Purslane —A Potential Vegetable Crop— RIRDC Publication No. 09/088
Purslane – A Potential Vegetable Crop by Dr Guijun Yan, Dr Nader Aryamanesh, Dr Shaofang Wang
July 2009 RIRDC Publication No 09/088 RIRDC Project No PRJ-000348
© 2009 Rural Industries Research and Development Corporation. All rights reserved.
ISBN 1 74151 887 3 ISSN 1440-6845 Purslane – A Potential Vegetable Crop Publication No. 09/088 Project No. PRJ-000348 The information contained in this publication is intended for general use to assist public knowledge and discussion and to help improve the development of sustainable regions. You must not rely on any information contained in this publication without taking specialist advice relevant to your particular circumstances. While reasonable care has been taken in preparing this publication to ensure that information is true and correct, the Commonwealth of Australia gives no assurance as to the accuracy of any information in this publication. The Commonwealth of Australia, the Rural Industries Research and Development Corporation (RIRDC), the authors or contributors expressly disclaim, to the maximum extent permitted by law, all responsibility and liability to any person, arising directly or indirectly from any act or omission, or for any consequences of any such act or omission, made in reliance on the contents of this publication, whether or not caused by any negligence on the part of the Commonwealth of Australia, RIRDC, the authors or contributors. The Commonwealth of Australia does not necessarily endorse the views in this publication. This publication is copyright. Apart from any use as permitted under the Copyright Act 1968, all other rights are reserved. However, wide dissemination is encouraged. Requests and inquiries concerning reproduction and rights should be addressed to the RIRDC Publications Manager on phone 02 6271 4165.
Researcher Contact Details Dr Guijun Yan School of Plant Biology Faculty of Natural and Agricultural Sciences The University of Western Australia Crawley, WA 6009 Phone: + 61 8 6488 1240 Fax: +61 8 6488 1108 Email:
[email protected] In submitting this report, the researcher has agreed to RIRDC publishing this material in its edited form. RIRDC Contact Details Rural Industries Research and Development Corporation Level 2, 15 National Circuit BARTON ACT 2600 PO Box 4776 KINGSTON ACT 2604 Phone: Fax: Email: Web:
02 6271 4100 02 6271 4199
[email protected]. http://www.rirdc.gov.au
Electronically published by RIRDC in July 2009 Print-on-demand by Union Offset Printing, Canberra at www.rirdc.gov.au or phone 1300 634 313
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Foreword This project evaluates variation in several promising cultivars of purslane (Portulaca oleracea) and identifies cultivars of potential for growing and processing in Australia. Purslane is a rich vegetable source of polyunsaturated fatty acids. This study confirmed the potential of purslane to be developed as a new vegetable source of omega 3 fatty acids (linolenic) as a potential alternative to fish oil. This study revealed that fatty acids content and genetic diversity were highly variable among P. oleracea accessions. High levels of unsaturated fatty acids (linoleic, linolenic and oleic acids) were detected in both leaves and stems. Overall, the amount of linoleic and oleic acids was highest in stem tissues and the amount of linolenic acid was highest in leaf tissues. The research found high levels of polymorphism not only between accessions but also within accessions, pointing to the potential for high gains in chemical yield and productivity through a careful selection and breeding program. The report also recommends a future direction of research that would optimise the potential for an industry based on purslane in Australia. It recommends: (i)
The further collection of accessions from different parts of Australia and the word and selection for accessions with high omega 3 fatty acid content,
(ii)
The breeding of high omega 3 purslane with other commercially useful characters using selected accessions in the breeding program to release commercial varieties,
(iii)
The development, marketing and commercialisation of omega 3 from P. oleracea plants.
This project was funded from RIRDC core funds which are provided by the Australian Government and from industry partner Dardin Agri-Holdings Pty. Ltd. This report, an addition to RIRDC’s diverse range of over 1800 research publications, forms part of our New Plant Products R&D program, which aims to facilitate the development of new industries based on plants or plant products that have commercial potential for Australia. Most of RIRDC’s publications are available for viewing, downloading or purchasing online at www.rirdc.gov.au. Purchases can also be made by phoning 1300 634 313.
Peter O’Brien Managing Director Rural Industries Research and Development Corporation
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Acknowledgements We would like to thank Ms Charissa Man for her research as a 4th year project student to work on the project, Prof. Avinoam Danin for supplying some Portulaca seeds, The Chemistry Centre of Western Australia for the chemical analysis of plant materials and SABC at Murdoch University for running our AFLP products.
Abbreviations AFLP
amplified fragment length polymorphism
ALA
α-linolenic acid
ANOVA
analysis of variance
DW
dried weight
GC
gas chromatography
GLA
gamma-linolenic acid
MAS
marker assisted selection
SABC
State Agriculture Biotechnology Centre (WA)
SDA
stearidonic acid
UPGMA
un-weighted pair group method with arithmetic average
WA
Western Australia
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Contents Foreword ................................................................................................................................................ ii Acknowledgments................................................................................................................................. iii Executive Summary.............................................................................................................................. vi Introduction ........................................................................................................................................... 1 Objectives ............................................................................................................................................... 2 Methodology........................................................................................................................................... 3 Plant materials................................................................................................................................... 3 Fatty acid analysis............................................................................................................................. 3 DNA extraction ................................................................................................................................. 4 AFLP analysis................................................................................................................................... 4 Statistical analysis ............................................................................................................................. 5 Results..................................................................................................................................................... 6 Fatty acid analysis results ................................................................................................................. 6 Genetic diversity ............................................................................................................................... 6 Discussion ............................................................................................................................................. 10 Implications.......................................................................................................................................... 12 Recommendations................................................................................................................................ 13 References ............................................................................................................................................ 14
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Executive Summary What the report is about This project evaluates variation in several promising cultivars of purslane (Portulaca oleracea) and identifies cultivars of potential for growing and processing in Australia. Who is the report targeted at? The report is initially targeted for use by RIRDC and potential industry participants that may be interested in developing a purslane industry. Background Omega-3 fatty acids belong to a group of polyunsaturated fatty acids essential for human growth, development, prevention of numerous cardiovascular diseases and maintenance of a healthy immune system. Purslane (Portulaca oleracea) has recently been identified as the richest vegetable source of omega-3 fatty acids. This study demonstrated that P. oleracea has a potential to be developed as a new vegetable source of omega 3 fatty acid. Aims and objectives This study aims to investigate the effect of genetic variability on poly-unsaturated fatty acids, linolenic (omega 3), in P. oleracea accessions, The second objective of the project is to study the genetic diversity of P. oleracea accessions and correlate the genetic diversity with the fatty acid content of different P. oleracea accessions sampled worldwide. Methods used The research team compared the amount of linolenic fatty acid between internationally and locally collected P. oleracea accessions. AFLP markers were successfully used to study the genetic diversity of 16 P. oleracea accessions along with gas chromatography for fatty acid composition analysis. Results/Key findings This study revealed that fatty acid content and genetic diversity were highly variable among P. oleracea accessions. The high level of diversity will give the breeders the opportunity for selecting P oleracea accessions with highly valuable fatty acid composition. There was a significant variation in the amount of saturated fatty acids (Palmitic and Stearic), monounsaturated fatty acid (oleic) and poly-unsaturated fatty acids (linoleic and linolenic) among the P. oleracea accessions in both leaves and stems. P. oleracea can be a good vegetable source of omega 3 fatty acid. Unsaturated fatty acids, linoleic, linolenic are high in P. oleracea in both leaves and stems. Accessions from Karnik in Poland (POPK2) and Hebei in China (POHB2) had the highest values for both linoleic (omega 6) and linolenic (omega 3) contents in leaves and stems. These accessions also had the highest values for mono-unsaturated fatty acid oleic in leaves and stems, respectively. Overall, the amount of linoleic and oleic was highest in stem tissue and linolenic was highest in leaf tissue. This study demonstrated that P. oleracea has a potential to be developed as a new vegetable source of omega 3 fatty acid. Existence of high level of diversity in fatty acid composition and DNA structure indicated that it is promising to select and breed this vegetable not only for omega 3 content but also for other desirable traits such as taste, leaf and stem structure, growth rate and tolerance to abiotic and biotic stresses.
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P. oleracea has the potential to become a perfect vegetable of high nutritional significance and is especially useful for vegetarian or vegan people who are not able to consume fish oils. Recommendations On the basis of the findings of this research, the following recommendations are made: •
Further collection of accessions from different parts of Australia and the word and selection of accessions with high omega 3 content, good agronomic performance and better taste.
•
Start breeding program to release commercial varieties.
•
Investigate the gene pathways involved in omega 3 production in order to manipulate the pathways for higher production of omega 3.
•
Marketing P. oleracea in all parts of the world as a fresh vegetable as a high omega 3 supplement.
•
Production and commercialization of omega 3 sourced from P. oleracea plants.
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Introduction Omega-3 fatty acids belong to a group of polyunsaturated fatty acids essential for human growth, development, prevention of numerous cardiovascular diseases and maintenance of a healthy immune system (Gill and Valivety 1995). Our bodies do not synthesise Omega-3 fatty acids, therefore Omega3 fatty acids must be consumed from a dietary source (Whelan and Rust 2006). Omega-3 fatty acids contain 18 to 24 carbon atoms and have three or more double bonds within its fatty acid chain (Whelan and Rust 2006). The number of double bonds is characteristic of polyunsaturated fatty acids, which gives the latter its beneficial attributes, distinguishing polyunsaturated fatty acids from the more harmful saturated fat (Gill and Valivety 1995). Saturated fat, as it is well known, is responsible for diseases such as obesity and coronary heart disease when consumed excessively (Hu et al. 2001; Zatonski and Willett 2005). Fish is the richest source of omega-3 fatty acids. Health authorities highly recommend that we consume fish regularly to meet our bodies’ requirements for Omega-3 fatty acids, as other sources are limited and do not supply nearly as much Omega-3 fatty acids (Nestel 1987). However, fish stocks are declining rapidly and will eventually deplete if over-fishing remains uncontrolled (Zhou et al. 2001). It is impossible to fully depend on the ability of farmed fish to supply the needs of an increasing population once fish stocks run out (Zhou et al. 2001). Purslane (Portulaca oleracea) has recently been identified as the richest vegetable source of αlinolenic acid (ALA), an essential omega-3 fatty acids (Simopoulos and Salem 1986). The lack of dietary sources of omega-3 fatty acids has resulted in a growing level of interest to introduce P. oleracea as a new cultivated vegetable (Liu et al. 2000; 2002; Palaniswamy et al. 2000; 2001; Yazici et al. 2007; Simopoulos et al. 2004). Portulaca oleracea is not only high in nutritional value, but is also highly adaptable to many different environments (Simopoulos et al. 1995). Danin et al. (1978) showed that P. oleracea flourishes in numerous biogeographical locations worldwide and is highly adaptable to many drought, saline and nutrient-deficient conditions. These characteristics give P. oleracea a competitive advantage over many other cultivated crops and has led some to consider P. oleracea as the ‘power food for the future’ (Levey, cited in Simopoulos et al. 1995).
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Objectives The amount of ALA in P. oleracea has been differentially quantified by several authors (Palaniswamy et al. 2001). There was a huge variation in ALA values reported by Omara-Alwala et al. (1991), Simopoulos et al. (1992), Liu et al. 2000 and Ezekwe et al. (1999). The variation in ALA values can be attributed to differences in experimental conditions, sampling material and analytical methods (Palaniswamy et al. 2001). This study aims to investigate the effect of genetic variability on ALA content in P. oleracea accessions, by comparing the amount of ALA between internationally and locally collected P. oleracea accessions. The accessions with the highest amount of ALA will then be chosen for the selection and breeding of P. oleracea as a new cultivated vegetable. The objectives of the project, in particular, were •
To study the genetic diversity of P. oleracea accessions collected from Western Australia and other parts of the world using AFLP markers.
•
To measure the fatty acid content among different P. oleracea accessions sampled worldwide.
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Methodology Plant materials Sixteen Portulaca oleracea accessions were used in this research (Table 1). Seven accessions were kindly provided by Prof Avinoan Danin from the Hebrew University of Jerusalem. The rest of the accessions were collected by Dr Guijun Yan from Western Australia and China. Each accession with 3 replicates were sown into 10 cm pots containing a peat-based potting mix (Richgro Garden Products), in a naturally-lit and air-conditioned glasshouse at School of Plant Biology, the University of Western Australia. After germination, seedlings were watered on alternate days and fertilised fortnightly with Thrive® soluble fertiliser. Table 1. List of plant materials used in this study and their origins Series No.
Code
Origin
Species
1
POUWA
The University of Western Australia (UWA)
P. oleracea
2
POUWA2
The University of Western Australia (UWA)
P. oleracea
3
POP
Perth City, Western Australia
P. oleracea
4
POA
Augusta, Western Australia
P. oleracea
5
POA2
Augusta, Western Australia
P. oleracea
6
POXJ
Xin Jiang, China
P. oleracea
7
POHB
Hebei, China
P. oleracea
8
POHY
Harvey, WA
P. oleracea
9
PGHB
Hebei, China
P. grandifolia
10
POIRR
Italy, Rome, Regina, Apostolorum
P. oleracea
11
PONA
Nir Akiva
P. oleracea
12
POPK
Poland, Karnik
P. oleracea
13
POIB
Israel, Bethlehem of the Galilee
P. oleracea
14
POAA
Algeria, Alger
P. oleracea
15
POSN
Sharon, Neranya
P. oleracea
16
POISP
Italy, Sicily, Porto
P. oleracea
Fatty acid analysis Fifty mg of dry leaves or stems were mixed with 20 ml of solvent (CH2CL2 : MeOH : H20; 3:6:1) at room temperature overnight. Samples were then centrifuged at 10,000rpm (Orbital 420). After centrifugation, 10 ml of solution was mixed with water and dichloromethane at a ratio of 1:1. Samples were centrifuged again, after which, the bottom dichloromethane solution was concentrated using a rotatory evaporator under vacuum. The resulting sample was then mixed with 2% sodium hydroxyl in methanol refluxing for 10 minutes. Then, borotrifluoride-methanol complex was added into samples and mixed for 2 minutes. Finally, hexane was added. The upper hexane solution was taken for fatty acid analysis by gas chromatography (GC). The GC column is BPX 70 with 60 m in length, 0.25 mm ID and 0.25 um (SGE). The initial oven temperature was set for 100°C, increasing at a rate of 2.5°C/minute to 150°C (held for 2 minutes). Temperature was then increased at 1.5°C/minute to 220°C (held for 1 minute). Finally, the temperature
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increased at 12°C/minute to 250°C (held for 8 minutes). The peak areas were determined with an integrator (Hewlett-Packard 5890) and identified by a comparison of retention times with those of a standard.
DNA extraction Fresh leaf material was collected for all accessions (three samples per accession) from one month old plants. Around 100mg of leaf tissue was broken down using a tissue homogeniser. Total DNA was extracted using a Nucleon Phytopure Genomic DNA Extraction Kit (GE Healthcare) according to manufacturer’s protocol. Extracted DNA was treated with RNAase - concentrations were determined and qualities were measured using a Nanodrop spectrophotometer (ND-1000, BIOLAB). The DNA was diluted to required concentration.
AFLP analysis AFLP was carried out using an IRDyeTM Fluorescent AFLP® kit ands following the manufacturer’s protocol for large plant genome analysis. For restriction digestion of genomic DNA, 100ng of DNA was mixed with 2.5 µl of 5x reaction buffer and 1 µl of EcoR1 enzyme mix in 12.5 µl reaction volumes in PCR tubes. After a short spin, the tubes were placed in a thermocycler (G-Storm, Gene Works Technologies) that was programmed for one cycle of 37°C for 2 hours followed by one cycle of 70°C for 15 minutes. For ligation of adapters, 12 µl of adapter mix and 0.5 µl of DNA ligase was added into each well and placed into the thermocycler which was programmed for 20°C for 2 hours. For the pre-amplification reaction, 2.5 µl of each ligation mixture (from previous step) was mixed with 20 µl of AFLP pre-amplification primer mix, 2.5 µl of PCR reaction buffer (10X) and 0.5 µl of Taq DNA polymerase (5 units/µl) (BIOTAQ™ supplied by Fisher Biotech) in a fresh PCR plate. After a short spin, the tubes were placed in the thermo-cycler programmed for hot start of 94°C for 2
minutes, then 20 cycles at 94°C for 30 seconds, 56°C for 30 seconds, and 72°C for 2 minute finishing at 4°C. For selective AFLP amplification, 3 µl of the diluted pre- amplification product (1/40) was added into a fresh PCR plate along with 6 µl of Taq DNA polymerase working mix (553 µl MQ water, 140 µl of 10x reaction buffer and 7 µl of Taq DNA polymerase), 1 µl of 5µM Mse1 primer and 1µl of 5µM IRDye (EcoR1 primer) in the following combinations: Mse1-CAG with EcoR1-AACFAM Mse1-CAG with EcoR1-ACTVIC Mse1-CAG with EcoR1-AGANED Mse1-CAG with EcoR1-ATGPET Mse1-CTA with EcoR1-AACFAM Mse1-CTA with EcoR1-ACTVIC Mse1-CTA with EcoR1-AGANED Mse1-CTA with EcoR1-ATGPET
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The plate was spun down briefly and placed in the thermocycler programmed for 1 cycle of 94°C for 30 s, 65°C for 30 s and 72°C for 1 minute ; 12 cycles of subsequently lowering the annealing temperature (65°C) by 0.7°C per cycle, while keeping at 94°C for 30 s (denaturation), and 72°C for 1 minute (elongation); 23 cycles of 94°C for 30 s, 56°C for 30 s, and 72°C for 1 minute. The final products for Mse1-CTA and Mse1-CAG with 4 different labels EcoR1 each were pooled separately. 3 µl of the products for each sample were transferred into a new tube. Then, 0.15 µl of LIZ500 size standard and 14.85 µl of formamide were added to the product in order to fragment analysis using AB3730xl capillary sequencer.
Statistical analysis The fatty acid content was calculated based on the amount of fatty acids (mg) in 100 grams of dried weight (DW) of plant tissue. GenStat (10th edition) were used for ANOVA test. Microsoft Excel 2007 was used to graph the diagrams. AFLP results obtained from the capillary sequencer were imported into GeneMapper software (version 3.7; Applied Biosystems) for analysis using an auto bin function. The quality of bands and size standard for each sample were examined before exporting the data. Microsoft Excel 2007 was used for sorting the data and changing the values into 1 (presence of a band) and 0 (no band). GenStat (10th edition) software was used for grouping the P. oleracea accessions based on the AFLP markers.
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Results Fatty acid analysis results There was a significant variation in the amount of poly-unsaturated fatty acids, linoleic and linolenic acids, among the P. oleracea accessions in both leaves and stems (P
