ENVIRONMENTAL AND INDUCED MUTATIONS INFLUENCES ON OIL CONTENT, OLEIC ACID AND ERUCIC ACID IN RAPESEED

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1 Sarhad J. Agric. Vol.29, No.2, 2013 ENVIRONMENTAL AND INDUCED MUTATIONS INFLUENCES ON OIL CONTENT, OLEIC ACID AND ERUCIC ACID IN RAPESEED IFTIKHAR ALI 1* and FARHATULLAH 2 1 Nuclear Institute for Food and Agriculture, Peshawar Pakistan 2 Department of Plant Breeding and Genetics, The University of Agriculture, Peshawar Pakistan. * iali63@yahoo.com ABSTRACT A comparative study was conducted to see the mutational analysis for monounsaturated fatty acids in rapeseed (Brassica napus L.) mutant populations. By using gamma irradiation, a range of variability generated for oil content and fatty acid composition in rapeseed seeds. The induced genetic variability was analyzed for two consecutive years in two contrasting environments at elevations of 510 and 2039 meters. In both cases induced variability differences were estimated for oil content as well as oleic Acid (C 18:1 ) and erucic Acid (C 22:1 ). Across radiation treatments, the direction and magnitude of induced variability were related to environmental influence. The gamma irradiation enhanced synthesis of oleic acid but induced different spectrum of variability for this desirable monounsaturated fatty acid at two locations. Desirable canaola quality modification i.e. low erucic and high oleic acid in rapeseed oil have been observed but with diverge pattern in same gamma dose mutant populations at two locations. The gamma dose 1200 Gys reduced oleic acid at Peshawar but same dose increased oleic acid at Kaghan. Gamma irradiation also affected in different way for erucic acid trait at two locations. These results demonstrated that variation in growth environments can cause differences in genetic variability for monounsaturated fatty acids in rapeseed through induced mutations. Furthermore, environment influence can significantly affect the selection index for oil yield and oil quality traits in rapeseed. Keywords: Induced mutations, gamma rays, rapeseed, oleic acid, erucic acid Citation: Ali, I. and Farhatullah Environmental and induced mutations influences on oil content, oleic acid and erucic acid in rapeseed. Sarhad J. Agric. 29(2): INTRODUCTION Mutations have played a significant part in the genetic improvement of oilseed crops and induced mutations have been employed for genetic modification of the fatty acid composition in rapeseed since long (Scarth and Tang, 2006). Successful performance of rapeseed mutant varieties with modified fatty acid composition on the whole links to the mutagen and the applied dose by which they were originally induced (Shah and Rahman, 2010; Bhatia et al., 1999). But there are hardly any investigations about the relative efficiency of induced mutations across diverse agro-climatic conditions. The early induced mutations studies concluded that the effects of many quantitative mutations on phenotype may be confined to only some environments despite the fact that the original genetic constitution remains unchanged in all conditions (Erkkz and Allen, 1961; James, 1961; Muller and James, 1961). Many mutation breeding experiments revealed that the fitness of a mutant may vary remarkably under environmental conditions of various kinds (Gustafsson, 1951). This crossover mutant and environment interaction is most important, which implies changes in the rankings of mutant lines across environments (James, 1961). In case of noncrossover interactions, mutant lines with superior means can be recommended for all environments. But crossover genotype and environment interaction complicates breeding, evaluation and selection of superior genotypes (Becker and Leon, 1988). Proper choice of environment for successful selection is of prime importance and this selection of environment can profoundly affect both the efficiency and outcome of selection scheme in a breeding program (Finlay and Wilkinson, 1963; Becker and Leon, 1988; Raj et al., 1997; Kang, 1998; Yadav et al., 1993; Larik and Rajput, 2000). The induced mutations used by plant breeders most likely concern those genes, which are involved in genetic and environmental interaction, regulating the response to triggers by signaling activation or inhibition to other genes responsible for particular reactions (Micke, 1999). Although there is an assortment of reactions among mutations, there is nevertheless a general trend toward protection by a good environment (Datta, 2005). A good environment can be expected to mask detrimental mutations; it is under poor conditions that selection works more effectively to eliminate detrimental types from a population (Muller, 1950). Selection and environment relationship indicated attribution of

2 Iftikhar Ali and Farhatullah. Environmental and induced mutations influences on oil content 188 adaptation to mutations in few genes with large effects (Bell, 2010; Orr, 2003) and many experimental studies confirmed responsibility of primary mutations and genetic variance of quantitative characters for adaptability in specific environments (Barrett, et al., 2006; Dykhuizen and Hartl, 1981; Notley-McRobb and Ferenci 1999; Kearsey and Farquhar 1998). The practical usefulness of rapeseed genetic stocks, with desirable modification in fatty acid composition, in a breeding program is dependent upon the stability of the mutant allele across different environment conditions (Tang and Scarth, 2004). The fatty acid profile in rapeseed oil depends on the genetic supposition of genotype. But environmental conditions are also of great influence (Pritchard et al., 2000). In rapeseed the climatic influences prevail over the genotypic influences during fatty acids synthesis has been confirmed (Werteker et al., 2010). The changing share of monounsaturated fatty acids (MUSFAs) at changing temperatures during the last days of seed maturity before harvest confirms the influence of controlled environments (Baux, et al. 2008). It is important for rapeseed breeders to understand the influence of environment on spectrum and direction of induced mutations concerning fatty acid composition for a successful execution of mutation breeding scheme. The present study was undertaken to establish the influence of contrasting environments on the dynamics of induced mutations pertaining to Oleic Acid (C 18:1 ) and undesirable Erucic Acid (C 22:1 ) in seed oil of rapeseed mutant population. MATERIALS AND METHODS The segregating M 2 population used for this study was developed from Brassica napus cv. Abasin-95 through gamma radiation doses of 800, 1000 and 1200 Gys following the procedure out lined in IAEA Technical Manual No.117 (1997). Field experiments for the generating and isolation of segregating populations were conducted at Himalyan Agricultural Research Institute (HARI), Kaghan, (elevation of 2039 m) (and Nuclear Institute for Food and Agriculture (NIFA), Peshawar, Pakistan elevation of 510 m) Pakistan. Two summer season (May-September) field experiments for isolation of M 2 segregating populations were carried out at HARI, Kaghan while two winter season (October-May) experiments were conducted at NIFA, Peshawar. The average agro-climatic features of two experimental locations are given in Table-1. At maturity fertile and vigorous individual plants containing or suspected to contain the mutant allele were harvested. In case of control (untreated) population fertile and healthy plants were also individually harvested. Table 1. Agro-climatic conditions of experimental locations of Kaghan and Peshawar Location Elevation (m) Crop maturity Minimum Temperature during Maximum Temperature during growth period Average annual rain (mm) (days) growth period ( 0 C) ( 0 C) Kaghan 2, Peshawar Biochemical Analysis The levels of Oleic Acid, (OA) and Erucic Acid (EA) with total oil content in seeds of normal and mutant populations in each season and environment were determined by near-infrared reflectance spectroscopy (NIRS). The reflectance spectra were collected from 5gm seed samples of each individual harvested plant with NIR System Model 6500 spectrometer installed in Oilseed Quality Laboratory of Plant Breeding and Genetics Division at NIFA, Peshawar. Simultaneous analyses were done for oil and fatty acid content as described by Reinhardt (1992). Statistical Analysis The average collected data was analyzed by numerical taxonomic techniques using the procedures of principal component analysis (Sneath and Sokal, 1973). The standardized and transformed data matrix was prepared using software STATISTICA (2004). The large dimensions of generated data were reduced to only two leading principal components (PC1 and PC2) which have extracted major share of variation in the data of three selected traits for rapeseed genetic variability. The PCA was used to compute variance in data and to determine the most variability-influencing trait and gamma radiation dose. The standard deviation and mean of the eigenvector of principal components were employed to study the interactions between environments, induced mutations and OA, EA and oil content in seeds of normal and mutant populations of rapeseed.

3 Sarhad J. Agric. Vol.29, No.2, RESULTS AND DISCUSSION Two principal components were identified which accounted for about or more than 20 % of the variation within the data set (Table 2). Cumulatively, these two vectors accounted for over 75% of the variation. The first principal component extract (PC-1) regarded as a spectrum of induced mutation vector. It has high loadings for two MUSFAs and for oil content in all data sets (Table 1). The second principal component extracted (PC-2) is an induced mutation direction vector. It has comparatively high loadings for OA (Table 2). These two principal components can be considered as modified traits containing information from all of the 24 data sets originally measured. The loadings of first two principal components on MUSFAs and oil content can be used as the basis for constructing indices which have an interpretation for environmental influence on the overall dynamics of induced mutation of gamma radiations in population of rapeseed crop. Table 2. Standardized loading and percentage of total variance within the data accounted for by the first two principal components for normal and mutant population of rapeseed at two locations. HARI, Kaghan NIFA, Peshawar PC-1 PC-2 PC-1 PC-2 Normal population Oil Oleic Acid Erucic Acid Variance Gys gamma rays Oil Oleic Acid Erucic Acid Variance Gys Gamma rays Oil Oleic Acid Erucic Acid Variance Gys Gamma rays Oil Oleic Acid Erucic Acid Variance PC-1 accounted for over 71.69% of the variation in MUSFA in the seed oil content among the plants from normal population of HARI, Kaghan and PC-2 expressed 19.53% variation (Table 2). These two vectors produced variation of 61.71% and 24.42% in MUSFA in normal population at NIFA, Peshawar (Table 2). The variances ratio of principal components was high for normal population at Peshawar (0.4) compared to the variances ratio (0.3) of population at Kaghan (Fig. 1). The difference in the variances ratio expressed the influence of divergent agro-climatic condition on at two experimental locations. Temperature claimed to be the main environmental factor responsible for affecting composition of fatty acids in oilseeds while other factors, such as photoperiod, intercepted radiation, and soil nutrients availability, have been also reported as responsible for such changes (Natalia 2006). High variability in MUSFA levels in rapeseed at Peshawar anticipates to more fluctuations in daily temperature during the growth period of crop as compared to Kaghan. The effects of the locations on the levels of fatty acids have been confirmed and reported (Hall, 2004). In the mutant population of 800 Gys gamma radiation, at Kaghan, 64.13% variation in PC-1, while 23.56% variation in PC-2 was recorded. The mutant population at Peshawar produced 58.26% and 29.29% portion of variation in PC-1 and PC-2 respectively (Table 1). The variances ratio of PC-2 to PC-1 (0.5) was high for mutant population at Peshawar (Fig. 1) than Kaghan. The variances ratios of principal components from

4 Iftikhar Ali and Farhatullah. Environmental and induced mutations influences on oil content 190 this group of mutant plants at both locations were relatively higher than the variances ratios of normal populations (Fig. 1). The gamma dose of 1000 Gys recorded 57.94% variation in PC-1 in mutant plants at Kaghan, while 29.42% counted in PC-2 (Table 1). At Peshawar, 1000 Gys radiation affected with the account of 44.38% and 30.82% variations in PC-1 and PC-2, respectively. The variances ratio of two principal components was higher at Peshawar again (Fig. 1). The ratios from 1000 Gys gamma dose at both locations were higher than the ratios of normal plants and mutant plants of 800 Gys. PC-1 accounted for 48.08% of the variation in the plants from 1200 Gys gamma dose at Kaghan and PC-2 showcased 38.50% variation (Table 1). These two principal components induced variation of 50.02% and 32.24% in MUSFA in mutant population at Peshawar. In case of higher dose of 1200 Gys the variances ratio for principal components at Kaghan was high as compared to Peshawar (Fig. 1). Collectively the variances ratio increased with the increase in gamma radiation doses. In case of mutant population at Kaghan this increase was curvilinear. The increase at Peshawar was linear only for 800 and 1000 Gys gamma doses (Fig. 1). The changed biochemical variability in rapeseed crop with increasing gamma rays doses is reported in literature (Datta, 2005; McVetty and Scarth, 2002; Schierholt et al., 2001; Velasco et al., 1999; Rucker and Robbelen, 1997; Velasco et al., 1997; Robbelen and Nitsch, 1975). Variation among mutant and normal populations was extensive for MUSFAs at both locations. Environment was significant factor for traits in lineages from two locations. The observed variation in first two principal components is plotted through biplot (Fig. 2). Two MUSFAs for normal populations were identified with low spectrum of variation but in opposite directional quarters. The desirable OA was found in upward direction quarter of biplot and undesirable EA was captured in downward directional quarter (Fig. 2). The assessment of MUSFAs in normal population was found consistent from two locations with minor disparity in case of EA. Gamma induced mutations synthesized significant variable MUSFAs in lineages from two locations. The highest gamma dose of 1200 Gys at Peshawar not only reduced the OA content sharply but also generated mutant population with a narrow range of variability for OA (Fig. 2). The high gamma dose 1200 Gys produced mutant population with high OA content and narrow spectrum at Kaghn. The 800 Gys of gamma radiation generated rapeseed mutant plants with high OA content and broad spectrum of variability at NIFA and HARI. The gamma dose of 1000 Gys induced mutations for increased OA content in rapeseed population with low spectrum at both locations. In the case of EA, 800 Gys at both locations and 1200 Gys at HARI, Kaghan increased the level of this highly undesirable fatty acid in mutant population. The gamma dose of 1000 Gys did not much affect the EA content at HARI and remained at low levels and low spectrum of variability as in case of normal population (Fig. 2). The same gamma dose induced a broad spectrum of variability for EA in mutant population at NIFA but with downward direction. CONCLUSIONS AND RECOMMENDATIONS Since the first report of induced mutants with modified fatty acid composition by Rakow (1973), successful mutation breeding for reduced or increased levels of fatty acids in rapeseed is in progress. Principal component analysis in combination with cluster analysis is the recommended technique for studying the interactions between genotypes, environments and attributes (McLaren, 1996). The current study emphasizes interpretation of the principal components in order to gain an understanding of the induced mutations to generate variability of MUSFAs among the populations raised under diversified agro-climatic conditions. The influence of environment has a major role in the modification of MUSFAs in rapeseed crop. The effects of agro-climatic conditions in conjunction with induced mutations for the genetic improvement in MUSFAs of rapeseed should be considered. Principal components analysis is an effective technique for gaining an understanding of the relationships between environment and breeding strategies.

5 Variance ratio Sarhad J. Agric. Vol.29, No.2, Normal 800 Gys 1000 Gys 1200 Gys HARI, Kaghan NIFA, Peshawar Fig. 1 Comparative variances ratio of PC-1 and PC-2 for MUSFAs in normal and mutant population at two locations Fig. 2 Spectrum and direction of induced mutations for oleic and erucic acids with oil content in normal and mutant populations of rapeseed. Normal (control) population encircled for three traits. Symbol represents HARI, Kaghan and represents NIFA, Peshawar data. 1=800 Gys, 2=1000 Gys, 3=1200 Gys gamma dose OL= Oil, OA= Oleic Acid, EA= Erucic Acid

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