Biochemical Investigation of 24 Prominent Mutants of Arachis Hypogaea (L.)

Groundnut (Arachis hypogaea L.) is an important oil seed crop and grain legume worldwide. It is self pollinat- ing and possesses limited variability. Thus this study investigates the improvement of groundnut oil quantitatively and qualitatively by induced mutations with Gamma rays and EMS mutagens. Pertaining to the oil quantity 20 kR and 10 kR doses of gamma rays is more effective in variety AK-159 and TAG-24 respectively. While in improving oil quality the 0.15% concentration of EMS and 0.10% concentration of EMS are more potent in variety AK-159 and TAG-24 respectively. This work has enabled us to conclude that the oil content, fatty acid composition and oil stability could get alter in positive direction through the application of mutagen.


Introduction:
Groundnut (Arachis hypogaea L.) is an important oil seed crop and grain legume worldwide. As an oilseed crop, groundnut rank fourth in world production. Although groundnut in India is primarily used as an oil seed, it is also consumed directly as food because of its palatability. Besides, it contain oil (48-52 %) and protein (22-30%) seed contain carbohydrates (10%), minerals (3%) and B complex vitamins especially thiamin (Rajgopal, et al., 2000). Groundnut oil contains 51% of monosaturated oil and 30% polyunsaturated and 19% saturated oil (Amarican heritages, 2007). It also has neutral flavor and odour. It does not absorb odours from other foods (Passera, 1981). This makes it the most preferred oil in India.
India is the third largest consumer after China and EU. It was observed that the area under cultivation, production and productivity of groundnut in Maharashtra and India have revealed continuous decreasing trend (Patil et al., 2009 andTalwar, 2003). However per capita consumption of edible oils is around 11 Kg per year. (Ramesh P & Murughan M. 2008). Oil quality and its stability are therefore very important for the consumers and application industries (Jambunathan et al., 1993). However, it is self pollinating and possesses limited variability. Thus this study investigate the improvement of groundnut oil quantitatively and qualitatively by induced mutations with physical (Gamma rays) and chemical (EMS) mutagens.
Mutation breeding offers the possibility of inducing desired attributes that either cannot be found in nature. There are examples where mutants have been identified for yield and quality traits in groundnut (Patil 1975).
Generally in groundnut, mutation breeding has done for the evolving species / varieties which are high yielding and resistant to leafy diseases. However, pertaining efforts has been made to incline secondary importance to the compositional and nutritional aspects of the final crop. In case of groundnut it would be desirable to evolve a variety that has not only high oil content but also nutritionally desirable fatty acid composition. Fatty acid composition of groundnut oil is an important trait with reference to human nutrition and oil stability during storage. Treated seeds of each variety were sown in field for raising M 1 generation following randomized block design (RBD) with three replicate having spacing of 15 X 35 cm. An untreated seed serves as a control. The experimental field situated at Latitude 19 0 48' 23" N and longitude 75 0 50' 42" E at an altitude of 507 meter above sea level. The experimental area for M 2 and M 3 and spacing are same as those used in M 1 generations.

Materials and
Critical screening was done though the M 1 , M 2 and M 3 generation and viable mutants were carefully screened from the population.

Oil content (%):
The seed oil percentage of 24 prominent mutants was estimated by soxhlet method according to pearson's (1981) 5 gm of each finely ground and sample was extracted with hexane (boiling point 65 -70 ).

Fatty acid profiling by GLC:
The fatty acid analysis was carried out by using Gas Liquid Chromatography (GLC) at quality control Laboratory, Teena oils and chemicals Ltd. MIDC, Latur (MS) a) Preparation of Fatty acid methyl esters: 300 mg of oil was mixed with 6 ml of methanolic NaOH in standard volumetric flask of 50 ml. Saponified it on water bath till no oil globules could remain in flask. After cooling the 8 ml of Boron trifluride (BF 3 ) was added for esterification and heated with continuous shaking for 2 minutes carefully, because BF 3 is toxic and explosive. Cooled the flask and 2 ml of petroleum ether was added. Then final volume (50 ml) was made with saturated NaCl. (AOAC 1990).

b) Determination of fatty acid composition of oil :
A layer of hexane was given on the fatty acid methyl ester solution and mixed well. Then the solution was allow to settle for 15 minutes and from clear supernatant, 0.6 of this solution was injected by sample syringe in gas chromatography. The fatty acid composition of oil was determined by gas liquid chromatography (GLC) performed on model Chemito GC-1000, armed with flame ionization detector (FID) and Polar capillary column BPX-70 (manufacturer-SEG capillary columns) having pore size 0.25 , Internal diameter 0.32mm and length 50 m (Part No. 054607). The oven, injector and detector temperatures was maintained at 100 , 250 and 280 respectively. Nitrogen was used as carrier gas, Split ratio was 1/36 and the run time was upto 18.00 minutes. Individual peak of fatty acid methyl eater (FAME) was identified by comparing their retention times with those of standards (Sigma Aldrich). Fatty acid composition of each sample was encoded by software (Chemito chrome card A/D for windows Reversion A-1.) in the form of peak in chromatogram.

Result and Discussion:
The oil content in prominent mutant ranged from 48.10% to 54.34% in variety AK-159 and 47.20% to 52.76% in variety TAG-24. The highest oil percentage was recorded 54.34% and 52.76% in Early maturing with dwarf Mutant (irradiated to 20 kR dose of gamma rays) of variety AK-159 and Faint seed coat mutant (10 kR of gamma rays) of var.TAG-24 respectively. Nadaf and et al., 2009 also recorded increased oil content after treating seeds with gamma rays and EMS in groundnut. Giriraj, et.al., (1990) have observed higher variability and shift in mean towards positive direction in M3 generation after EMS and gamma ray treatments in sunflower. Similar results were recorded by Badere and Choudhary (2004) after using SA and EMS in linseed.
Generally high oleic content oil is preferred. It is a monounsaturated fatty acid. It lowers the heart attack risk and arthrosclerosis and aids in cancer prevention (Rotella, 2004). David, T.W. (2005) examined the major role of oleic acid in prevention of breast cancer. The evidence that long-chain fatty acids e.g. arachidic and behenic, in groundnut oil may be implicated in heart disease has been noted (Kies et al., 1978). The oil produced from high oleic acid groundnuts had a substantially higher resistance to oxidation than that from conventional groundnuts (Talcott et al 2005a)

# OSI-Oxidative stability index #NQI-Nutritional quality index
The final quality of groundnut oil depends on its fatty acid composition and in present investigation the acid composition among the mutants revealed significant alternation in the linoleic and oleic acid and in regard to other fatty acids only slight change could be noticed in both the varieties of groundnut as shown in table 1. The major fatty acid component in groundnut seed oil is Palmetic(C 16:0), Stearic (C 18:0), Oleic (C 18:1), Linoleic (C 18:2), Arachidic(C 20:0) and Erucic (   navar (2010) reported the variation in fatty acid composition in groundnut after mutagenesis.
The seed oil stability is again important parameter regarding the shelf-life of products made from oil. It can be determine from the ratio of 18:1/18:2 in groundnut (Young et al 1974).
Higher this ratio, the higher is the oil stability and longer is the shelf-life of groundnut products. In present investigation the oil stability among 24 prominent mutants of groundnut is highest in late flower mutant (0.15% of Ems) and Large leaf mutant (0.10% EMS) of variety AK-159 and variety TAG-24 respectively. However higher unsaturation in an oil affects seed oil stability and lowers the shelf life of such oil derived products (Lea, 1962).  Groundnut oil is a source of valuable nutrients. The ratio of unsaturated fatty acid to saturated fatty acid indicates NQI of oil. These health parameters have been recommended by WHO for the dietary use of vegetable oils. It was found that, majority of the mutants possessed the NQI above 4. In variety AK-159 the highest NQI value was recorded in the large leaf and high yielding mutant, while in case of variety TAG-24 the highest NQI value was demonstrated by bold seeded mutant.

Conclusion:
This work has enabled us to conclude that the oil content, fatty acid composition and oil stability could get alter in positive direction through the application of physical (Gamma rays) and chemical (EMS) mutagen. Pertaining to the oil quantity 20 kR and 10 kR doses of gamma rays is more effective in variety AK-159 and TAG-24 respectively. While in improving oil quality the 0.15% concentration of EMS and 0.10% concentration of EMS are more potent in variety AK-159 and TAG-24 respectively. In conclusion, Induced mutation can enhance the oil yield, nutritional value and stability of groundnut (Arachis hypogaea L.)