Screening of Zea mays L. Germplasm for the High Carotenoid Endosperm using Biochemical and Molecular Approaches

Retinol is a key essential vitamin required by the humans which cannot be synthesized and needed to be given as a food supplement as provitamin A carotenoids which include β-carotene, α-carotene and β-cryptoxanthin which are the key precursors for the retinol biosynthesis. One of the best methods to improve the retinol content in the food is to breed the plant varieties for high β-carotene (Vignesh et al., 2015). In this scenario, the best suitable crops for the enrichment of β-carotene are maize.
       
Zea mays L. is the third largest cultivated crop in the world after wheat and paddy and is termed as “Queen of all the cereals” (Sukumar et al., 2023). It is a staple meal for millions of people and it is grown in an area of 168 million hectares with a total output of 984 million tons with an average output of 3.41 tons/ha. In India, maize is cultivated across 9.4 million hectares in 2013-14 with a total yield of 16.8 million metric tons and an average output is 2.5 tons/ha. The major compounds in the maize grains are β-carotene, β-cryptoxanthin, zeaxanthin and lutein. Among which b-carotene has the major provitamin A content and is present in very low concentrations in maize grains. So, screening of germplasm for high β-carotene maize inbred varieties and transferring the high β-carotene alleles from superior lines to the adapted cultivars through introgression breeding are much encouraged (Harjes et al., 2008).
       
Maize kernels have different colors like white, yellow and orange which depends on the presence or absence of carotenoids. Yellow and orange varieties of maize contain carotenoids in its endosperm whereas the white variety lacks the carotenoid. Recent studies have also led to the elucidation of the carotenoid biosynthetic pathway in plants and identification of candidate genes and favorable alleles that influence the synthesis of carotenoids (Babu et al., 2013). Among several genes influencing the carotenoid biosynthesis, three genes viz., Phytoene syntheses 1(Y1 or Psy1), lycopene epsilon cyclase (lcyE) and b-carotene hydroxylase 1 (crtRB1) play a crucial role in the final accumulation of carotenoids in the endosperm (Yan et al., 2010).
       
The present study aims in studying the β-carotene content in the maize lines and identification of β-carotene alleles.

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 blankand 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 togetherand 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 addedand 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 zeaxanthinand 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 parentsand 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.

Extraction of total carotenoids
 
The total carotenoids from the maize cultivars were assessed. A total of 8 maize lines were assessed for total carotenoids content. Among all the cultivars, 5732 cultivar showed 24.47 mg/g followed by 3003 cultivar (20.41 mg/g), whereas low carotenoid content was recorded in 3896 cultivar (7.72 mg/g) Fig 1.
 

 
Quantitative analysis of BC and BCX using HPLC
 
The in-house maize lines were tested for the presence of BC and BCX using HPLC and the components were quantified. Along with the BC and BCX, zeaxanthin and lutein were also quantified. Among all the maize lines tested, high lutein and zeaxanthin were recorded in 5732 i.e., 7.548 mg/g and 3.21 mg/g. Similarly, high β-cryptoxanthin and β-carotene were recorded in 5732 with 5.691  mg/g and 8.455  mg/g Fig 2. The chromatogram of 5732 showed the peak for β-carotene and had secreted high β-carotene content when compared to the maize lines Fig 3.
 

 

       
Most of the maize lines showed very less β-carotene content when compared to the 5732-maize line. When total carotenoid content was estimated, 5732 showed high total carotenoid content with 24.925 mg/g followed by 3003 with 19.325 mg/g Fig 4. The results of total carotenoid content obtained in the extraction process matches with the HPLC results. β-carotene is the key component which acts as a precursor for provitamin A. The HPLC analysis shows that the 5732 cultivar has high content of β-carotene and β-cryptoxanthin which are the core precursors in the retinol biosynthesis. Comparatively, the 5732-cultivar showed high synthesis of β-carotene and β-cryptoxanthin when compared to the other cultivars.
 

 
Screening of germplasm for the of b–carotene favourable alleles
 
Screening of germplasm was carried out for the high β–carotene maize lines which has β–carotene favorable genes. Screening process was carried out using SSR markers and percentage of polymorphism observed. Among all the chromosomes (CHR) tested, CHR-5 showed high percentage of polymorphism with 62% when tested using 71 markers, overall 670 markers we tested entire chromosome-1 (Chr) to chromosome-10, we observed average of 44% polymorphism between elite line (3001) and carotenoid donor line (5732), Table 1.
 

 
Molecular markers for foreground marker-assisted selection of b-carotene accumulating alleles from donors
 
Marker-assisted selection of b-carotene accumulating alleles from donors was carried using ε-LCY gene and β-HYD 3. Molecular markers used for the introgressing were ε-LCY gene along with β-HYD 3. The test showed high favorable alleles in class D i.e., 1 followed by class B (8 favorable alleles) (Table 2 and Fig 5).
 

 

       
Carotenoids play a key role in the metabolic activities of humans, further improving the growth and development in the humans. For the Vit A biosynthesis, β-carotene, β-carotene and β-cryptoxanthin act as precursor molecules. Lutein and zeaxanthin act as free radical scavengers which can be taken as an antioxidant dietary supplement to reduce the risk of degenerative diseases (Vignesh et al., 2015).  In order to increase the yield of b-carotene in the maize cultivars the inbreeding techniques are the best suitable methods. In the present study, total 8 maize lines were selected and the total carotenoid content was tested using the conventional method and also by using HPLC. Among all the maize lines, 5732 cultivar showed 24.47 mg/g when compared to the other maize cultivars. The obtained results are in correlation with the previous results obtained in the Hungarian maize lines which has the total kernel carotenoid in between 12.20-30.10 mg/g (Anshuman et al., 2012). The low total carotenoid in the maize lines show that the cultivars ack the carotenoid favorable alleles. Similarly, the results quantified using the HPLC showed lutein 7.548 mg/g, zeaxanthin 3.21 mg/g, β-cryptoxanthin 5.691 mg/g and b- carotene 8.455 mg/g in 5732 maize cultivar. Among all the 8 maize lines studied for the total carotenoid content, 5732 was selected as high carotenoid containing maize cultivar which indirectly reciprocates the presence of β-carotene favorable alleles. Similar results were obtained in the previous research studies which showed the zeaxanthin 10.22 mg/g, β-cryptoxanthin 1.93 mg/g and β- carotene 2.12 mg/g. The average range of β-carotene is 0-4.58 mg/g (Changan et al., 2017). Similarly, yellow maize lines showed total carotenoid 14 µg/g and 1.69 mg/g for β-carotene (Tura et al., 2012). In the present study the 5732 maize line showed 5.455 mg/g of β- carotene which was much higher than the required level. There are few research reports outlining the genetics of carotenoids in yellow maize, despite the fact that natural genetic diversity in carotenoids has been discovered in yellow maize lines and hybrid types in the temperate zone and in the tropics. Few research reports also indicated the inheritance of gene action in maize (Alhassan et al., 2016; Karuganti et al., 2013).
       
The maize lines were further screened for the β-carotene favorable genes. Initial screening was carried out using SSR markers and the polymorphism was observed. Among all the chromosomes, CHR-5 showed high percentage of polymorphism with 44%. In the current study, ε-LCY gene and β-HYD 3 were used for screening the best carotenoid maize elite line. Research reports showed that the polymorphism at the ε-LCY loci will increase the β- carotene in the maize (Alhassan et al., 2016). Results obtained in the present study showed that the 5732-cultivar showed high similarity for both ε-LCY gene and β-HYD 3 genes which shows its ability to secrete high β- carotene content. The maize line 5732 was further used for the inbreeding techniques.

The present research was carried out to screen best b- carotene maize line. The results obtained were encouraging and the 5732 was screened as the best β-carotene secreting maize line which was characterized by using biochemical and molecular analysis. The 5732 maize line is promising and can be used for the inbreeding.

I express my sincere gratitude to the Science and Engineering Research Board (SERB) and the Confederation of Indian Industry (CII) for providing me with the Doctoral scholarship under the PM fellowship scheme. Additionally, I extend my heartfelt appreciation to the Department of Biotechnology, Nuziveedu Seeds Limited for their invaluable assistance and technical support in facilitating my research study at their location.

Authors have no conflict of interests.