Title: Determination of Essential Fatty Acids Composition among Mutant Lines of

Transcription

1 Title: Determination of Essential Fatty Acids Composition among Mutant Lines of Canola (Brassica Napus L.,) through High Pressure Liquid Chromatography Running Title: Essential Fatty Acid Analysis of Brassica Napus L. Mutants Authors: Ghulam Raza 1*, Aquil Siddique 1, Imtiaz Ahmad Khan 1, Muhammed Yasin Ashraf 2 and Abdullah Khatri 1 1 Agriculture Biotechnology Division, Nuclear Institute of Agriculture (NIA), Tando Jam, Pakistan. 2 Nuclear Institute of Agriculture and Biology, Faisalabad, Pakistan *Authors for Correspondence Cell No <lalian77@yahoo.com>

2 Abstract This study aimed to quantify the methyl esters of Lenoleic Acid (LA), γ-lenolenic acid (LNA) and Oleic acid (OL) in the oil of Brassica napus mutants. Five stable mutants (ROO-75/1, ROO-100/6, ROO-125/12, ROO-125/14, and ROO-125/17) of Rainbow (P) and three mutants (W97-95/16, W /11 and W /13) of Westar (P) at M6 stage exhibiting better yield and yield components were analyzed for essential fatty acids. The highest seed yield was observed in the mutant (ROO-100/6) followed by ROO- 125/14 of Rainbow i.e., 34 and 32 % higher than parent, respectively. Westar mutant W97-75/11 also showed 30 % higher seed yield than parent. HPLC analysis regarding fatty acids composition indicated that OL was the most dominant fatty acid, ranging from 42 to 52%, LNA was the second (18-27% ) and third was LA (18-29%). Mutant ROO- 125/14 showed higher OL contents than parent (Rainbow). Mutants W /11 & W /16 of Westar are in advanced yield trial due to better yield and oil contents however their oil quality is not better than ROO-125/14. These results are expected to support ROO-125/14 mutant in the National Uniform Varietal Yield Trials (NUVYT) for approval as variety on better oil quality basis. Key words: Brassica; HPLC; lenoleic acid; lenolenic acid; oleic acid.

3 The oil quality in rape and mustard is a complex and qualitatively inherited character and its improvement is a key factor in the success of rapeseed as a new source of high quality edible oil. (Larik et al. 1999). The fatty acid composition of Brassica oil determines its physical/chemical properties and nutritional quality. The rapeseed oil contains the lowest amount of saturated fatty acids as compared to other vegetable oils and its fatty acid composition is considered by many nutritionists as ideal for human nutrition and superior to that of many other plant oils (Rakow and Raney 2003). It contains nutritionally desired oleic acid along with two essential fatty acids, lenoleic and lenolenic acids. High oleic acid oils have been shown to have equivalent heat stability to saturated fats and are therefore suitable replacements for them in commercial food-service applications that require long life stability and it can be heated to a higher temperature without smoking, so that the cooking time is reduced and food absorbs less oil (Miller et al. 1987). Additionally, high oleic acid oil has cholesterollowering properties; whereas saturated fatty acids tend to raise blood cholesterol levels (Rakow and Raney 2003). Vegetable oils with a high content of C18:1 is of interest for nutritional and industrial purposes. The reduction of the levels of polyunsaturated fatty acids and their substitution for the monounsaturated C18:1 is an important goal for the development of higher quality mustard oil (Scarth & McVetty 1999). A diet containing a high content of C18:1 can reduce the content of the undesirable low-density lipoprotein cholesterol in blood plasma (Grundy 1986), and monounsaturated fatty acids more effectively prevent arteriosclerosis than polyunsaturated fatty acids (Chang & Huang 1998).

4 Improvement in edible oil by modification of fatty acid composition is current objective of plant breeders. To achieve this, induced mutations and hybridization can play a vital role. The use of mutagenesis permitted the development of Ethiopian mustard lines with specific fatty acid profiles such as lines with a high oleic acid content and some lines with a low C18:1 content (Velasco et al. 1997) and also lines with low and high C22:1 contents whose genetics have been reported in previous studies (Del Rı o et al. 2003; Velasco et al. 1997; Velasco et al. 1998; De Haro et al. 2001). These oils with specific fatty acid profiles are in demand because of their improved nutritional and/or technological properties (Kinney 1994). Therefore, efficient analytical methods are a pre-requisite for analysis of desired components. In this study, HPLC (high performance liquid chromatography) method was used to analyze the mutant lines which are already in advanced stage and exhibiting better agronomical traits. In future, this programme will be used for selection of desired genotypes from mutant lines which are among a large number of segregating populations. Therefore, efforts have been made to develop genotypes exhibiting high oleic acid content and 2: 1 ratio of linoleic to linolenic acid contents using mutagenesis. Results Agronomic performance of mutants Mean performance and chi-square regarding seed yield and its components in advanced yield trial for rapeseed mutants is shown in Table 1. Significant difference among the agronomic traits was observed. Among the Rainbow mutants, ROO-100/6 showed the highest seed yield performance (1665 kg ha -1 ) and produced over 34% more

5 than parent (1242 kg ha -1 ) followed by mutant ROO-125/14 (1635 kg ha -1 ) which has 32% more seed yield than the parent. On the other hand, mutant ROO-125/17, ROO- 125/12 & ROO-75/1 exhibited lower yield as compared to the parent. Among the Westar mutants, mutant W97-75/11 gave the 30% higher yield (1525 kg ha -1 ) than the parent (1175 kg ha -1 ) followed by the mutant W /16 which produced 1266 kg ha -1 seed yield that is only 8 % more than the parent. All the Rainbow & Westar mutants exhibited more 1000 grain weight than their respective parents. The mutant ROO-125/12 and ROO-125/14 showed the highest 1000 grain weight among all. No difference was observed regarding the plant height among the Rainbow mutants except for mutant ROO-75/1 which was dwarf than the parent. All Westar mutants were shorter than the parent. All the mutants matured earlier than their parent. Mutant ROO-75/1 matured 23 days earlier than parent followed by ROO-100/6 which matured 20 days earlier than the parent. No correlation was observed for days of maturity with seed yield. Some mutants (ROO-75/1, ROO-125/12 & ROO-125/17) matured earlier and also exhibited lower yields while some mutants like ROO1-100/6 & ROO-125/14 exhibited higher seed yield and also matured earlier than the parent. Oil contents: Among the Rainbow mutants, the highest oil contents (46%) have been observed in ROO-125/14 and ROO-75/1, which is 4.5 % more than the parent (44%) While mutants ROO-100/6 produced 45 % oil content that is 2.2% more than the parent. Among the Westar mutants, W /16 showed the highest oil contents (46%), which was 9 % more than the parent (42%) while mutant W /13 exhibited 45.5 % oil which was 8 % higher than the parent (Table 2).

6 HPLC Analysis The composition of unsaturated fatty acids (lenolenic acid, Lenoleic acid, Oleic acid) in mutants of Rainbow and Westar was determined by HPLC method using Ezi Chrome Elite software. For obtaining desired peaks, flow rate, oven temperature and solvent ratio parameters were optimized. The best results were obtained using following conditions; flow rate: 1.00 ml min -1, Mobile phase ratio: Methanol: 0.1% H 3 PO 4: 85:15, Oven temperature: 45 o C. The fatty acids (Lenolenic Acid, Lenoleic Acid & Oleic Acid) chromatographic profile is given in Fig. 1, wherein the retention time of these fatty acids is also shown in Fig 1. Lenolenic acid eluted with in min followed by lenoleic acid which was eluted after min. Oleic acid was eluted later after min. The retention time for these fatty acids was identified by using standards of these fatty acids with known retention time. Fatty Acid Analysis Composition of unsaturated fatty acids was expressed in relative concentrations rather than absolute (Table 2). HPLC analysis of the fatty acids in the seed oil of five mutants of Rainbow (P) and three mutants of Westar (P) showed that oleic acid (C18: 1) was the most dominant fatty acid, ranging between 39.1% and 66.3%, lenoleic acid (C18: 2) was the second most dominant fatty acid and ranged between ( %) while lenolenic acid ranged between 18.1 to 28.9%. Among the Rainbow mutants, ROO-125/14 showed the highest oleic acid content (66.3%), which is 28% more than the parent (51.8%) while ROO-125/12 showed the least oleic acid content (39.1%) which was 24.51% lower than the parent (51.8%). On the

7 other hand all the Westar mutants had the lower oleic acid content with respect to the parent. ROO-125/17 showed the highest lenolenic acid content (28.9%), which was 16.53% more than the parent (24.8%) while ROO-125/14 showed the least lenolenic acid content (18.5%) which was 25.4% lower than the parent (24.8%). Mutant ROO-125/12 had the highest percentage (41.6%) of lenoleic acid being 78% more than the parent (23.3%) followed by mutant ROO-75/1 having 24.6% which was 5.5% more than the parent. The lowest percentage (15.3) was shown by the mutant ROO-125/14. Among the Westar mutants, W /13 had the highest lenoleic acid contents (23.7%), which was 5% more than the parent (22.5%). Among the Rainbow mutants, mutant ROO-125/14 is already in NUVYT on the basis of better yield/yield components and oil contents against parent. From this study, it was observed that mutant ROO-125/14 showed the highest oleic acid contents as compared to the parent which makes crop resistant to shattering and with more stable oil content. So, these results are quite encouraging and will be helpful for the approval of this mutant as a variety in future while among the Westar mutants, mutants W /11 & W /16 are in advanced yield trial due to better yield and oil contents but their oil quality is not better than the mutant ROO-125/14. The analysis of canonical variates was conducted in order to create a graphic image of the arrangement of the genotypes (mutant lines, Parental lines) on the plane (Figure 2).The graphic image of the distribution of mutant lines characterized by the three fatty acids in the space of first two canonical variates reflects both their variability and relation to the parental lines. The closer the distance between points representing

8 individual genotypes, the closer the similarity with regard to the contents of the three fatty acids. The graphic image showed that mutant lines ROO-75/1, ROO-100/6 and ROO- 100/17 were found closest to Rainbow parent while mutants ROO125/12 and ROO- 125/14 were far from the respective parents. This represented the uniqueness of these mutants and also reflects the presence of variation. On the other hand among the Westar mutants, W97-75/11 and W97-75/13 are near to their respective parent while W97-75/16 is far away from the parent. Discussion Mutation breeding is a technique through which new crop varieties with desirable characteristics can be developed in shorter time. It is more sophisticated technique than conventional pedigree breeding. Mutagenesis was plasticized with not only seeds but also with single cell (Hoffmann 1980). Now this method is under routine practice and numerous reports are present in which mutation breeding was used to improve the yield and yield components and other attributes of rapeseed (Olejniczak & Adamska 1999; Maluszynski et al. 2000; Abdullah et al. 2005; Shah et al. 2005). Similarly, this technique we also used in the present study to get mutants with high yield and yield components and better oil quality than parents. Results of the present study indicated that Mutant ROO-100/6 exhibited 34% more yield than the parent but this mutant showed almost similar oleic acid content (51%) like the parent (51.8%) while the mutant ROO-125/14 showed 32% higher yield than the parent and this mutant exhibited the highest oleic acid contents among all the mutants. So, it is better both from qualitative and quantitative

9 point of view and can be recommended to have higher seed yield and better oil quality. The existing HPLC method can be used to screen genotypes with respect to desired fatty acid composition which is very important for oil quality. Similar approach was used to analyze underivatized very long chain fatty acids (C16-C26) and other apolar compounds such as triacylglycerols (Kornel et al. 2004). The major advantages of HPLC method over GC are lower temperatures during analysis, which reduces the risk of isomerization of double bonds, and the possibility of collecting fractions for further investigations (Czauderna & Kowalczyk, 2004; ). Similarly, Sanches-Silva et al 2004 compared a reversed-phase high performance liquid chromatographic (RP-HLPC) method with gas chromatography flame ionization detection (GC FID) method for determining fatty acids in potato crisps. They observed that both methods presented good precision and recovery but precision using HPLC method was slightly better than for GC FID and they also observed that HPLC method (LOD 0.74 µg/ml) was more sensitive than GC method (LOD 5.00 µg/ml) for all compounds studied. Mutation breeding has a potential in creating lines with a modified fatty acid composition. In particular, as these results showed, the development of line ROO125/14 with high oleic acid content seems promising and line ROO125/12 has lenoleic and lenolenic acid with 2:1 which is acceptable for nutrition having lower polyunsaturated fatty acids. The similar breeding strategy has been used by a lot of scientists and obtained promising lines with modified fatty acids (Schierholt et al. 2001; Velasco et al. 2003; Del Rio-celestino et al. 2006). Therefore, the mutant lines selected in the study could be used in rapeseed breeding for further genetic modification of seed quality by changing the proportion of fatty acids suitable for nutritional purposes. In the present study, line

10 ROO125/14 exhibited the desired amount of oleic acid contents so it can be recommended for cultivation to achieve the high quality oil and in maintaining the high level of monosaturated fatty acids and reduced level of polyunsaturated fatty acids successfully. It may have higher oxidative stability and reduced oxidation products in the oil. Schierholt et al. (2001) analyzed eight mutants which showed significant increase in oleic acid content in the seeds. Admska et al also reported that two DH lines H5-30 and H5-43 had higher oleic acid contents. Mollers and Schierholt, (2002) concluded that rapeseed oil which is high in C18:1 has a lower content of the saturated fatty acids making the oil more suitable from the nutritional point of view. In this study, line ROO125/14 has oil with high oleic acid content so it may be recommended for commercial use. It was also observed that mutant ROO had lenoleic and lenolenic acid in 2:1 ratio. This ratio is also acceptable from nutrition point of view. Similar study was also reported by Admska et al Moreover, remaining mutants of Rainbow and all the mutants of Westar have not any differences for the studied fatty acids in comparison to their respective parents. These results suggest that substitution of canola oil for other dietary lipid sources may assist in reducing the likelihood of a transient ischemic event leading to life-threatening cardiac arrhythmias. Increasing the proportion of oleic acid in the diet in exchange for saturated fats has previously been shown to have little influence on either the fatty acid composition of the myocardial cell membrane or the vulnerability of the heart to develop serious arrhythmias (McLennan 1993).

11 Material and methods Homogeneous seeds of rapeseed (Brassica napus L.) cv. Rainbow and Westar treated with three dozes of gamma rays viz; 750, 1000 and 1250 Gy, three concentrations of Ethyl Methyl Sulfonate (EMS) i.e. 0.75, 1.00 and 1.25% solution separately and 4 treatments of these mutagenes in combinations (750 Gy+0.75%EMS, 750 Gy % EMS, 1000Gy+0.75% EMS and 1000 Gy+1.00% EMS). The treated seeds along with untreated (control) seeds were grown in the field with Randomized Complete Block Design (RCBD) having 3 replications to raise M 1 generations. After thorough selection in successive generation, five mutants (ROO-75/1, ROO-100/6, ROO-125/12, ROO-125/14, and ROO-125/17) of Rainbow (P) and three mutants (W97-95/16, W /11 and W /13) of Westar (P) at advanced stage exhibiting better yield and yield components were analyzed for essential fatty acids as shown in Table 1. Ten-gram seed of these mutants were dried overnight at 55 o C and ground to powder in a Moulinex coffee grinder. Seed powder was mixed with 100 ml petroleum ether and the lipid fraction was extracted in a Soxhlet apparatus at 60 o C for 12 h. The solvent was evaporated and the weight of lipid extraction residues was determined, from which oil percentage was calculated. Fatty acid methyl esters were obtained as described by Garce s & Mancha (1993).The extracted oil was filtered by using 0.45µM pore size filter. In a sample tube, the solution of purified oil and heptane (1ml of each), was mixed with methanol based on 0.1ml of a 2N KOH solution, then the tube was caped and screwed tightly. The tube was shaken vigorously for 15 seconds and kept at room temperature until the upper layer became clear.

12 The upper layer was separated and 20µl of the supernatant was injected in HPLC and analyzed on a Hitachi HPLC system (Hitachi, Japan) equipped with a refractive index detector and reverse phase intersil column. The analysis was done under conditions mentioned under results. HPLC method was established by using Ezi Chrome Elite software. Calibration curves were prepared by injecting mixtures of standards (Lenolenic acid, Lenoleic acid and Oleic acid) of 2.5, 5, 10 and 20.0% under previously mentioned conditions. The fatty acid content was expressed as the percentage of total fatty acid. Statistical analysis: Statistical analysis was carried out using uni-variate and multi-variate techniques. Through principle component analysis plane was reduced to two canonical variants, which were used as a coordinates for plotting genotypes. Genotypic means for agronomic traits were compared using least significant difference test at 5 % probability level (Steel et al., 1997). Acknowledgment: The statistical analysis was done by Mr. Tanveer Mustafa, Junior Scientist, NIBGE, Faisalabad for which I would like to express my deep gratitude to him. References Abdullah K, Khan IA, Sidique MA, Raza S, Nizamani GS (2004). Evaluation of high yielding mutants of Brassica Juncea cv. S-9 developed through Gamma rays and EMS. Pak. J. Bot. 37(2),

13 Adamska ET, Cegielska-Taras, Kaczmarek Z, Szala L (2004). Multivariate approach to evaluating the fatty acid composition of seed oil in a doubled haploid population of winter oilseed rape (Brassica napus L.,). J. Appl. Genet. 45 (4), Chang NW, Huang PC (1998). Effects of the ratio of polyunsaturated and monounsaturated fatty acid to saturated fatty acid on rat plasma and liver lipid concentration. Lipids 33, Czauderna M, Kowalczyk J (2001). Separation of some mono-, di- and tri-unsaturated fatty acids containing 18 carbon atoms by high-performance liquid chromatography and photodiode array detection. Journal of Chromatography 760, De Haro A, Del Ri O M, Velasco L, Dominguez JJ, Ferna Ndez-Marti Nez JM (2001). Registration of one low, two medium, and one high erucic acid Ethiopian mustard genetic stocks. Crop Sci. 41, Del Ri O M, De Haro A, Ferna Ndez-Marti Nez JM (2003). Transgressive segregation of erucic acid content in Brassica carinata A. Braun. Theoretical and Appl. Genet. 107, Garce S R, Mancha M (1993). One step lipid extraction and fatty acid methyl esters preparation from fresh plant tissues. Analytical Biochemistry 211, Grundy SM (1986). Composition of monounsaturated fatty acids and carbohydrates for lowering plasma cholesterol. New Eng. J. of Medicine 314, Hoffmann F (1980). Mutation and selection of haploid cell culture systems of rape and rye. Proc. Int. Symp. Plant cell culture Ges. Straheln- u. Umweltforschg. Munchen 319. Kinney AJ (1994). Genetic modification of the storage lipids of plants. Current Opinion in Biotech. 5,

14 Larik AS, Rajpot M, Kakar AA, Bukhari SS, Shaikh MA (1999). Effect of weedicide Afalon on character association in Brassica juncea and Eruca sativa L. Sarhad J. Agri. 15, Maluszynski MK, Nichterlein L, Van-Zanten, Ahloowalia BS (2000). Officially released mutant varieties. The FAO/IAEA. Database. Mut. Breed. Rev., No. 12, pp.1-84, IAEA, Vienna, Austria. McLennan PL (1993). Relative effects of dietary saturated, monounsaturated, and polyunsaturated fatty acids on cardiac arrhythmias in rats. Am. J. Clin. Nutr. 57, Miller JF, Zimmerman DC, Vick BA Genetic control of high oleic acid content in sunflower oil. Crop Sci. 27, Mollers Ch, Schierholt A (2002). Genetic variation of palmitic and oil content in a winter oilseed rape doubled haploid population segregating for oleate content. Crop Sci. 42, Olejniczak J, Adamska E (1999). Achievement of mutation breeding of cereal and oilseed crops in Poland. Proc. 3rd Int. Symp. New Genetical Approaches to Crop Improvement. (eds. Arain et al.,) Nuclear Institute of Agriculture, Tando Jam, Allied Printing Corporation, Hyderabad, Pakistan, pp Kornel N, Annamaria J, Jeno F, Karoly V (2004). An HPLC-MS Approach for Analysis of Very Long Chain Fatty Acids and Other Apolar Compounds on Octadecyl- Silica Phase Using Partly Miscible Solvents Anal. Chem. 76, Rakow G, Raney (2003). Present status and future perspectives of breeding for seed quality in Brassica oilseed crops. Proc 11th Int. Rapeseed Congress, Copenhagen, Denmark 1,

15 Sanches-Silva A, Rodríguez-Bernaldo de Quirós A, López-Hernández J, Paseiro- Losada P (2004). Comparison between high-performance liquid chromatography and gas chromatography methods for fatty acid identification and quantification in potato crisps. Journal of Chromatography A, 1032, Schierholt AB, Rucker, Becker HC, (2001). Inheritance of high oleic acid mutations in winter oilseed rape (Brassica napus L.). Crop Sci. 41, Scarth R, McVetty PBE (1999). Designer oil canola a review of new food-grade Brassica oils with focus on high oleic, low lenolenic types. Proc 10th Int. Rapeseed Congress, Canberra, Australia, CD-ROM. Shah SA, Iftikhar A, Rahmkan K, Mumtaz A, (2005). NIFA mustard canola - First mutant variety of oil seed mustard ( Brassica juncea COSS & CZERN.) in Pakistan. Mutation Breeding Newsletter and Reviews, No.1. Steel RGD, Torrie JH, Deekey DA, (1997). Principles and procedures of statistics: A Biometrical Approach. 3 rd ed. cgraw Hill Book Co. Inc. New York. Takagi Y, Rahman SM (1996). Inheritance of high oleic acid content in the seed oil of soybean mutant M23. Theoretical and Applied Genetics 92, Velasco L, Ferna Ndez-Marti Nez JM, De Haro A (1997). Induced variability for C18 unsaturated fatty acids in Ethiopian mustard. Canadian Journal of Plant Sci, 77,

16 Figure legends Figure 1. Representative profile of studied fatty acids in oil of mutants Brassica napus L. Conditions: flow rate: 1.00 ml/min; Mobile phase ratio: Methanol: 0.1% H 3 PO 4, 85:15; Oven temperature: 45 o C. Peak numbers and the correspondent representative fatty acid are: (1) Solvent peak, (2) Lenolenic Acid, (3) Lenoleic Acid, (4) Oleic Acid and (5 & 6) Unidentified peaks. Figure 2. Configuration of rapeseed mutants lines and parents characterized by three fatty acids in the space of the two first canonical variates V1 and V2.

17 Tables Table 1. Performance of rapeseed (B. napus) mutants in Advanced Yield Trial of Rainbow& Westar Varieties Number of days Plant height 1000 grain wt. Grain yield to maturity (cm) (g) (kg ha -1 ) 1)R00-75/ c 130c 4.73b e 2) R00-100/ bc 156b 4.76b a 3)R00-125/ b 150b 4.93a d 4)R00-125/ b 156b 4.90a a 5)R00-125/ b 156b 4.81a d 6)Rainbow(P) 138.0a 156b 4.04d c 7)W97-75/ ab 157b 4.38c b 8)W97-75/ a 154b 3.99d c 9)W / b 152b 4.38c c 10)Westar(P) 137.0a 160a 3.68e d Values in the same row sharing same letter did not differ significantly (P 0.05)

18 Table 2: Oil content and three fatty acids in rapeseed mutants and two parents cv. Rainbow and Westar Fatty acids (% of total unsaturated fatty acids) S.No. Mutant name Oil contents (%) Lenolenic acid Lenoleic acid Oleic aci d (C18:3) (C18:2) (C18:1) 1 ROO100/ ROO125/ ROO125/ ROO125/ ROO75/ Rainbow(P) W970.75/ W97.075/ W / Westar(P)

19 Figure 1 Figure R W ) %.8 2 (3 2 V R75-1 R100-6 R W Rainbow-P Wester-P W R V1(50.4%) 1 2