mL of 70% v/v

125

methanol/water (of HPLC grade). The sealed vials were sonicated and then stored at

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-20 °C until further analysis. Two individual plants per variety were sampled.

127 128

Analysis. Kale leaf extracts were obtained after thermal deactivation of myrosinase as

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aqueous methanolic extracts. Extracts were profiled using an adapted liquid

130

chromatography–mass spectrometry method (LC-MS) from the literature.28, 29 Five

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glucosinolates (i.e., glucobrassicin, glucoraphanin, gluconasturtiin, gluconapin and

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progoitrin; Fig. 1) were detected and quantified with high sensitivity by HPLC-qTOF-

133

MS with negative ion electrospray ionization (ESI). A Bruker impact HD qTOF mass

134

spectrometer (Bruker Daltonik GmbH, Bremen, Germany) coupled to an Agilent 1260

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Series HPLC system was used (Agilent Technologies, Santa Clara CA, USA),

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consisting of: Online Degaser G1322A, Bin Pump G1312B, High Performance

137

Autosampler HiP-ALS G1367E, Thermostatted column compartment G1316A, Diode

138

Array Detector DAD-VL G1315D. Before analysis, 2 mL of the extracted samples were

139

filtered (0.45 μm pore size) into 2 mL screwtop vials. The HPLC-column was a 150 mm

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x 2 mm (2.8 μm particle size) Pursuit XRs Ultra 2.8 Diphenyl column (Agilent

141

Technologies, Santa Clara CA, USA). The mobile phase was 0.005% formic acid

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(HCOOH) with an acetonitrile/water gradient (CH3CN) over 75 min at a flow rate of 0.5

143

mL/min. The gradient profile increased from 5 to 75% linearly in 45 min followed by a 7 ACS Paragon Plus Environment


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return to 5% and 30 min isocratic to re-equilibrate. The eluate absorbance was

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monitored by an UV-detector at 229 nm. MS-scans were performed in the negative ion

146

mode on the glucosinolate [M-H]- ions and spectra were recorded between m/z (mass-

147

to-charge ratio) 100 and 700 (capillary voltage: 4500 V, nebulizer: 1.8 bar, dry gas

148

temperature: 200 °C, dry gas flow: 9 L/min). For quantification eight point calibration

149

curves (see Supplementary Figure 1) were obtained for each glucosinolate from

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authentic reference substances (analytical standard glucosinolates from PhytoLab

151

GmbH & Co. KG, Vestenbergsgreuth, Germany). Quantification was carried out in the

152

negative ion mode on extracted ion chromatograms of each respective glucosinolate m/z

153

value with isolation width of 0.002 Da. All Pearson correlation coefficients R2 were

154

found to be above 0.99 and repeat injections yielding a relative standard deviation

155

(RSD) below 5%. The lowest quantifiable amount is indicated for each glucosinolate by

156

the signal-to-noise ratio (S/N) (see Table 3). Mass calibration was carried out using a

157

0.01 mM sodium formate solution injected prior to each chromatographic run using an

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enhanced quadratic calibration algorithm. Spiking of the kale extract with authentic

159

reference compounds was carried out with a RSD below 5% indicating that no

160

appreciable matrix effect was operating.

161 162

Data Evaluation. For data evaluation, ESI Compass 1.3 Data Analysis software was

163

used (Version 4.0.234.0, Bruker Daltonik GmbH, Bremen, Germany). Statistical

164

analyses were conducted using the software R (v3.0.3, R Core Team30), specifically

165

Analysis of Variance (ANOVA), Tukey’s HSD test and Pearson’s product-moment

166

correlation analysis.

167 168

Phylogenetic Analyses Using KASP-Assay


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DNA-Extraction and KASP Assay Analysis. For each variety, one intact, fully

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developed leaf was cut, placed in a plastic bag with silica beads inside and stored until it

171

was completely dried. DNA was extracted from leaves using the innuPREP Plant DNA

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Kit (Analytik Jena AG, Jena, Germany) and afterwards stored at -20 °C until further

173

analysis. The subsequent KASPTM assay, developed by KBioscience/LGC Genomics

174

(Hoddesdon, Hertfordshire, UK), was carried out by TraitGenetics (TraitGenetics

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GmbH, Gatersleben, Stadt Seeland, Germany). Each sample was analyzed with 100

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KASP markers well distributed over the genome of Brassica oleracea. KASP assays

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rely on allele-specific oligo extension and Fluorescence Resonance Energy Transfer

178

(FRET) for signal generation.31 In the KASP cycle, an allele-specific primer matches a

179

target SNP and, with a common reverse primer, amplifies the target region. In a second

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PCR round, the reverse primer binds to the previously generated product and thus

181

makes a complementary copy. In a third PCR round, a fluorescence-labeled oligo (FAM

182

or HEX from FRET cassette) binds to the amplicon from the previous round.32, 33

183 184

Data Analysis. Phylogenetic relationships were inferred using Bayesian Inference (BI)

185

and Neighbor-Net methods. The BI analysis was conducted using MrBayes (v3.2.4,

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Ronquist et al.34) sampling across the entire GTR (general time reversible) model space,

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including gamma. Two independent runs of 10,000,000 generations were completed

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with four chains each (three heated, one cold). Trees were sampled every 100

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generations, the first 25% of generations were discarded as burn-in. A majority-rule

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consensus of the remaining trees from the two runs was produced with posterior

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probabilities. Siberian kale (B. napus var. pabularia DC.) was used as outgroup taxon.

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Compared to a phylogenetic tree, phylogenetic networks can often better illustrate the

193

evolutionary history, e.g. in the case of hybridization. They display the extent of



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conflicting signals in the data.35 Neighbor-Net is a distance based method for

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constructing phylogenetic networks, based on the Neighbor Joining (NJ) algorithm.36 A

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Neighbor-Net was constructed using SplitsTree (v4.13.1, Huson & Bryant37) with

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uncorrected P-distances and ambiguous states were matched. A detailed description of

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the underlying mechanism of the method is given by Bryant & Moulton.38

199 200

Results

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Quantification of the Glucosinolate Content

202

The five glucosinolates previously described in kale gluconapin, progoitrin,

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glucoraphanin, gluconasturtiin and glucobrassicin were identified in the chromatogram

204

based on their high resolution m/z value of their [M-H] pseudomolecular ion, which all

205

showed errors below 5 ppm. Additionally the structures were confirmed using a tandem

206

MS measurement with all glucosinolates showing characteristic fragment ions at m/z

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259.01 (C6H12O9S). Based on this characteristic fragmentation mechanism a search for

208

previously unreported glucosinolates was carried out by construction of extracted ion

209

chromatograms in All MSn mode (see Supplementary Figure 2). Isothiocyanate products

210

(e.g., erucin, sulforaphane) are absent from the LC-MS chromatograms (see

211

Supplementary Figure 4).

212 213

The content of five glucosinolates were analyzed in different kale and cabbage samples,

214

with two individual plants being measured for each variety with analytical duplicate

215

measurements. The resulting levels, received with the help of reference standard

216

measurements, are summarized in the Supplementary Table 1. The gluconapin content

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varied between 0.005 mg/100g fresh weight (FW) (Redbor) and 19.6 mg/100g FW

218

(Vates) (Fig. 2). The American varieties (especially Georgia Southern, Champion and


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Vates) showed significantly higher gluconapin contents compared to all others (p
0.05). Additionally, the general lack of coherence

408

among German landraces and German commercial cultivars indicates multiple origins

409

of German commercial cultivars from various Northern German landraces. Holterfehn

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and Jellen as well as Buss Bunde, Rote Palme and Diepholzer each have very similar

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glucosinolate contents. However, looking at the phylogenetic results, none of them are

412

placed in a sister-group relationship. A lot of these Northern varieties have their native

413

region around the Northern German city “Leer” (Holterfehn, Jellen, Buss Bunde,

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Lammertsfehn, Schatteburg, Neuefehn). In the BI phylogeny (Supplementary Figure 3)

415

they cluster nearly perfectly together. In the Neighbor-Net (Fig. 4) we find them also

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quite near to each other.

417


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Flower-Sprout, a hybrid between kale and Brussels sprouts, is a newly developed hybrid

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cultivar originating in England (Gärtner Pötschke GmbH, Kaarst, Germany). It is not

420

known which kale variety was used as parent for hybridization, but with reference to

421

Flower-Sprout’s red colored leaves it is imaginable that the parental variety also had red

422

leaves (like Redbor or Rote Palme).

423

Siberian kale was used as outgroup taxon in phylogenetic analyses because it belongs to

424

Brassica napus ssp. pabularia. The leaves of Siberian resemble those of kale a bit,

425

because it is believed that Brassica napus originated from hybridization between turnip

426

(Brassica rapa) and kale. Carlson et al. reported a high progoitrin and glucobrassicin

427

content in this variety.48 We instead found a high gluconasturtiin content, which is

428

consistent with studies on Brassica rapa.49, 50

429 430

Implications for health-oriented breeding

431

While we have assembled data on different aspects of the glucosinolate content and

432

relationships of kale and demonstrated differences between regional origins, a number

433

of mechanisms may explain why glucosinolate levels differ between varieties. Several

434

sets of genes regulate the content of specific glucosinolates in different cultivars. Also

435

additional factors may influence the glucosinolate composition (soil properties, drought,

436

temperature, light etc.12). Since some of the glucosinolate metabolites exhibit cancer

437

chemopreventive or even anticancerogenic properties and others have rather detrimental

438

effects and can decrease food quality through bitter taste (due to high progoitrin levels,

439

for example23), there has to be a trade-off between beneficial and unfavorable

440

properties. A combination of different metabolites in plants should be aimed at. Most

441

potent breakdown products in terms of anticancerogenic effects are the ones of

442

glucoraphanin, like the isothiocyanate sulforaphane.13, 51 Therefore kale varieties with



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elevated glucoraphanin levels are desirable as functional food with added health benefit.

444

Frostara and Black Tuscany as well as red cabbage and Romanesco produce much

445

glucoraphanin. In comparison typical broccoli varieties contain around 10 mg

446

glucoraphanin per 100 g FW.28 As our quantitative data show glucoraphanin levels in

447

some kale varieties can be as high as ten times that of an average broccoli, which is

448

currently the gold standard with respect to provision of glucoraphanin dietary exposure.

449

In contrast, the American varieties, Palmizio and Lerchenzungen have rather low

450

amounts. Frostara at the same time produces comparatively much progoitrin, and so do

451

Siberian kale, the American kale, red and wild cabbage. These ones may therefore taste

452

more bitter. The aromatic gluconasturtiin is mostly present in low amounts in Brassica

453

oleracea, Siberian kale (B. napus) instead contains much higher volumes. The wide

454

range of variability in glucosinolates content among kale can be used as an information

455

for breeding and developing new varieties with enhanced benefits.

456 457

Abbreviations Used

458

AFLP, amplified fragment-length polymorphism; ANOVA, analysis of variance; BI,

459

bayesian inference; Da, Dalton; ESI, electrospray ionization; FRET, fluorescence

460

resonance energy transfer; FW, fresh weight; GTR, general time reversible; HD, high

461

definition; HPLC, high-pressure liquid chromatography; KASP, kompetitive allele

462

specific polymerase chain reaction; MS, mass spectrometry; m/z, mass-to-charge ratio;

463

NJ, neighbor joining; PCR, polymerase chain reaction; ppm, parts per million; qTOF,

464

quadrupole time of flight; RSD, relative standard deviation; S/N, signal-to-noise ratio;

465

SNP, single nucleotide polymorphism

466 467


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468

Acknowledgements

469

We thank Vera Mageney for help with DNA extraction, Reinhard Lühring for seeds of

470

local Northern German kale and Holger Ihler and Gertrud Genz for assistance in the

471

cultivation of the plants.

472

Funding / Notes

473

The authors declare no competing financial interest.

474 475

Supporting Information Available

476

Supplementary Figure 1: calibration curves of glucoraphanin and glucobrassicin

477

Supplementary Figure 2: base peak chromatogram (BPC) and extracted ion

478

chromatograms (EIC) of kale variety Frostara

479

Supplementary Figure 3: Bayesian Inference phylogeny

480

Supplementary Figure 4: extracted ion chromatogram (EIC) of sulforaphane and

481 482

glucoraphanin in kale variety Buss Bunde Supplementary Table 1: glucosinolate values of analyzed kale and cabbage samples



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(1) FAOSTAT. Food and Agriculture Organization of the United Nations – Statistics

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Division; http://faostat3.fao.org/download/Q/QC/E (accessed December 15, 2015).

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(3) Wittstein, G. C. Die Naturgeschichte des Cajus Plinius Secundus; Gressner &

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Figure captions

Figure 1. Chemical structures of glucosinolates quantified. The structures were obtained by using ChemDraw software (v15.0.0.106, PerkinElmer Informatics, Massachusetts, USA).

Figure 2. Glucosinolate content (mg/100g FW) of different kale and cabbage varieties. Two individual plants per variety were measured (black and grey bars). Note the different scales.

Figure 3. Differences in glucosinolate content (mg/100g FW) between the groups of kale and cabbage varieties: (A) gluconapin content, (B) progoitrin content, (C) glucoraphanin content, (D) gluconasturtiin content, (E) glucobrassicin content. Note the different scales. Different letters above each boxplot indicate significant differences in a post-hoc Tukey’s HSD test (p < 0.05).

Figure 4. Neighbor-Net of different kale and cabbage varieties based on the KASPanalysis. Sample names are color coded according to their origin. Numbers beside branches are Bayesian posterior probabilities (support >70) for the respective clade. Leaf margin curliness of the varieties is indicated.



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Tables

Table 1. The Glucosinolates Analyzed In This Study, Listed With Their Relevant Characteristics. Molecular Formulas Were Obtained From The Royal Society Of Chemistry52. group

gluconapin

m/z experimental [M-H] 372.0399

m/z theoretical [M-H] 372.0418

error [ppm] 4.9

molecular formula C11 H19 N1 O9 S2

retention time [min] 7.0

progoitrin

388.0368

388.0366

2.5

C11 H19 N1 O10 S2

4.4

glucoraphanin

436.0396

436.0400

3.5

C12 H23 N1 O10 S3

4.3

aromatic

gluconasturtiin

422.0578

422.0574

1.7

C15 H21 N1 O9 S2

17.0

indole

glucobrassicin

447.0496

447.0537

2.6

C16 H20 N2 O9 S2

15.8

aliphatic

trivial name


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Table 2. Summary Of The Kale And Cabbage Varieties Used In This Study. The Assignment To The Analyses Is Displayed As Well As The Origin Of The Seeds (e.g., Company The Varieties Have Been Obtained From). included in glucosinolate analysis

variety

non-kale cabbage cultivars

local Northern German kale

American varieties

Italian varieties

German kale varieties (B. oleracea convar. acephala var. sabellica)

Lerchenzungen

x

Vitessa

included in phylogenetic analyses x x

Vitessa

x

Lage Fijngekrulde

x

Niedriger Grüner Krauser

x

Reflex F1

x

x

Redbor F1

x

x

Frostara

x

x

Winnetou F1

x

x

Flower-Sprout "Petit Posy Mix" (Brussels sprouts × kale) Black Tuscany

x

x

x

x

Palmizio Senza Testa

x

x

Negro Romano

x

x

Georgia Southern

x

x

Morris Heading

x

x

Champion

x

x

Vates

x

x

Buss Bunde

x

x

Niedriger von Rosenweide

x

x

Rote Palme

x

x

Rote Palme Holterfehn

x

x

Schatteburg

x

x

Jellen

x

x

Neuefehn Hainwatjes

x

x

Lammertsfehn

x

x

Diepholzer Dickstrunk

x

x

Red cabbage "Roodkop" B. oleracea convar. capitata var. rubra Savoy "Vertas 2" B. oleracea convar. capitata var. sabauda Butter cabbage B. oleracea var. costata Kohlrabi "Delikateß blauer" (blue) B. oleracea convar. acephala var. gongylodes Romanesco broccoli B. oleracea convar. botrytis var. botrytis Brussels sprouts "Hilds Ideal" B. oleracea convar. gemmifera var. gemmifera Marrow-stem kale "Westfälischer Furchenkohl" B. oleracea convar. acephala var. medullosa Marrow-stem kale "Walking Stick" B. oleracea convar. acephala var. medullosa Galician cabbage B. oleracea convar. acephala Wild cabbage „Helgoländer“ B. oleracea L. Siberian kale B. napus ssp. pabularia

x x x x x x x

x

x

x

x

x

x

x

x

x

seed origin Kiepenkerl, Bruno Nebelung GmbH, Everswinkel, Germany N.L.Chrestensen Erfurter Samen- und Pflanzenzucht GmbH, Erfurt, Germany Centrala Nasienna, Saatbau Polska Sp. z o.o., Środa Śląska, Poland N.L.Chrestensen Erfurter Samen- und Pflanzenzucht GmbH, Erfurt, Germany PNOS (Polskie Nasiennictwo Ogrodnictwo Szkółkarstwo Sp. z o.o.), Ożarów Mazowiecki, Poland Dürr Samen S. Schwenk e.K., Reutlingen, Germany Kiepenkerl, Bruno Nebelung GmbH, Everswinkel, Germany Kiepenkerl, Bruno Nebelung GmbH, Everswinkel, Germany Kiepenkerl, Bruno Nebelung GmbH, Everswinkel, Germany Gärtner Pötschke GmbH, Kaarst, Germany Thompson & Morgan (UK) Ltd., Ipswich, UK Thompson & Morgan (UK) Ltd., Ipswich, UK Reinhard Lühring, private farmer, Rhauderfehn, Germany

Sustainable Seed Company, Covelo CA, USA

Reinhard Lühring, private farmer, Rhauderfehn, Germany

Gartenland GmbH Aschersleben, Essen, Germany toom, Centor-Warenhandels-GmbH, Köln, Germany Magic Garden Seeds, Andreas Fái-Pozsár, Regensburg, Germany N.L.Chrestensen Erfurter Samen- und Pflanzenzucht GmbH, Erfurt, Germany Magic Garden Seeds, Andreas Fái-Pozsár, Regensburg, Germany toom, Centor-Warenhandels-GmbH, Köln, Germany Kiepenkerl, Bruno Nebelung GmbH, Everswinkel, Germany Thompson & Morgan (UK) Ltd., Ipswich, UK Reinhard Lühring, private farmer, Rhauderfehn, Germany Botanical Garden of the University of Oldenburg, Oldenburg, Germany Exotic-Samen Wolfgang Meier, Fürstenwalde, Germany



Page 32 of 37

Table 3. Parameters For Calibration Curves: Slope, Axis Intercept, Pearson Correlation Coefficient R2, As Well As Relative Standard Deviation Of Analytical Repeat Injections (RSD) And Signal-To-Noise Ratio (S/N) Of The Five Glucosinolate Standards. glucosinolate

slope

axis intercept

gluconapin progoitrin glucoraphanin gluconasturtiin glucobrassicin

0.0012 0.0006 0.0039 0.0005 0.0012

-0.1089 -0.3175 0.0663 -0.0386 0.1321

coefficient of determination R2 0.9998 0.9980 0.9998 0.9999 1.0

RSD (%)

S/N

3.97 3.01 4.16 4.37 3.23

45 230 45 195 55


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Figure graphics

Figure 1:



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Figure 2:


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Figure 3:



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Figure 4:


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Graphic for table of contents


Diversity of Kale (Brassica oleracea var. sabellica): Glucosinolate

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