Maydica 55 (2010): 95-100

SPECTROPHOTOMETRIC AND TLC BASED CHARACTERIZATION OF KERNEL CAROTENOIDS IN SHORT DURATION MAIZE P. Mishra, N.K. Singh* Department of Genetics and Plant Breeding, G. B. Pant University of Agriculture and Technology, Pantnagar-263145, Distt. Udhamsingh Nagar (Uttarakhand) India

Received August 10, 2009

ABSTRACT - Thirty seven lines of short duration maize comprised of 30 inbred lines and 7 improved populations were characterized for kernel carotenoids content using spectrophotometer and thin layer chromatography (TLC). Analysis of variance revealed significant differences in genotypes for carotenoids content. Carotenoids content was found to vary from minimum of 0.027 µg/g to maximum of 25.75 µg/g dry weight with overall mean of 18.11 µg/g dry weight. Banding intensities on thin layer chromatogram were visually observed and relative provitamin A and non-provitamin A carotenoids were determined for each genotype. The grain colour was found to be poor determinant of either total kernel carotenoids or relative provitamin A carotenoids. Poor relation with very low coefficient of determination (R2) was also observed between total kernel carotenoids and relative provitamin A carotenoids. Thus, the result revealed that selection based on kernel colour may not always be a true reflection for high or low carotenoids in maize. KEY WORDS: Maize; Zea mays; Kernel carotenoids; Spectrophotometric; TLC.

INTRODUCTION Maize (Zea mays L.) is one of the important cereal crops and used as staple food for human being, feed for animals and for diverse processed products across the continents. Amongst the three major cereals, only maize kernel contains coloured carotenoid compounds that can be converted into vitamin A in the human and animal body (WURTZEL, 2004). Carotenoids are natural pigments that occur widely in plants and serve several functions in plants. They are essential in photosynthesis, acting both in light collection and photo-protection. They also serve in a non-photosynthetic role by con-

* For correspondence (email: [email protected]).

tributing to flowers and fruit color as well as being part of the pathway for the biosynthesis of abscisic acid (ABA), an essential plant hormone involved in kernel dormancy. Carotenoids play an important role in human and animal nutrition, providing the major source of vitamin A and antioxidants. In addition to pro-vitamin A activity, carotenoids have also been found to involve in essential biological functions in human health, particularly in reducing the risk of degenerative diseases such as cardiovascular, cancers, cataract and macular degeneration (FRASER and BRAMLEY, 2004). Carotenoids can be grouped into two classes i.e. provitamin A and non-provitamin A carotenoids. Provitamin carotenoids serve as a dietary source of vitamin because it can release one molecule of each α-carotene and β-cryptoxanthin or two β-carotene molecules of retinal. Vitamin A deficiency (VAD) now is a global problem. However, most of the cases are in developing countries whose populations rely on single staple crop for their sustenance. Poor infrastructural development and limited access to direct vitamin supplementation in developing countries necessitates as well as highlight the significance of biofortification. Biofortification is process by which staple crops are purposely bred for higher nutritional density, to make available essential nutrient in diet of common people living in accessible or inaccessible areas having poor purchasing capacity without adding any additional cost. Maize is one of the potential crops for biofortification because of its widespread use and also because of the presence of considerable natural variability for kernel carotenoids. The dominant carotenoids in maize kernels, in decreasing order of concentration, are leutin, zeaxanthin, β-carotene, βcryptoxanthin and α-carotene. Among the diverse panel of inbred lines analysed, β-carotene level was found up to 13.6 µg/g whereas most of the yellow maize grown and consumed throughout the world

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has only 0.5 to 1.5 µg/g β-carotene (HARJES et al., 2008). Yellow kernel maize is thought to be associated with high levels of β-carotene, a source of vitamin A (BUCKNER et al., 1990) and constitute main source of provitamin A in food and feed (BRUNSON and QUACKENBUSH, 1962). However, comparisons between β-carotene and total carotenoids with grain colour (scaled according to shade of yellow) revealed poor correlations with low R2 values, which indicated that marker assisted selection (MAS) may prove much more efficient than selection based on colour alone (HARJES et al., 2008). Considering significance of carotenoids and its natural variability, the present investigation was undertaken to characterize a set of potential inbred lines and populations for carotenoids content using spectrophotometer and thin layer chromatography (TLC).

MATERIALS AND METHODS Seed materials Fifty grams seeds of 37 short duration (below 90 days maturity) maize genotypes comprised of 30 inbred lines and 7 released composites were taken from germplasm maintained by Maize Breeding group at the University. Seeds of each genotype were visually screened and uniform representative seeds were taken for grinding.

plate using micropipette. The TLC plate was run in PE+DE+Acetone (40:10:10 v/v/v) for only 4-5 cm (SCHAUB et al., 2004; Maize quick carotenoid extraction protocol http://www.cropsci.uiuc. edu/faculty/rocheford/quick_carotenoid_analysis_ protocol.pdf). Spectrophotometric observation and determination of carotenoids One ml aliquots from each sample were taken in quartz cuvette and optical density (OD) at 450 nm against petroleum ether: diethyl ether (2:1, v/v) as blank, was recorded using UV-Visual dual beam spectrophotometer. The OD value of each sample was used to determine the carotenoids concentration using LambertBeer equation (as described in Rocheford’s Lab protocol): E=εcd Where, E = extinction (Spectrophotmeter reading) ε = molar extinction coefficient c = concentration d = distance =1, to determine concentration c = E/ε Lutein, zeaxanthin and beta- carotene are the major carotenoids in maize. So an average value for ε and for the molecular mass was used. ε lutein = 122688 l/mol cm; ε zeaxanthin = 133480 l/mol cm; ε β-carotene = 134000 l/mol cm; ε average = 130056 l/molcm

M = 568 g/mol M = 568 g/mol M = 537 g/mol Maverage = 557.7 g/mol

Hence, c = OD / 130056 x 557.7 x 1000 x 10 / w (µg/g)

Grinding of maize kernel into fine flour About 20 g maize grains from each line were milled to fine flour in laboratory miller and further the maize flour were sieved through the fine muslin cloth. The sieved fine flour of each genotype was packed in water proof butter paper bag and kept at 4 °C before the processing for extraction of carotenoids.

Where,

Extraction of total carotenoids 0.5 g of fine powder was taken in Falcon tube and 6 ml of EtOH:BHT was added to each sample and mixed by vortexing. Samples were incubated at 85 °C in water bath for 5 minutes and were mixed again after 3 minutes by vortexing. Freshly prepared 120 µl KOH (1g KOH/ml H2O) was added to each sample. Saponification was done by incubating samples for 5 minutes at 85 °C in water bath, vortexed for 10 seconds and further incubated at 85 °C for 5 minutes. Samples were cooled down on ice and 4 ml of H2O was added. Further 3 ml of Petroleum Ether (PE):Diethyl Ether (DE) (2:1, v/v) was added to each samples. Samples were mixed by vortexing and thereafter centrifuged at 1400 g for 10 minutes. Upper phase (supernatant) of the mixture was pipette out and transferred to fresh Falcon tube. Mixing of 3 ml of PD:PE (2:1, v/v) and centrifugation steps were repeated thrice. Finally, approximately 8 ml of extract was recovered. Equal volume of PE:UDE (2:1, v/v) was added to each samples and mixed by inverting the tube. Optical density (OD) was recorded immediately at 450 nm using dual bean spectrophotmeter against the PD:PE (2:1, v/v) as blank. Kernel carotenoids were quantified using Lambert-Beer equation. For TLC, 2 ml aliquot were air dried, resuspended it in 50 µl chloroform and loaded it to a silica TLC

Each sample was analysed three times and the data were subjected to statistical treatment in completely randomized design (SNEDECOR and COCHRAN, 1989).

c = concentration of total carotenoids content (µg/g) in a given sample on dry weight basis OD = optical density taken at 450 nm wavelength using spectrophotometer W = wt of sample (0.5 g).

Thin layer chromatography (TLC) Thin layer chromatography (TLC) was performed for the separation of different components of kernel carotenoids. For this purpose, Silica TLC plates were prepared by applying silica gel G (with binder) paste on glass plates with the help of applicator making 1.5 mm thick layer. Silica gel TLC plates were activated by incubating at 110 °C for 2 h prior to loading of samples. Two ml of aliquot (extracted using Maize Quick Carotenoids Extraction Protocol described earlier) was taken, air dried and re-suspended into 50 µl chloroform and immediately loaded to silica TLC plate using micro-pipette. TLC plates were run in solvent mixture of Petroleum Ether + Diethyl Ether + Acetone (40+10+10 v/v/v) for about 15 minutes when solvent cover the 6-7 cm of the silica gel. Each chromatogram was analyzed visually for banding pattern. The thickness of upper two bands in relation to the lowest band was determined visually and the relative value of provitamin A and non-provitamin A carotenoids were determined in each sample.

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TABLE 1- Analysis of variance for carotenoids content in maize. ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– Source of variance Degree of freedom Sum of squares Mean square F- value ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– Genotype 36 3486.77 96.85 45.03** Error 74 159.15 2.15 ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– Total 110 3645.92 ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– ** Significant at 1% probability level.

Association among kernel colour, total carotenoids and relative provitamin A carotenoids The kernel colour of each line was recorded visually. The 37 lines were grouped according to kernel colour such as white (0), dark yellow (1), yellow (2), light yellow (3), orange (4) and light orange (5). The regression analysis of visual kernel colour, relative provitamin A carotenoids and total carotenoids content along with coefficient of determination (R2) was also performed (SNEDECOR and COCHRAN, 1989) to study the pair wise association of these parameters.

RESULTS AND DISCUSSION The data on kernel carotenoids were analyzed in completely randomized design (CRD) to test the statistical validity of variance among different genotypes for maize kernel carotenoids content. Analysis of variance revealed significant differences for total carotenoids content which indicated that 37 lines chosen for the investigation were genetically different for total kernel carotenoids content (Table 1). The quantitative estimation revealed wide range of carotenoids content among 37 genotypes (Table 2). The mean value for kernel carotenoids content was found to vary from minimum of 0.027 µg/g dry weight in inbred line CM-300 to the maximum of 25.75 µg/g dry weight in CM-125 with population mean of 18.11 µg/g. When those lines which possessed yellow or different shades of yellow colour kernel were only considered, the range of kernel carotenoids content was found to vary from minimum of 8.72 µg/g in composite variety ‘Tarun’ to maximum of 25.75 µg/g dry weight in CM-125 with population mean of 18.61 µg/g. Among the inbred lines, CM-300, a white kernel maize exhibited minimum amount of total carotenoids (0.027 µg/g) whereas CM-125 possessed highest amount of carotenoids (25.75 µg/g). The inbred lines CM-125 followed by YHPA-3, Pop31-7, YHPA-4, CM-139-3, Pop31-6, LPP-1 and YHPA-2 were among the lines which possessed more than

23.0 µg/g of carotenoids on dry matter basis. The mean carotenoids content of all the inbred lines was 19.02 µg/g whereas 19.68 µg/g of carotenoids was observed when white kernel inbred line CM-300 was excluded. Among the composite, the variety ‘Amar’ was found to have highest amount of carotenoids (21.38 µg/g) followed by ‘D-131’ (19.28 µg/g) whereas the variety ‘Tarun’ exhibited minimum of 8.72 µg/g of carotenoids which was statistically similar to the second lowest carotenoids containing line ‘Kanchan’ (8.90 µg/g). The average carotenoids content in all the composite varieties was 14.24 µg/g. HARJES et al. (2008) reported wide range of variability for carotenoids (5.5 µg/g to 66.0 µg/g) with mean value of yellow lines (23 µg/g). BERARDO et al. (2009) analyzed 1245 samples of maize and reported range of carotenoids from 1.09 µg/g to 61.10 µg/g dry weight. EGESEL et al. (2003) and CHANDER et al. (2008) found average total carotenoids of 31.9 µg/g and 10.298 µg/g in maize, respectively. Thin layer chromatography analysis (TLC) was performed to separate the different constituents of total carotenoids (Fig 1). TLC is basically deployed for visual screening and assessing relative value of provitamin A carotenoids and non-provitamin A carotenoids in total carotenoids of maize kernel. Silica gel thin layer plates with the solvent system separate total kernel carotenoids according to the number of their functional (-OH) groups in the following pattern of separation: • Carotenes, such as β-carotene have no retention and migrate with the solvent front. • Monohydroxylated compounds (cryptoxanthins) migrate at an intermediate distance. • Dihyroxylated compounds (lutein and zeaxanthin) remain close to the origin. The upper two bands are related to provitamin A carotenoids whereas the lowest band is related to non-provitamin A carotenoids (SCHAUB et al., 2004). Thickness of upper two bands in relation to the

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FIGURE 1- Chromatograms showing banding patterns of total carotenoids (Numbering is not as per Table 2).

lowest band give an idea about the relative content of provitamin A and non-provitamin A content. Banding thickness and ratio of upper two bands with the lowest band of each line on the chromatogram was visually assessed and relative value of provitamin A carotenoids (high or low) was determined for all the lines (Fig. 1). Thin layer chromatogram revealed that 20 lines namely YHPA-2, Pop 31-2, YHPA-3, CML 139-2, YHPP-9, LPP-1, YHPP-5, D131, YHPA-1, Amar, D765, Kanchan, Tarun, Pop31-4, Surya, YHPP-6, Pop446-1, YHPP-7, Pop31-1 and YHPP-3 possessed relatively high proportion of pro-vitamin A carotenoids while the remaining 16 lines namely CM-125, Pop31-3, Pop31-7, Pob446-3, YHPP-10, YHPA-4, Pop31-5, YHPP-4, Pop31-6, Pob446-2, CM139-1, CM139-3, YHPP-8, YHPP-1, YHPP-2 and Pragati had relatively high proportion of non-provitamin A carotenoids. No banding pattern was observed in case white kernel in bred line CM-300. Maize kernel colour of each line was recorded and broadly grouped into Dark yellow, yellow, light yellow, orange, light orange and white colour to assess relation between kernel colour and carotenoids content (Table 2). Eight lines with dark yellow kernel colour had carotenoids content from 10.64 to 25.75 µg/g whereas 11 lines with yellow kernel colour varied in carotenoids content from 8.72 to 25.69 µg/g. The 14 lines with light yellow kernel

colour had carotenoids content varied from 9.23 to 23.96 µg/g. Two lines with orange kernel colour had carotenoids content of 17.84 and 25.59 µg/g where as one line with light orange kernel colour had 24.33 µg/g carotenoids. The kernel colour score generated from 0 to 5 were regressed over to carotenoids content. The regression analysis revealed non-significant relation between kernel colour and carotenoids content. The coefficient of determination of kernel colour on carotenoids content was also very low (R2 = 0.002; Fig. 2). The results of the present investigation, therefore, indicated that kernel colour did not have any strong correlation with total kernel carotenoids content. HARJES et al. (2008) also reported poor coefficient of determination of total carotenoids with kernel colour (scaled according to shade of yellow). Different kernel colours recorded on 37 genotypes were also analysed in relation to TLC based relative provitamin A content (Table 2). Maize kernel with different shades of yellow consisted of lines both with relatively high as well as relatively low provitamin A carotenoids whereas three lines of orange/light orange colour had relatively low provitamin A carotenoids in total kernel carotenoids. Thus, we did not observe any impact of kernel colour on relative provitamin A carotenoids in our study. HARJES et al. (2008) also observed poor association of these two parameters.

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TABLE 2 - Total carotenoids content, kernel colour and relative pro-vitamin A content in maize. ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– Carotenoids Relative Carotenoids Relative Kernel colour Genotypes content (µg/g) pro-vitamin A Kernel colour Genotypes content (µg/g) pro-vitamin A dry weight content dry weight content ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– Dark yellow CM-125 25.75 Low Light yellow Pop31-6 23.96 Low YHPA-2 23.47 High YHPA-1 22.59 High Pop31-3 19.84 Low Amar 21.38 High Pob446-3 19.07 Low Pob446-2 19.53 Low D-765 13.04 High Surya 17.73 High Pragati 10.64 Low YHPP-6 16.90 High Pop31-2 18.84 High YHPP-1 14.78 Low YHPP-10 18.69 Low YHPP-2 14.63 Low Yellow YHPA-3 25.69 High Pob446-1 14.21 High YHPA-4 24.36 Low YHPP-7 13.75 High Pop31-5 22.72 Low Pop31-1 09.23 High CM-139-2 21.01 High YHPP-3 15.75 High YHPP-9 19.40 High Orange Pop31-7 25.59 Low LPP-1 23.70 High CM-139-1 17.84 Low YHPP-5 22.21 High Light orange CM-139-3 24.33 Low Kanchan 08.90 High White CM-300 0.027 Nil Tarun 08.72 High CD at 1% 03.16 YHPP-4 18.78 Low CD at 5% 02.38 Pop31-4 18.04 High CV (%) 8.09% Light yellow D-131 19.28 High YHPP-8 15.69 Low –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––

FIGURE 2 - Relation of kernel colour and total carotenoids in maize (0- white, 1- Dark yellow, 2- Yellow, 3- light yellow, 4- Orange, 5- Light orange).

FIGURE 3- Relation between TLC based relative provitamin A carotenoids (1- high, 2-low) with total kernel carotenoids in maize.

Investigation was also made to establish relation between total carotenoids content with TLC based relative provitamin A carotenoids. Ten lines, out of the 37 studied, had high total carotenoids (>average)

as well as relatively high provitamin A carotenoids whereas 5 lines possessed both low carotenoids (