Journal of Integrative Plant Biology 2009, 51 (12): 1080–1085

Determination of Essential Fatty Acid Compositionamong Mutant Lines of Canola (Brassica napus),through High Pressure Liquid Chromatography

Ghulam Raza1∗, Aquil Siddique1, Imtiaz Ahmad Khan1, Muhammed Yasin Ashraf2

and Abdullah Khatri1

(1Plant Genetic Division, Nuclear Institute of Agriculture (NIA), Tando Jam, 70060, Pakistan;2Nuclear Institute for Agriculture and Biology, Faisalabad, 38000, Pakistan)

Abstract

The present study aimed to quantify the methyl esters of lenoleic acid (LA), γ-lenolenic acid (LNA) and oleic acid (OL) inthe 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 B. napus cv. ‘Rainbow’ (P) and three mutants (W97-95/16, W97-0.75/11 and W97-.075/13) of B. napus cv. ‘Westar’ (P) atM6 stage, exhibiting better yield and yield components, were analyzed for essential fatty acids. The highest seed yield wasobserved in the mutant (ROO-100/6) followed by ROO-125/14 of Rainbow, that is, 34% and 32% higher than their parentplants, respectively. Westar mutant W97-75/11 also showed 30% higher seed yield than its parent plant. High performanceliquid chromatography analysis of the composition of fatty acids indicated that OL was the most dominant fatty acid, rangingfrom 39.1 to 66.3%; LA was second (15.3–41.6%) and LNA was third (18.1–28.9%). Mutant ROO-125/14 showed higher OLcontents than parent (Rainbow). These results are expected to support the approval of ROO-125/14 in the National UniformVarietal Yield Trials (NUVYT) as a new variety based on high oil quality.

Key words: Brassica; high performance liquid chromatography; lenoleic acid; lenolenic acid; oleic acid.

Raza G, Siddique A, Khan IA, Ashraf MY, Khatri A (2009). Determination of essential fatty acid composition among mutant lines of canola (Brassicanapus L.), through high pressure liquid chromatography. J. Integr. Plant Biol. 51(12), 1080–1085.

Available online at www.jipb.net

The oil quality in rape and mustard is a complex and qualita-tively inherited character and its improvement is a key factor inthe success of rapeseed as a new source of high-quality edibleoil. (Larik et al. 1999). The fatty acid composition of Brassicaoil determines its physical/chemical properties and nutritionalquality. Rapeseed oil contains the lowest amount of saturatedfatty acids as compared with other vegetable oils, and its fattyacid composition is considered by many nutritionists as idealfor human nutrition and superior to that of many other plant oils(Rakow and Raney 2003).

Brassica oil contains the nutritionally desirable oleic acid,along with two essential fatty acids; lenoleic acid and lenolenic

Received 5 Mar. 2009 Accepted 31 Aug. 2009∗Author for correspondence.

Tel. +92 34 5676 6959;

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E-mail: <lalian77@yahoo.com>.

C© 2009 Institute of Botany, the Chinese Academy of Sciences

doi: 10.1111/j.1744-7909.2009.00880.x

acid. Oils high in oleic acid have been shown to have equivalentheat stability to saturated fats and are therefore suitable replace-ments for them in commercial food-service applications thatrequire long life stability. It can also be heated to higher temper-ature without smoking, so that the cooking time is reduced andfood absorbs less oil (Miller et al. 1987). Additionally, high oleicacid oil has cholesterol-lowering properties; whereas saturatedfatty acids tend to raise blood cholesterol levels (Rakow andRaney 2003). Vegetable oils with a high content of C18:1 is ofinterest for nutritional and industrial purposes. The reduction ofthe levels of polyunsaturated fatty acids and their substitutionfor the monounsaturated C18:1 is an important goal for thedevelopment of higher quality mustard oil (Scarth and McVetty1999). A diet containing a high content of C18:1 can reduce thecontent of the undesirable low-density lipoprotein cholesterol inblood plasma (Grundy 1986), and monounsaturated fatty acidsmore effectively prevent arteriosclerosis than polyunsaturatedfatty acids (Chang and Huang 1998).

Improvement in edible oil by modification of fatty acid com-position is a current objective of plant breeders. To achievethis, induced mutations and hybridization can play a vital role.

Essential Fatty Acid Analysis of Brassica napus Mutants 1081

The use of mutagenesis permitted the development of Ethiopianmustard lines with specific fatty acid profiles such as lines witha high oleic acid content and some lines with a low C18:1content (Velasco et al. 1997) and also lines with low and highC22:1 contents whose genetics have been reported in previousstudies (Velasco et al. 1997, 1998; De Haro et al. 2001; DelRio et al. 2003;). These oils with specific fatty acid profilesare in demand because of their improved nutritional and/ortechnological properties (Kinney 1994).

Therefore, efficient analytical methods are a pre-requisitefor the analysis of desired components. In the present study,high performance liquid chromatography (HPLC) was used toanalyze the mutant lines that are already in advanced stagesand exhibiting better agronomical traits. In future, this programwill be used for the selection of desired genotypes from mutantlines, which are among a large number of segregating popula-tions. Therefore, efforts have been made to develop genotypesexhibiting high oleic acid content and a 2:1 ratio of linoleic tolinolenic acid contents using mutagenesis.

Results

Agronomic performance of mutants

Mean performance and χ2-test regarding seed yield and itscomponents in advanced yield trial for rapeseed mutants areshown in Table 1. Significant difference among the agronomictraits was observed. Among the Rainbow mutants, ROO-100/6showed the highest seed yield performance (1 665 kg/ha) andproduced over 34% more than the parent plant (1 242 kg/ha)followed by mutant ROO-125/14 (1 635 kg/ha), which has 32%more seed yield than the parent. On the other hand, mutantROO-125/17, ROO-125/12 and ROO-75/1 exhibited lower yield

Table 1. Performance of rapeseed (Brassica napus) mutants in ad-

vanced yield trials of Rainbow and Westar

Number Plant 1 000 Grain

Varieties of days height grain yield

to maturity (cm) weight (g) (kg/ha)

(1) R00-75/1 115.0c 130c 4.73b 1 075.7e

(2) R00-100/6 118.8bc 156b 4.76b 1 664.8a

(3) R00-125/12 120.0b 150b 4.93a 1 152.1d

(4) R00-125/14 122.0b 156b 4.90a 1 635.4a

(5) R00-125/17 125.3b 156b 4.81a 1 161.1d

(6) Rainbow (P) 138.0a 156b 4.04d 1 242.0c

(7) W97-75/11 130.6ab 157b 4.38c 1 525.0b

(8) W97-75/13 133.6a 154b 3.99d 1 236.4c

(9) W97-0.75/16 128.0b 152b 4.38c 1 266.0c

(10) Westar (P) 137.0a 160a 3.68e 1 175.0d

Values in the same row sharing same letter did not differ significantly

(P ≤ 0.05).

as compared with the parent. Among the Westar mutants,W97-75/11 gave the 30% higher yield (1 525 kg/ha) than theparent (1 175 kg/ha) followed by W97-0.75/16, which produced1 266 kg/ha seed yield that is only 8% more than the parent.

All of the Rainbow and Westar mutants exhibited more 1 000grain weight than their respective parents. The mutant ROO-125/12 and ROO-125/14 showed the highest 1 000 grain weightamong all plants. No difference was observed regarding theplant height among the Rainbow mutants except for mutantROO-75/1, which was shorter than the parent. All Westarmutants were shorter than the parent. All of the mutants maturedearlier than their parent. Mutant ROO-75/1 matured 23 daysearlier than the parent, followed by ROO-100/6, which matured20 days earlier than the parent. No correlation was observedfor days of maturity with seed yield. Some mutants (ROO-75/1, ROO-125/12 and ROO-125/17) matured earlier and alsoexhibited lower yields, whereas some mutants, like ROO1-100/6and ROO-125/14, exhibited higher seed yield and also maturedearlier than the parent.

Oil contents

Among the Rainbow mutants, the highest oil contents (46%)were observed in ROO-125/14 and ROO-75/1, which was 4.5%more than the parent (44%), whereas mutants ROO-100/6produced 45% oil content, which is 2.2% more than the parent.Among the Westar mutants, W97-0.75/16 showed the highestoil content (46%) which was 9% more than the parent (42%),whereas mutant W97-0.75/13 exhibited 45.5% oil content, whichwas 8% higher than the parent (Table 2).

HPLC analysis

The composition of unsaturated fatty acids (lenolenic acid,lenoleic acid and oleic acid) in mutants of Rainbow and Westarwas determined by the HPLC method using Ezi Chrome Elitesoftware (Scientific Software Inc., Pleasanton, CA, USA). Forobtaining desired peaks, the flow rate, oven temperature andsolvent ratio parameters were optimized. The best results wereobtained using the following conditions; flow rate: 1.00 mL/min,mobile phase ratio: methanol: 0.1% H3PO4: 85:15, oven tem-perature: 45 ◦C. The fatty acids (lenolenic acid, lenoleic acid andoleic acid) chromatographic profile is given in Figure 1, whereinthe retention time of these fatty acids is also shown. Lenolenicacid was eluted within 6.227 min, followed by lenoleic acid,which was eluted after 7.410 min. Oleic acid was eluted laterafter 9.057 min. The retention time for these fatty acids wasidentified by using standards of these fatty acids with knownretention times.

Fatty acid analysis

Composition of unsaturated fatty acids was expressed in relativeconcentrations rather than absolute (Table 2). HPLC analysis

1082 Journal of Integrative Plant Biology Vol. 51 No. 12 2009

Table 2. Oil content and three fatty acids in rapeseed mutants and two parent cultivars: ‘Rainbow’ and ‘Westar’

Fatty acids (percentage of total unsaturated fatty acids)Mutant Oil

Sample no.name contents (%) Lenolenic Lenoleic acid Oleic acid

acid (C18:3) (C18:2) (C18:1)

1 ROO100/6 45.0 27.7 20.7 51.0

2 ROO125/14 46.0 18.5 15.3 66.3

3 ROO125/17 44.3 28.9 22.5 48.5

4 ROO125/12 44.0 19.3 41.6 39.1

5 ROO75/1 46.0 27.6 24.6 47.8

6 Rainbow (P) 44.0 24.8 23.3 51.8

7 W970.75/11 45.5 26.5 19.8 53.6

8 W97.075/13 44.0 24.0 23.7 52.3

9 W97-0.75/16 46.0 24.3 22.7 53.1

10 Westar (P) 42.0 18.1 22.5 59.4

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 between39.1% and 66.3%, lenoleic acid (C18:2) was the second mostdominant fatty acid and ranged between (15.3% and 41.6%),while lenolenic acid ranged between 18.1% and 28.9%.

Among the Rainbow mutants, ROO-125/14 showed the high-est oleic acid content (66.3%), which is 28% more than theparent (51.8%), while ROO-125/12 showed the least oleic acidcontent (39.1%), which was 24.51% lower than the parent(51.8%). On the other hand, all of the Westar mutants hadlower 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%), whichwas 25.4% lower than the parent (24.8%). Mutant ROO-125/12had the highest percentage (41.6%) of lenoleic acid, being78% 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-

Figure 1. Representative profile of studied fatty acids in oil of mutants of Brassica napus L.

Conditions: flow rate: 1.00 mL/min; mobile phase ratio: methanol: 0.1% H3PO4, 85:15; oven temperature: 45 ◦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.

125/14. Among the Westar mutants, W97-0.75/13 had thehighest lenoleic acid content (23.7%), which was 5% more thanthe parent (22.5%).

Among the Rainbow mutants, mutant ROO-125/14 is alreadyin NUVYT on the basis of better yield/yield components andoil contents compared with the parent plant. From the presentstudy, it was observed that mutant ROO-125/14 showed thehighest oleic acid contents as compared with the parent. So,these results are quite encouraging and will be helpful forthe approval of this mutant as a future variety. Among theWestar mutants, mutants W97-0.75/11 and W-97-0.75/16 arein advanced yield trials due to good yield and oil contents, buttheir oil quality is not better than the ROO-125/14 mutant.

The analysis of canonical variates was conducted in order tocreate a graphic image of the arrangement of the genotypes(mutant lines and parental lines) on the plane (Figure 2). Thegraphic image of the distribution of mutant lines characterized bythe three fatty acids in the space of first two canonical variatesreflects both their variability and relation to the parental lines.The closer the distance between points representing individual

Essential Fatty Acid Analysis of Brassica napus Mutants 1083

210-1-2

2.0

1.5

1.0

0.5

0.0

-0.5

-1.0

V1 (50.4%)

V2

(3

2.8

%)

Wester-P

W97-75-16

W97-75-13

W97-75-11Rainbow-P

R75-1

R125-12

R100-17

R125-14

R100-6

Figure 2. Configuration of rapeseed mutant lines and parents charac-

terized by three fatty acids in the space of the two first canonical variates

V1 and V2.

genotypes, the closer the similarity with regard to the contentsof 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 the Rain-bow parent, while mutants ROO125/12 and ROO-125/14 werefar from their respective parents. This represented the unique-ness of these mutants and also reflects the presence of varia-tion. On the other hand among the Westar mutants, W97-75/11and W97-75/13 are near to their respective parents, while W97-75/16 is far away from the parent.

Discussion

Mutation breeding is a technique through which new crop vari-eties with desirable characteristics can be developed in shortertime, and it is a more sophisticated technique than conventionalpedigree breeding. Mutagenesis was plasticized with not onlyseeds but also with single cells (Hoffmann 1980). Now thismethod is under routine practice and numerous reports existin which mutation breeding was used to improve the yield andyield components and other attributes of rapeseed (Olejniczakand Adamska 1999; Maluszynski et al. 2000; Abdullah et al.2004; Shah et al. 2005). Similarly, we also used this techniquein the present study to obtain mutants with high yield and yieldcomponents and better oil quality than their parents. Our resultsindicated that mutant ROO-100/6 exhibited 34% more yield thanthe parent, but this mutant showed a similar oleic acid content(51%) as the parent (51.8%), while the mutant showed 32%higher yield than the parent and also exhibited the highest oleicacid contents (66.3%) among all of the mutants. Hence, ROO-125/14 is superior both from qualitative and quantitative pointsof view. The existing HPLC method can be used to screengenotypes with respect to desired fatty acid composition, which

is very important for oil quality. A similar approach was usedto analyze underivatized very long chain fatty acids (C16–C26)and other apolar compounds such as triacylglycerols (Kornelet al. 2004). The major advantages of the HPLC method overgas chromatography (GC) are lower temperatures during anal-ysis, which reduces the risk of isomerization of double bonds,and the possibility of collecting fractions for further investigations(Czauderna and Kowalczyk 2001). Similarly, Sanches-Silvaet al. 2004 compared a reversed-phase HPLC (RP-HLPC)method with a gas chromatography-flame ionization detection(GC-FID) method for determining fatty acids in potato crisps.They observed that both methods presented good precisionand recovery, but precision using the HPLC method was slightlybetter than for GC-FID, and they also observed that the HPLCmethod (level of detection [LOD] ≤ 0.74 μg/mL) was moresensitive than the GC method (LOD ≤ 5.00 μg/mL) for allcompounds studied.

Mutation breeding has potential in creating lines with amodified fatty acid composition. In particular, as the presentresults showed, the development of line ROO125/14 with higholeic acid content seemed promising and line ROO125/12 haslenoleic and lenolenic acid with 2:1, which is acceptable fornutrition, having lower polyunsaturated fatty acids. The similarbreeding strategy has been used by several scientists andobtained promising lines with modified fatty acids (Schierholtet al. 2001; Velasco et al. 2003; Del Rio-Celestino et al. 2007).Therefore, the mutant lines selected in the study could beused in rapeseed breeding for further genetic modification ofseed quality by changing the proportion of fatty acids suitablefor nutritional purposes. In the present study, line ROO125/14exhibited the desired amount of oleic acid contents so it can berecommended for cultivation to achieve the high quality oil andin maintaining the high level of monosaturated fatty acids andreduced level of polyunsaturated fatty acids successfully. It mayhave higher oxidative stability and reduced oxidation productsin the oil. Schierholt et al. (2001) analyzed eight mutants thatshowed a significant increase in oleic acid content in the seeds.Adamska et al. (2004) also reported that two DH lines H5-30 andH5-43 had higher oleic acid contents. Mollers and Schierholt(2002) concluded that rapeseed oil, which is high in C18:1 hasa lower content of the saturated fatty acids making the oil moresuitable from a nutritional point of view. Xue Wen et al. (2006)reported that Gracilaria lemaneiformis (Bory) contained linoleicacid and oleic acid in higher amounts as unsaturated fatty acids,while it possessed only small amounts of polyunsaturated fattyacids. In the present study, line ROO125/14 has oil with higholeic acid content, so it may be recommended for commercialuse.

It was also observed that mutant ROO125-12 had lenoleicand lenolenic acid at a 2:1 ratio. This ratio is acceptable fromthe nutrition point of view. Similar results were also reportedby Adamska et al. (2004). Moreover, the remaining mutants ofRainbow and all of the mutants of Westar do not demonstrate

1084 Journal of Integrative Plant Biology Vol. 51 No. 12 2009

any differences in the fatty acids studied in comparison withtheir respective parents. These results suggest that substitutionof canola oil for other dietary lipid sources may assist in reducingthe likelihood of a transient ischemic event, which could lead tolife-threatening cardiac arrhythmias. Increasing the proportionof oleic acid in the diet in exchange for saturated fats haspreviously been shown to have little influence on either thefatty acid composition of the myocardial cell membrane orthe vulnerability of the heart to develop serious arrhythmias(McLennan 1993).

Materials and Methods

Homogeneous seeds of rapeseed (Brassica napus L.) cv.Rainbow and Westar treated with three doses of gamma rays,that is 750, 1 000 and 1 250 Gy, three concentrations ofethyl methyl sulfonate (EMS), that is, 0.75, 1.00 and 1.25%solution separately and four treatments of these mutagenes incombinations (750 Gy + 0.75% EMS, 750 Gy + 1.00% EMS,1 000 Gy + 0.75% EMS and 1 000 Gy + 1.00% EMS). Thetreated seeds, along with untreated (control) seeds were grownin the field with randomized complete block design (RCBD),having three replications to raise M1 generations. After thoroughselection in a successive generation, five mutants (ROO-75/1,ROO-100/6, ROO-125/12, ROO-125/14, and ROO-125/17) ofRainbow (P) and three mutants (W97-95/16, W97-0.75/11 andW97-.075/13) of Westar (P) at the advanced stage exhibitingbetter yield and yield components were analyzed for essentialfatty acids as shown in Table 1.

Ten gram seeds of these mutants were dried overnight at55 ◦C and ground to powder in a Moulinex coffee grinder.Seed powder was mixed with 100 mL petroleum ether and thelipid fraction was extracted in a Soxhlet apparatus at 60 ◦Cfor 12 h. The solvent was evaporated and the weight of lipidextraction residues was determined, from which oil percentagewas calculated. Fatty acid methyl esters were obtained asdescribed by Garces and Mancha (1993).The extracted oil wasfiltered by using a 0.45 μM pore size filter. In a sample tube, thesolution of purified oil and heptane (1 mL of each), was mixedwith methanol based on 0.1 mL of a 2 N KOH solution, thenthe tube was capped and screwed tightly. The tube was shakenvigorously for 15 s and kept at room temperature until the upperlayer became clear.

The upper layer was separated and 20 μL of the supernatantwas injected in HPLC and analyzed on a Hitachi HPLC system(Hitachi, Japan) equipped with a refractive index detector andreverse phase intersil column. The analysis was done underconditions mentioned under results. HPLC method was estab-lished by using Ezi Chrome Elite software. Calibration curveswere prepared by injecting mixtures of standards (lenolenic acid,lenoleic acid and oleic acid) of 2.5, 5.0, 10.0 and 20.0% under

previously mentioned conditions. The fatty acid content wasexpressed as the percentage of total fatty acid.

Statistical analysis

Statistical analysis was carried out using univariate and multi-variate techniques. Through principle component analysis, theplane was reduced to two canonical variants, which were usedas coordinates for plotting genotypes. Genotypic means foragronomic traits were compared using least significant differ-ence test at the 5% probability level (Steel et al. 1997).

Acknowledgements

We express our deep gratitude to Mr Tanveer Mustafa, JuniorScientist, Nuclear Institute of Agriculture and Biology, Faisal-abad for helping us with the statistical analysis.

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