Experiment conducted
The experiment was conducted in the year 2021-August, in the R and D laboratory of Nuzuveedu Seeds Private Limited, Hyderabad.
Extraction of total carotenoids
Total carotenoid was estimated from the individual genotypes using the standard protocol
(Menkir et al., 2008). After kernel maturation, ears with the husk were manually harvested and were dried under the shade till 14% moisture was obtained. 50 kernels from each genotype were randomly sampled. Sample was ground and 0.5 g of each triplicate per sample was collected. Each step prior to and during biochemical analysis was carried out under the dim yellow light to prevent the photo-oxidation of carotenoid.
0.5 g of fine powder was taken and 6 ml of EtOH:BHT was added to each sample and mixed by vortex mixer. Samples were incubated at 85°C in water bath for 5 min and were mixed again after 3 min by vortex. Freshly prepared 120mL KOH (1g KOH/ml of water) was added to each sample. Saponification was done by incubating samples for 5 min at 85°C in water bath, vortexed for 10 sec and further incubated at 85°C for 5 min. Samples were cooled down on ice packs and 4 mL of water was added. Further 3 mL of petroleum ether (PE):diethyl ether (DE) (2:1 v/v) was added to each sample. Samples were mixed by vortex and thereafter centrifuged at 3600 rpm for 10 min. Upper phase (supernatant) of the mixture was pipetted out and transferred to fresh 50 ml centrifuge tube. Mixing of 3 ml of PE:DE (2:1 v/v) and centrifugation steps were repeated thrice. Approximately, 8 ml of extract was recovered in the final step to which equal volume of PE:DE (2:1 v/v) was added to each sample and mixed by inverting the tube. The absorbance of total carotenoid was measured at 450 nm using UV Vis Spectrophotometer (Elico SL 218) by taking PE:DE (2:1, v/v) as blank
and the total carotenoid concentration was calculated based on the formulae as suggested by Harvest plus equation (
Rodriguez-Amaya and Kimura, 2004).
Sample preparation for the estimation of individual carotenoids
The seeds were cleaned and shade dried. The grains from ears of same families were equally shelled and bulked together
and were stored in deep freezer (-20°C) for further biochemical analysis. Extraction and quantification of b-carotene (BC), β-cryptoxanthin (BCX), lutein (LUT)
and zeaxanthin (ZEA), from kernels was done by utilizing standard procedure
(Vignesh et al., 2015; Kurilich and Juvik, 1999). Under dark conditions, samples of maize seeds were powdered and used for further analysis. Carotenoids from maize samples were extracted by taking 1 g of powder to which 6 mL of ethanol with 0.1% b - hydroxytoulene (BHT) (w/v) was added
and kept in water bath at 85°C for 5 min with intermittent vortex. To the above hot mixture, 120 mL of potassium hydroxide (KOH) was mixed to saponify the potentially interfering oils. Samples were vortexed and kept in water bath (85°C) for 10 min with intermittent vortex.
After saponification process, samples were kept in the ultra-low freezer and 3 mL of chilled deionized water was added to the samples. 3 mL of petroleum ether (PE): diethyl ether (DE) (2:1 v/v) was added for carotenoids extraction. Samples were centrifuged at 2700 rpm for 10 min. Supernatant was transferred to the new 50 mL of centrifuge tubes and the above steps were repeated thrice for each sample. The organic layers were dried in rotary vacuum evaporator it was dissolved in the tert-butyl-methyl ether
(Vignesh et al., 2015).
Quantitative analysis of BC and BCX using HPLC
BC and BCX were quantified using Waters and Alliance W2690/5 model HPLC system incorporated with Waters Empoer 3 software with W2998 photodiode array detector. The column used was YMC C30 column (5 mm, 4.6×250 mm). The standards of lutein, zeaxanthin, β-carotene, β-cryptoxanthin were sourced from Sigma Aldrich, Mumbai
(Zunjare et al., 2017). The mobile phase used was methanol: TBME (tert-butyl methyl ether) (80:20 v/v)
and 1 mL/min was the flow rate. Standards were purchased from Sigma Aldrich, India and dissolved in acetone and desired dilutions were made. The concentrations of BC and BCX in each sample was detected by standard regression. To improve the detection of BC and BCX, the absorbance was recorded at 450 nm and the chromatogram obtained was compared with the standards. The pro-A concentration was estimated as sum of BC and half the BCX concentration while non-pro-A estimated as sum of LUT and ZEA concentration
(Babu et al., 2013).
Screening of germplasm for the of β–carotene favourable alleles
Maize germplasm from the Nuziveedu seeds Limited, Hyderabad was screened for the b– carotene donors which delivers the superior/favorable genes. Similarly, recurrent parents of high zeaxanthin were also screened and identified. 8 lines of high lutein, high zeaxanthin, β-cryptoxanthin and β-carotene were analyzed. Lutein and zeaxanthin are the 2 compounds which add up to the total carotenoid content as they are abundant in endosperm. Accumulation of β-cryptoxanthin and β-carotene is rare, these compounds get converted to zeaxanthin
and downstream products towards ABA.
Identifying maize lines with accumulation of β-carotene is a critical factor, with corresponding dominant alleles as they would serve as donors, introgressing superior carotenoid alleles, for controlling the accumulation of β-carotene into high yielding maize elite germplasm lines. As orange color is highly preferred by farmers, most of the germplasm selected were of orange color,
i.e., high in zeaxanthin and only very few are yellow in color.
Among those yellow lines, few lines are of enhanced β -carotene, but they are not the best parents in the hybrid crossing program. So, in an obvious sense, the enhanced β-carotene lines were used as donors in crossing program. The orange germplasm high in zeaxanthin was selected as recurrent parents
and if they are introgressed with β-carotene accumulating alleles, the lines should accumulate β-carotene.
Molecular markers for foreground marker-assisted selection of β-carotene accumulating alleles from donors
PCR assays on both the donors and recurrent parents were performed for target polymorphisms, such as 5'TE, 3'TE and DEL4 present on the genes ε-LCY and β-HYD respectively. PCR products thus amplified were loaded on agarose gels. ε-LCY had 2 polymorphisms such as 5'TE and 3'TE. There were two classes of 5'TE (2 and 3), of which class 2 was favorable
(Harjes et al., 2008). An insertion of 8 bp brings a polymorphism of LCY 3'TE. Both the polymorphisms favor an increase in the ratio of zeaxanthin over lutein. However, in recent studies, it was indicated LCYE is not as effective as the b-carotene hydroxylase and it was very obvious, because it had no direct effect on β–carotene accumulation, whereas β-HYD is directly involved in controlling the flux between the conversion of β–Carotene and zeaxanthin. In β-HYD, there were 3 polymorphisms, with 5'TE being most effective
(Yan et al., 2010). 5'TE holds two classes, of which class 2 was favorable. Among 3'TE type class 1 with 543 bp was favorable, followed by DEL4 with 12 bp insertion. These markers were used to perform marker assisted foreground selection.