Characterization of maize population for pro-vitamin A carotenoids using TLC

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1 [Vol. 13 (2), May - August 2015] International Journal of Basic and Applied Agricultural Research 181 Characterization of maize population for pro-vitamin A carotenoids using TLC MAHAK TUFCHI 1, RASHMI 2, N. K. SINGH 2 and ANIL KUMAR 1 1 Department of Molecular Biology and Genetic Engineering, College of Basic Sciences and Humanities 2 Department of Genetics and Plant Breeding, College of Agriculture, G. B. Pant University of Agriculture and Technology, Pantnagar (U.S Nagar, Uttarakhand) ABSTRACT: In the present investigation the pro-vitamin A carotenoids in a population of 85 individuals derived from biparental crossing using thin layer chromatography (TLC) was determined. Based on the analysis of chromatogram, 54 lines were characterised to be high whereas 31 lines were relatively low in pro-vitamin A carotenoids. In general, both the group categorised as relatively high and low pro-vitamin A carotenoids were consisted of lines with high, medium and low total carotenoids and thereby indicating no definite pattern between relative pro-vitamin A carotenoid and total carotenoids. However, three recombinant promising lines namely BC 2 :60; BC 2 :62 and BC 2 :66 identified with high relative pro-vitamin A carotenoid and total carotenoids may be useful in maize biofortification programme. Key words: Biofortification, maize, Pro-vitamin A, thin layer chromatography. Micronutrient enriched maize (Zea mays. L) seems to complement significantly in minimizing micronutrient malnutrition due to its cosmopolitan availability and cheap price as compared to other cereals. Hidden hunger, a popular phrase to denote micronutrient malnutrition, has become one of the alarming problems afflicting an estimated three billion people around the world (UNSCN, 2004). In India, 230 million people were reported to be undernourished, accounting for more than 27% of the world s undernourished population (Lodha et al., 2005). To feed the weaning children and malnourished section of the society, there is need to estimate and enrich quality and quantity of important nutrient components consumed in the daily diet. Vitamin A deficiency (VAD) causing blindness, weakened immune systems and even death is a threatening problem in children across the globe. Incorporating carotenoid rich diet can minimize VAD. Plant based naturally occurring carotenoids have been the major source of vitamin A in the human diet particularly in the developing countries, where access to animal food is restricted due to poor economic conditions (Sivaranjani et al., 2013). A number of methods such as food fortification and supplementation have been tried all over the world for alleviating micronutrient deficiencies. However, these measures in general have been either partly or largely unsuccessful for several reasons. Biofortification, a breeding approach to enhance the concentration of essential minerals and vitamins in crops, seems to be a rational approach to ensure the availability of biofortified food through dietary intake. Among the three major cereals, maize is considered to be the most potential candidate for biofortification of vitamin A since it is having carotenoids in its kernel. In addition to being a precursor of vitamin A, carotenoids also act as antioxidant thereby minimising the ill effect of reactive oxygen species. Maize provides over 20% of total calories in human diets in 21 countries, and over 30% in 12 countries that are home to a total of more than 310 million people (Shiferaw et al., 2011). White kernel maize lacks ß-carotene in the endosperm due to the presence of yellow1 (y1) recessive allele in homozygous conditions (Buckner et al., 1990). The dominant version of this allele, Y1 produces enough ß-carotene and makes the kernel yellow in colour. Although yellow maize is rich in ß carotene concentration as compared to other cereals, the concentration is below the prescribed daily requirement by human. However, wide variability for carotenoids present in maize germplasm (Weber et al., 1987; Wong et al., 2004; Hulshof et al., 2007, Harjes et al., -2008; Safawo et al., 2010; Mishra and Singh, 2010; Das and Singh, 2012; and Chandler et al., 2013) may help in breeding maize for higher carotenoids. In general, yellow coloured kernel contain more pro-vitamin A carotenoids and low total carotenoid whereas orange coloured kernel had more amount of total carotenoid, but most of the higher total carotenoids concentration results from non pro-vitamin

2 182 International Journal of Basic and Applied Agricultural Research [Vol. 13 (2), May - August 2015] A carotenoids (Weber, 1987). Even though considerable variation exists for total carotenoids levels in maize kernels, Harjes et al. (2008) showed that the majority of yellow kernel maize inbred lines from a global collection do not have enough pro-vitamin A (average of 1.7 ìg/g ß- carotene). It is therefore necessary to develop lines with high pro-vitamin A carotenoids so that biofortified maize hybrids could be developed to minimize VAD through maize based dietary approach. The present investigation was therefore planned to screen maize population consisted of 85 recombinant lines for pro-vitamin A carotenoid and to identify promising lines for maize biofortification programme. MATERIALS AND METHODS The 85 recombinant lines (BC 2 :1-85) derived from cross between Pant10k1375 (P1-JP25W ) and CML161(P2), followed by two generation of backcrossing with Pant10k1375 and two generation of selfing, were used to analyse relative pro-vitamin A carotenoids. Both parental lines along with CM400, a white kernel colour inbred line, were also included in the study. Colour categorization was done according to the colour Fan 1 of The Royal Horticultural Society, London (Anonymous. 2001). Thirty seeds of each of the 85 samples were ground to a very fine powder using grinding mill. 0.5 g fine flour was taken in 50 ml polypropylene tube and 6 ml EtOH: BHT (in which 6 mg of BHT is dissolved in 6 ml ethanol) was added to every sample and the samples were mixed by vortexing. The samples were incubated at 85 C in a water bath for 6 min, and were mixed by vortexing after 3 min for 10 seconds. 120 µl KOH (1g/ml H 2 O) prepared freshly was added and vortexed thoroughly for 20 seconds. The samples were again incubated for 5 minutes at 85 C, vortexed for 10 seconds and incubated another 5 minutes at 85 C. The samples were cooled down on ice and 4ml H 2 O with 3 ml PE: DE (2+1, v/ v) were added. Vortexed the samples and further centrifuged for 10 minutes at 1400 x g for phase separation. The distinct upper yellow phase was transferred to a fresh 50 ml tube. Phase separation step was repeated twice to ensure complete extraction of carotenoids. A total amount of 8-9 ml of extract was added with equal volume of PE: DE (2+1, v, v) and mixed the samples. 2 ml aliquot from each sample were dried and resuspended in 50 µl chloroform. Samples were immediately loaded with micropipette on to the activated (60 0 C for 1h) silica thin layer chromatography (TLC) plate. Sample loaded TLC plate was run in PE+ DE+ acetone ( v, v/v) for only 4-5 cm (about 5 minutes) as described in Rocheford s Lab protocol (Schaub et al., 2004). Chromatographic patterns so obtained were documented using digital camera for further analysis. RESULTS AND DISCUSSION Thin-layer chromatography is an extremely valuable analytical technique used for rapid separation of compounds, and thereby gives an indication of the number and nature of the components of a mixture. Banding pattern of the chromatographic plate reveals the relative amount of pro-vitamin A carotenoids and non pro-vitamin A carotenoids in total carotenoids of maize kernel. Carotenes including α, β and γ carotenes showed highest mobility i.e. minimum Rf (retention factor) value with band at the top whereas mono-hydroxy compounds mainly cryptoxanthins showed a relatively lower mobility on the silica coated plate and acquired an intermediate position. The third group of compounds having 2-OH groups mainly lutein and zeaxanthin moved slowest with maximum Rf value (Fig 1). The upper two bands are related to pro-vitamin A carotenoids whereas the lowest band is related to non pro-vitamin A carotenoids (Schaub et al., 2004). Banding thickness and ratio of upper two bands Carotenes Mono-hydroxyl Di -hydroxyl BC 2-59 BC 2 :67 BC 2 :60 BC 2 :73 Pant10k1375 BC 2 :77 BC 2 :69 BC 2 :66 BC 2 :63 BC 2 :61 BC 2 :64 CML 161 Fig 1: Chromatogram showing banding patterns of total carotenoids

3 [Vol. 13 (2), May - August 2015] International Journal of Basic and Applied Agricultural Research 183 with the lowest band of each line on the chromatogram was visually assessed and relative value of pro-vitamin A carotenoids (high or low) was determined for all the lines. A total of 54 lines showed high relative pro-vitamin A while 31 lines had low relative provitamin A of the total 85 lines analysed. Of the 54 lines with high relative provitamin A carotenoid, 3 lines had total carotenoids more than P2 while 12 lines had total carotenoid level in between the P1 and P2. The remaining 39 lines exhibited total carotenoids less than parent lower in total carotenoids (P1). On the other hand, out of 31 lines with low relative pro-vitamin A, 24 had total kernel carotenoids less than P1, 6 had total carotenoids in between the P1 and P2 whereas only one possessed total kernel carotenoids more than the parent higher in total carotenoids (P2). No banding pattern was observed in case of white kernel inbred line CM-400. Based on Colour Fan 1 of the Royal Horticultural Society, the 85 lines were grouped into different shades of yellow orange and different shades of orange. Out of the 54 lines scored to be relatively high in pro-vitamin A carotenoids, 37 had different shades of yellow orange and 17 exhibited different shades of orange (Table 2). Thus, around 69% of relatively high pro-vitamin A containing lines exhibited kernel colour of yellow orange. Further, it is interesting to note that all the yellow orange colour lines with high relative pro-vitamin A carotenoids had total carotenoids less than the parent lower in total carotenoids (P1). Harjes et al., (2008) note similar observations while analysing diverse maize germplasm. However, presence of high relative pro-vitamin A lines in both the kernel colour groups declines the strong association of kernel colour with pro-vitamin A carotenoid. Further analysis of relation between kernel colour (1-13) and relatively low (1) and high provitamin A carotenoid indicated poor correlation with very low coefficient of determination (R 2 ) (Fig. 2). The present observation therefore indicates that kernel colour cannot be used as criteria of selecting lines with high provitamin A carotenoid. Based on high provitamin A and total carotenoids, three lines namely BC2F3:60; BC2F3:62 and BC2F3:66 have been identified to be promising and can serve as potential parents in carotenoids biofortification programme in maize. Observance of three bands on thin layer chromatographic silica plate is evident from the fact that presence of the hydroxyl groups (polar groups) renders the surface of silica gel highly polar and functionality in the organic analyte interacts strongly with the surface of the gel particle and nonpolar functionality interacts only weakly. Thus, an analyte that displays multiple polar groups in position to interact with the surface of the stationary phase will interact more strongly than an analyte that displays the same polar functionality in a way that does not permit multidentate binding. As a consequence of which we observed such type of banding pattern where dihydroxy derivatives stick more firmly to the silica gel and have slower mobility with lowest band Table 2: Grouping of 85 lines based on relative pro-vitamin A, total carotenoids and kernel colour Kernel colour High Pro-Vitamin A Low Pro-Vitamin A TC * <P1 TC= (P1-P2) TC>P2 TC<P1 TC= (P1-P2) TC>P2 Yellow orange 14A 2 Yellow orange 15A 1 1 Yellow orange 17A Yellow orange 17B 1 Yellow orange 19A 1 Yellow orange 20A 3 Yellow orange 21A Yellow orange 22A 1 7 Yellow orange 23A Orange 24A Orange 25A Orange 25B 2 Orange 26A 1 *TC denotes total carotenoids; P1: Pant10k1375; P2: CML 161

4 184 International Journal of Basic and Applied Agricultural Research [Vol. 13 (2), May - August 2015] Fig. 2: Relation between kernel colour and relative provitamin A carotenoid in maize while monohydroxy derivatives giving a little higher banding pattern on the silica plate. Non polar components move much faster like β-carotene due to least affinity to polar functional groups on silica plate. Thus, TLC analysis of pro-vitamin A carotenoid is easy, fast and therefore can be used in determination of pro-vitamin A carotenoid and selection of promising progenies out of the large breeding population within the limited time period. HPLC is the most reliable technique for absolute quantification of pro-vitamin A carotenoid however it is costly and time consuming. Thus, the promising lines in terms of high pro-vitamin A identified using TLC can further be validated using HPLC for use in future biofortification programme of maize. CONCLUSION Eighty five recombinant lines of a maize population characterised for relative pro-vitamin A carotenoids using TLC and divided lines into high and low relative provitamin A carotenoids groups. Most of yellow orange kernel colour lines with high pro-vitamin A carotenoids were low in total carotenoids. However, no definite pattern of pro-vitamin A with either total carotenoids or kernel colour was observed. Three lines having high relative pro-vitamin A and total carotenoids were obtained that could be used in breeding programme for biofortification of maize. ACKNOWLEDGEMENTS Director Experiment Station, G. B. Pant University of Agriculture & Technology, Pantnagar (India) and AICRP Maize are duly acknowledged for support during the course of investigation. Indian Council of Agricultural Research (ICAR) New Delhi is duly acknowledged for awarding Ph. D. Fellowship to senior author. REFERENCES Anonymous. (2001) Fan 1 yellow, yellow orange, orange, orange red, red groups. The Royal Horticultural Society London, 1-56 Buckner B., Kelson T. L., Robertson D. S. (1990) Cloning of the y1 locus of maize, a gene involved in the biosynthesis of carotenoid. Plant Cell, 2: Chandler, K., Lipka, A. E., Owens, B. F., Li, H., Buckler, E. S., Rocheford, T., Gore, M. A. (2013) Genetic Analysis of Visually Scored Orange Kernel Color in Maize. Crop Sci., 53: Das, A.K. and Singh, N.K. (2012). Carotenoid and SSR marker-based diversity assessment among short duration maize (Zea mays L) genotypes. Maydica, 57:

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