Quantification of intracellular seed compounds of ...

Quantification of intracellular seed compounds of Moringa oleifera Lam. using digital image analysis Q.E. Muhl1, F.H.J. Kanfer2, E.S. du Toit1 and J.M. Steyn1 1Department

of Plant Production and Soil Science, University of Pretoria, Pretoria, 0002, South Africa;

2Department of Statistics, University of Pretoria, Pretoria, 0002, South Africa.

Abstract Histochemical staining of sectioned seed for the identification of various intracellular compounds has successfully been used in the past. The estimation of storage compound content through the quantification of a specific compound selecting stain through digital image analysis using Adobe® Photoshop® has however not been explored to date and therefore, became the main aim of this study. Moringa oleifera seeds of various developmental stages were prepared for microscopic analysis and stained with oil (Sudan III), protein (Light Green SF, Orange G) and starch (Periodic Acid Schiff) detecting stains. The percentage coverage of a specific stain in digital images of sectioned seed was determined using Adobe® Photoshop® and compared to analytical data. Protein stain: Orange G and oil detecting stain: Sudan III demonstrated the best correlation to the measured seed protein and oil content throughout seed development. For simple comparisons between storage compound contents at different developmental stages, image stain quantification proved reasonably effective. Keywords: Adobe® Photoshop®, histochemical staining, Sudan III, Light Green SF, Orange G, Periodic Acid Schiff INTRODUCTION The use of histochemical staining of biological material for the identification of various compounds has successfully been used for nearly two centuries (Wick, 2012). Although such stains are best suited for qualitative images analyses, they offer very little quantitative data to perform accurate scientific comparisons. Commercial photo-editing software has in the past been used for the quantification of histological samples, predominantly in the medical field of histopathology (Dahab et al., 2004; Lahm et al., 2004), although its application in plant sciences has been limited to date. The main aim of this study was thus to determine whether commercial photo-editing software such as Adobe® Photoshop® could be used to quantify the amount of stain in seed sections through colour selection within a slide. MATERIALS AND METHODS Seed for the purpose of this trial were harvested from six-year-old Moringa oleifera trees at the field trial section on the Experimental Farm of the University of Pretoria (25°45’S; 28°16’E) at an altitude of 1372 m a.s.l. and an average annual rainfall of 674 mm. Trees for the purpose of this trial were grown from ‘PKM1’ cultivar seeds, sourced in India and transplanted into the field. Moringa oleifera is becoming increasingly popular as an industrial crop due to its multitude of uses such as water purifier, nutritional supplement and biofuel feedstock (Fuglie, 2001; Anwar et al., 2007; Rashid et al., 2008). The trees bear 20 to 30 cm long fruit that once mature, change colour from green to brown, revealing numerous round or triangular seeds with three papery wings (Folkard et al., 1999). Seed developmental stages were based on fruit width, grouped into 2 mm intervals starting at 8 mm (8, 10, 12, 24 mm) up to 24 mm in diameter. Light microscopy Due to continuous flower initiation and development, fruit of different developmental

Acta Hortic. 1125. ISHS 2016. DOI 10.17660/ActaHortic.2016.1125.50 XXIX IHC – Proceedings of the Symposia of the V World Congress on Medicinal and Aromatic Plants and International Symposium on Plants, as Factories of Natural Substances, Edible and Essential Oils Eds.: B. Patil et al.

383

stages could be found on the trees throughout the growing season. Ten seeds of at least nine developmental stages were randomly sampled from all three irrigation treatments and prepared for light microscopy according to O’Brien and McCully (1981). After harvesting, seeds were immediately fixed in FAA (80% ethanol:37% formaldehyde:100% acetic acid, in proportions 8:1:1 v/v/v) for at least 24 h, before being dehydrated in an ethanol in water solution series (30, 50, 70, 100% v/v) for 24 h at each concentration. Then, ethanol was extracted from the specimens through a series of xylene in ethanol concentrations (30, 50, 70, 100% v/v) prior to placing them into paraffin wax (60°C). Embedded seed samples were cut into 10 μm thick sections, using a Reichert-Jung semi-thin rotary microtome, and placed onto microscope slides before being de-waxed in a series of xylene concentrations. The histochemical staining of seed sections differed depending on the compound that was stained for; hence they are discussed individually below. Protein Intracellular protein was detected by staining seed sections in duplicate using two different protein stains namely, Light Green SF (Figure 1A) and Orange G (Figure 1B) according to James and Tas (1984). The same staining procedure was followed for both these stains. After de-waxing, sections were stained in either Orange G or Light Green SF for 30 min, followed by three rinses of 1% acetic acid and two rinses of distilled water. Sections were then briefly rinsed in three changes of tert-butanol, followed by two changes in xylene, before being mounted with synthetic resin. Protein stained bright green with the Light Green SF and bright orange with Orange G.

Figure 1. Moringa oleifera endosperm stained with different compound detecting stains. A – Light Green SF, B – Orange G, C – Periodic Acid-Schiff, D – Sudan III. Starch Starch reserves were detected using Periodic Acid-Schiff (PAS) (Figure 1C) reagent according to Merck (2011). De-waxed sections were rinsed in distilled water and placed into Sodium periodate solution for 5 min each. After rinsing sections in distilled water, they were stained with PAS reagent for 15 min and rinsed with sulfite water (6 min), followed by distilled water (10 min). Sulfite water was prepared by adding a mixture of 10 mL sodium disulfite solution (10%) and 10 mL of hydrochloric acid (1 mol L-1) to 200 mL of distilled water. Finally, sections were stained with hemtoxylin solution (2 min) and rinsed with distilled water (3 min) before being mounted. Starch (polysaccharides) stained purplemagenta with PAS.

384

Oil Seed used for the lipid staining could not be embedded using the conventional procedure used for light microscopy as discussed above, given that seed lipids are xylene soluble. Alternatively, under such circumstances fresh seed specimens would merely be sectioned by hand. With this method, section thickness would have become inconsistent and this would in turn have affected staining, making different samples incomparable. Moringa seed posed an additional challenge in that they are relatively hard once mature and difficult to section uniformly thin. For this reason, the following customized method was developed for Moringa oleifera seed in order to perform lipid staining. Seed samples were fixed in FAA (80% ethanol:37% formaldehyde:100% acetic acid, in proportions 8:1:1 v/v/v) for 7-14 days, followed by 24-48 h in distilled water to remove excess fixative prior to sectioning. Samples were then thinly sectioned using a freeze-microtome and transferred onto microscopy slides prior to staining. Preliminary trials found Sudan III (Sudan red) (Figure 1D) to be the best lipid stain for Moringa oleifera seed, when compared to Sudan IV and Sudan black B. Hence Sudan III was used for all subsequent intracellular lipid staining according to Culling (1974). Sections were submerged in propylene glycol (2 min) and then transferred into Sudan III solution (10 min). Then, excess Sudan III was removed by rinsing sections in two changes of 85 and 50% propylene glycol. Lipids stained red/orange with Sudan III. Image analysis Once stained, seed sections were viewed using a Leitz Biomed light microscope, while photographs were taken using a Canon PowerShot A630 digital camera. Digital images of the sectioned seed stained with Orange G, Light Green SF, Sudan III and PAS were analysed using Adobe® Photoshop® CS6, Version 13.0 x32. Digital images were individually uploaded into Adobe® Photoshop® and the colour (RGB ratio) set to the specific stain in question (Figure 2). The RGB ratios for the different stains used during analysis are given in Table 1.

Figure 2. Screenshot of Adobe® Photoshop® CS6, with uploaded seed cross-section stained with the protein stain Orange G. Table 1. RGB ratios used for the color range selection for various histochemical stains. R G B

Green SF 10 165 157

Orange G 173 131 23

Periodic Acid-Schiff 151 61 157

Sudan III 211 87 1 385

Subsequently, the “Color Range” command was selected under the “Select” drop-down menu. To ensure the selection was not limited to the set colour but included additional shades around the set colour, the “fuzziness” was set to 100. This meant that all pixels within 100 brightness levels (both lighter and darker) from then set colour (RGB ratio) were included in the selection. The selection area remained visible in the set colour range, while the unselected areas appeared black in the main window (Figure 3). Once all pixels within the set colour range were selected, the pixel count was displayed under the Histogram section (Figure 4). The percentage coverage of a certain colour within an image was calculated using the equation below. %

=

(

( ) )

× 100

(1)

The difference between a sample section stained with Orange G before and after selective extraction using Adobe® Photoshop® CS6 is illustrated in Figure 5.

Figure 3. Screenshot of Adobe® Photoshop® CS6, illustrating the colour specific selection process using the colour range function.

Figure 4. Screenshot of Adobe® Photoshop® CS6, displaying the total selection area within the set colour range as well as the histogram section revealing the number of pixels that have been selected. 386

Figure 5. Moringa oleifera seed sections stained with Orange G before (A) and after (B) selective extraction using Adobe® Photoshop® CS6. RESULTS AND DISCUSSION Stained seed sections offer very little tangible data as they can essentially only be used for visual comparisons. Quantification of these sections enabled differences between developmental stages to be statistically assessed. The percentage coverage of the oil, protein and starch detecting stains in digital images of sectioned seed was determined using Adobe® Photoshop®, results of which are illustrated in Figure 6.

Figure 6. Average stain coverage (%) of seed sections from various developmental stages, stained with starch, oil and protein detecting stains. PAS – Periodic Acid-Schiff. Vertical bars (±) indicate standard errors. Results obtained from the quantification of stained seed sections were very similar to the storage compound content (g 100 g-1) measured in seeds across the various developmental stages (Muhl et al., 2014). Despite only slight differences between storage compounds (oil, protein and starch) during the initial growth stages, significant increases across all compounds were measured during the early reserve deposition phase, between 387

the fruit diameters of 16 to 18 mm. While both starch (PAS) and protein (Green SF and Orange G) continued to increase significantly beyond these growth stages, differences in oil (Sudan III) levels on the other hand, levelled off after seed reached a diameter of 18 mm, with only slight increases until maturity. CONCLUSIONS Since stained seed sections viewed under the microscope offer no tangible data for comparison, the quantification of these sections using digital imaging software enabled seed samples from different developmental stages to be compared. The estimation of a storage compound content (g) based on the relative stain intensity was not equally effective for all stains. Orange G (protein stain) and Sudan III (oil stain) demonstrated a better correlation between the percentage stain coverage and the actual compound content (g) when compared to Periodic Acids Schiff reagent (starch stain). Nonetheless, stain quantification was used successfully to determine the onset of storage compound biosynthesis and the differentiation between storage compound amounts at different developmental stages. Although the success of digital image analysis might be stain dependant, it certainly is a cost effective technique for compound content estimation. ACKNOWLEDGEMENTS National Research Foundation (NRF). Literature cited Anwar, F., Latif, S., Ashraf, M., and Gilani, A.H. (2007). Moringa oleifera: a food plant with multiple medicinal uses. Phytother Res 21 (1), 17–25 http://dx.doi.org/10.1002/ptr.2023. PubMed Culling, C.F.A. (1974). Handbook of Histopathological and Histochemical Techniques (including museum techniques) (Butterworth). Dahab, G.M., Kheriza, M.M., El-Beltagi, H.M., Fouda, A.M.M., and El-Din, O.A.S. (2004). Digital quantification of fibrosis in liver biopsy sections: description of a new method by Photoshop software. J. Gastroenterol. Hepatol. 19 (1), 78–85 http://dx.doi.org/10.1111/j.1440-1746.2004.03183.x. PubMed Folkard, G., Sutherland, J., and Shaw, R. (1999). Water clarification using Moringa oleifera seed coagulant (Loughborough University). Fuglie, L. (2001). Natural nutrition for the tropics. In The Miracle Tree: the Multiple Attributes of Moringa (Dakar, Sé né gal: CTA/CWS), p.103–115. James, J., and Tas, J. (1984). Histochemical Protein Staining Methods (Oxford University Press; Royal Microscopical Society). Lahm, A., Uhl, M., Lehr, H.A., Ihling, C., Kreuz, P.C., and Haberstroh, J. (2004). Photoshop-based image analysis of canine articular cartilage after subchondral damage. Arch Orthop Trauma Surg 124 (7), 431–436 http://dx.doi. org/10.1007/s00402-004-0701-6. PubMed Merck. (2011). Microscopy - Schiff’s Reagent Pararosaniline (chloride) (C.I. 42500), MERCK (Darmstadt, Germany: Merck). Muhl, Q.E., Du Toit, E.S., and Steyn, J.M. (2014). Irrigation amounts affect the compositional changes of Moringa oleifera seeds throughout different developmental stages. Int. J. Agric. Biol. 16, 201–206. O’Brien, T.P., and McCully, M.E. (1981). The Study of Plant Structure: Principles and Selected Methods (Melbourne: Termarcarphi Pty. Ltd.). Rashid, U., Anwar, F., Moser, B.R., and Knothe, G. (2008). Moringa oleifera oil: a possible source of biodiesel. Bioresour. Technol. 99 (17), 8175–8179 http://dx.doi.org/10.1016/j.biortech.2008.03.066. PubMed Wick, M.R. (2012). Histochemistry as a tool in morphological analysis: a historical review. Ann Diagn Pathol 16 (1), 71–78 http://dx.doi.org/10.1016/j.anndiagpath.2011.10.010. PubMed

388

XXIX International Horticultural Congress on Horticulture: Sustaining ...

1 of 4

http://www.ishs.org/ishs-book/1125

Apply for membership Manage/Renew your membership

International Society for Horticultural Science T h e wo rl d 's l e a d i n g i n d e p e n d e n t o r g a n i za t i o n o f h o rt i cu l tu ra l sci e n ti st s

5184 | Log out

Home » Acta Horticulturae

XXIX International Horticultural Congress on Horticulture: Sustaining Lives, Livelihoods and Landscapes (IHC2014): V World Congress on Medicinal and Aromatic Plants and International Symposium on Plants, as Factories of Natural Substances, Edible and Essential Oils Number 1125

ISBN 9789462611313

ISSN 0567-7572

Availability This title is available both in print and ActaHort CD-rom format.

Publicaton date October 2016

Select Language ▼

Price € 99

Buy this book

Symposium V World Congress on Medicinal and Aromatic Plants (WOCMAP) and International Symposium on Plants, as Factories of Natural Substances, Edible and Essential Oils

Number of articles 53

Groups involved Section Medicinal and Aromatic Plants Section Nuts and Mediterranean Climate Fruits Workgroup Olive Culture

Volumes 1 Pages 406 Symposium venue Brisbane (Australia) Symposium date August 17, 2014 Conveners M.A. Deseo K.H.C. Baser V. Sergeeva

M. Heinrich A. Mathé

Editors B. Patil L. Craker

S. Gafner S.R. Mentreddy

Online articles Metabolomic profiling of antiviral Scaevola spinescens extracts by high resolution tandem mass spectrometry I.E. Cock

B. Matthews

Comparative HPLC analysis of phenolic compounds in the roots of Acanthopanax trifoliatus and Acanthopanax senticosus P. Sithisarn

S. Jarikasem

S. Muensaen

1H NMR-based metabolomics differentiation and real time PCR analysis of medicinal Cannabis organs N. Happyana

O. Kayser

Strategies for the conservation of Warburgia salutaris (family: Canellaceae), a red data list species - development of propagation methods K. Hannweg

A. Sippel

M. Hofmeyr

L. Swemmer

W. Froneman

Biomass production and photosynthetic efficiency of turmeric grown in different shade conditions M.I. Ferreira

G.P.P Lima

L.F.O.S. Rodrigues

M.B. Silva

C. Jadoski

G.G. Gonçalves

L.C. Ming

Pharmacognosy of Centipeda cunninghamii, an endemic Australian traditional medicinal plant D.N. Leach

R.N. Spooner-Hart

K.D. Beattie

Investigating sustainable production of the Australian medicinal plant Centipeda cunninghamii (DC.) A. Braun & Asch. (Asteraceae) R.N. Spooner-Hart

D.N. Leach

Antibacterial activity of eucalypt gum M. Nobakht

S.J. Trueman

H.M. Wallace

P.R. Brooks

M. Katouli

2017/01/20 12:04 PM


2 of 4


Phenology of Hyptis pectinata and effects of cattle manure and cutting height in management of its cultivation M.F. Arrigoni-Blank

R.B. Feitosa

A.F. Blank

T.M. Andrade

R. Silva-Mann

Harpagide and related iridoid glycosides in vegetative tissues of cultivated Scrophularia lanceolata and Scrophularia marilandica K.J. Brownstein

A.L. Thomas

G.E. Rottinghaus

B.A. Lynch

D.R. Gang

W.R. Folk

Prioritization of the ecologic cultivation of certain species which are less spread, but have multiple potential uses (culinary, medicinal, melliferous, decorative) C. Brezeanu

P.M. Brezeanu

S. Ambarus

T. Robu

A. Dobrescu

A comparative evaluation: phytochemical composition and antioxidant capacity of three roselle (Hibiscus sabdariffa L.) accessions K.L. Chin

J. Zhen

Y. Qi

S.L. Chin

M. Breithaupt

Q.L. Wu

J. Simon

J. Henson

V. Ferchaud

Early development and gas exchange of Ficus adhatodifolia Schott under different levels of shading G.G. Gonçalves

W.H.S. Takata

L.C. Ming

R.A. Campos

E. Ribeiro

M.I. Ferreira

Agronomic production and essential yield of Lavandula dentata L. in different systems and fertilization J.M.Q. Luz

S.M. Silva

G.R. Marquez

P.A.M. Nogueira

P.B. Alves

M.F. Alves

I.L. Matos

Effect of short periods of heat stress during early flowering period on flower development and pyrethrin accumulation in pyrethrum D.D. Suraweera

M.E. Nicolas

T. Groom

Allicin and total phenolic content and antioxidant activity in conventional and organic garlic P. Wongsa

W. Spreer

P. Sruamsiri

J. Müller

In vitro activity of essential oil of Ocimum selloi and its major chemical compound against Moniliophthora perniciosa, causal agent of witches’ broom disease in cacao L.C.B. Costa

J.C.B. Costa

J.E.B.P. Pinto

S.K.V. Bertolucci

P.B. Alves

E.S. Nicolau

Bioactive volatiles from Geijera parviflora Lindl. (Rutaceae): evidence for coumarin chemotypes N.J. Sadgrove

D. Lyddiard

G.L. Jones

The anti-inflammatory and analgesic effects of star anise, an aromatic herb in South China Jiagang Deng

Lizhen Huang

Yan Xie

Zhengcai Du

Erwei Hao

Xiaotao Hou

Anti-hemorrhoid activities from Cissus quadrangularis L. P. Ahmadi Pirshahid

C. Thisayakorn

T. Hemthanon

T. Suntorntanasat

C. Banchonglikitkul

J. Eiamwat

S. Sookwhan

Brahmi saponins inhibit proliferation of Hep G2 cells by blocking cell cycle progression and inducing apoptosis M. Kalachaveedu

D. Adapala

A.M. Punnoose

S. Kuruvilla

Genifuranal and other derivatives: smoking desert plants N.J. Sadgrove

D. Lyddiard

T.L. Collins

B.W. Greatrex

G.L. Jones

Antibacterial activity of Capsicum annuum L. oleoresin S. Nurjanah

Z. Sudaryanto

A. Widyasanti

H. Pratiwi

Antimicrobial activity of citrus oil against Salmonella and Escherichia coli growth I.R. Maldonade

V.C. Ginani

E.J. Sanjinez-Argandoña

Sustainable development of medicinal and aromatic plants as potential cash crops for the welfare of rural people in eastern India M. Ghosh

S.K. Chatterjee

Effect of drying methods on anti-oxidative potential of garlic (Allium sativum L.) P. Wongsa

W. Spreer

J. Müller

P. Sruamsiri

Chemical composition and antimicrobial activity of the essential oil from the leaves and flowers of Aloysia gratissima F.M. Santos

J.E.B.P. Pinto

S.K.V. Bertolucci

A.A. Alvarenga

M.N. Alves

M.C.T. Duarte

A. Sartoratto

Effect of selected commercial bio-additives on nutritional value of basil (Ocimum basilicum) T. Kopta

J. Vabkova

Medicinal and aromatic plant collection of Baccharis dracunculifolia DC. germplasm bank C.M.B. Belini

M.I. Zucchi

M.M. Bajay

M.O.M. Marques

R. Facanali

M.A.R. Vieira

G.M. Figueira

L.L. Haber

Dioscoridean herbals: comparisons of images provide a clue to their relationships J. Janick

Medicinal plants in the Dominican Republic and their possible role in public health care C. Roersch

Preclinical evidence of Malay traditional herbal supplement for diabetes F.A. Abdul Majid

L.C. Teh

S.H.A. Sayid Abdullah

R. Adawiyah Ahmad

M. Taher

M.E. Abd Wahid

Seed conservation methods and evaluation of germination rate in Baccharis dracunculifolia DC. (Asteraceae) C.D.L. Santos

G.M. Figueira

C.M.B. Belini

An examination of the medicinal potential of Planchonella queenslandica: toxicity, antibacterial, and antiviral activities

2017/01/20 12:04 PM


3 of 4

F.R. Kalt


I.E. Cock

The antioxidant activity of Vitis vinifera L. and Vaccinium myrtillus L. extracts J. Poracova

V. Sedlak

Z. Gogalova

J. Ondekova

M. Mydlarova Blascakova

J. Fejer

D. Grulova

Breeding of German chamomile, Matricaria recutita L., with a high content of α-bisabolol J. Fejer

I. Salamon

Germination and emergence of twenty-five Calendula officinalis cultivars M. Intervera

D.C. Scown

P. Cherry

Reviewing the importance of aromatic medicinal plants in the traditional pharmacopoeia of Australian Aboriginal people N.J. Sadgrove

G.L. Jones

Phytochemical variability of Pittosporum angustifolium Lodd. (Pittosporaceae): a traditional and contemporary Aboriginal Australian medicine N.J. Sadgrove

G.L. Jones

Indigenous plants for ethnoveterinary uses in the Pondoland, South Africa L. Kambizi

Morphological characterisation of cultivated Pelargonium sidoides in the northern KZN P.G. Mthiyane

S. Mavengahama

A. Opoku

R. Kleynhans

The effect of fertilizer to growth and quality of perennial sow-thistle (Sonchus arvensis L.) H. Nurhayati

Economic issues of Malagasy horticultural and beekeeping fields facing the emerging of the varroa S.I. Andriamanalina

S. Ramananarivo

J. Razafiarijaona

R.M. Rindra

R. Ramananarivo

Evaluation of turmeric (Curcuma longa L.) cultivars for growth and yield to determine regional specificity J. Venkatesha

S. Siddalingayya

Propagation of Melaleuca cajuputi by stem cuttings on the central coast of Vietnam Thi Ngoc Diep Huynh

P. Kristiansen

I. Yunusa

Minh Duc Tran

Plants, as factories of natural substances and edible oils Á. Máthé

Use of plant extracts and essential oils in modern plant protection V. Sergeeva

Effects of N fertilization on trichome density, leaf size and artemisinin production in Artemisia annua leaves I. Bilkova

A. Kjaer

F. van der Kooy

W.J.M. Lommen

Plant nutrition, growth and polygodial production of Tasmannia lanceolata M.D. Wilson

R.C. Menary

D.C. Close

Quantification of intracellular seed compounds of Moringa oleifera Lam. using digital image analysis Q.E. Muhl

F.H.J. Kanfer

E.S. du Toit

J.M. Steyn

Antioxidant activities and phenolic contents of the extracts in different loquat (Eriobotrya japonica) cultivars Somi Lee

Moonyoung Park

Eun-sik Kim

Younsup Cho

Hyesung Cho

Influence of postharvest treatments on the artemisinin content of leaves of Artemisia annua X. Simonnet

M. Quennoz

C. Carlen

High pressure biorefinery of essential oil yielding plants into valuable ingredients P.R. Venskutonis

K. Jonusaite

RELATED ACTA HORTICULTURAE

1149

1139

II International Workshop on Bacterial Diseases of Stone Fruits and Nuts

III Balkan Symposium on Fruit Growing

1130

1120

1116

1109

XXIX International Horticultural



XXIX International Horticultural Congress on Horticulture: Sustaining Lives, Livelihoods and Landscapes (IHC2014): International Symposia on the Physiology of Perennial Fruit Crops and Production Systems and Mechanisation, Precision Horticulture and Robotics

2017/01/20 12:04 PM


4 of 4

Congress on Horticulture: Sustaining Lives, Livelihoods and Landscapes (IHC2014): International Symposia on Postharvest Knowledge for the Future and Consumer and Sensory Driven Improvements to Fruits and Nuts

Home Sitemap About ISHS FAQ Flickr Photostream


Congress on Horticulture: Sustaining Lives, Livelihoods and Landscapes (IHC2014): III International Jujube Symposium

Congress on Horticulture: Sustaining Lives, Livelihoods and Landscapes (IHC2014): International Symposium on Nut Crops

1099

1098

1089

II International Symposium on Horticulture in Europe

International Symposium on Medicinal Plants and Natural Products

III International Symposium on Pomegranate and Minor Mediterranean Fruits

Facebook Careers YouTube

Contact Legal Cookies Logo VAT

Copyright © 2016 International Society for Horticultural Science.

Webdesign Desk02

2017/01/20 12:04 PM