The percentage of RPGR was calculated following the procedure of Sundaram et al. The seeds obtained from the recurrent and donor parents and the improved lines were subjected to β-carotene estimation.

Estimation of β-carotene was carried out by adopting the Harvest plus protocol The carotenoid compounds were extracted by grinding the seed samples using ice-cold acetone until a fine powder was obtained. Since the carotenoid compounds undergo photo-oxidation, further processing was done under yellow light 19 , The samples extracted were concentrated in a rotary evaporator at 45°C and made up to 2 ml using methanol prior to separation.

The β-carotene concentration was quantified by high-performance liquid chromatography HPLC. Samples were eluted by C18G A column × 4. Sigma Aldrich, India reconstituted in methanol to five different concentrations 0.

The β-carotene concentration was identified by their characteristic spectra and comparison of their retention times with known standard solutions. Four improved lines from UMI × HP and two improved lines from UMI × HP were crossed during Kharif to generate the five hybrids.

These hybrids were evaluated along with the commercial hybrid NK and CO6 during the Rabi and Kharif at the Experimental Farm, Agricultural Research Station, Vaigai dam, Tamil Nadu Agricultural University, India and Maize Research Station, Tamil Nadu Agricultural University, Vagarai, India under comparative yield trial CYT.

Further, one superior hybrid identified from CYT was tested in multi-location trials MLT during Kharif and in diverse maize growing environments of Tamil Nadu along with eight maize hybrids including commercial hybrids. Kharif included four environments viz.

Hybrids [namely NK G1 , CO 6 G2 , ACM-M G3 , ACM-M G4 , CMH G5 , VaMH G6 , VaMH G7 , M GOLD G8 , and CMH G9 ] were used in MLT. All the field trials were conducted in the irrigated condition. The spacing followed was 60 × 25 cm with a plot size of 5 × 3. Trials were carried out using a randomized complete block design RCBD with two replications.

Grain yield data were subjected to a combined analysis of variance ANOVA using GEA-R statistical software Additive Main Effect and Multiplicative Interaction AMMI model 22 and Genotype and Genotype by Environmental Interaction GGE effects biplot 23 were employed to analyse the Genotype by Environment GE interaction and to assess grain yield stability based on the principal component analysis PCA.

The crtRB1 allele was introgressed into UMI and UMI using a recurrent backcrossing procedure, combined with foreground and background selection Figure 1.

Parental polymorphism screening was conducted between the recurrent and donor parents using a set of SSR markers. In the cross combinations UMI × HP and UMI × HP, among the SSR markers, and SSR markers were found to be polymorphic markers.

These polymorphic SSR markers were used for background selection and RPGR analysis. The F 1 plants were produced from crosses UMI × HP and UMI × HP After confirming heterozygosity with a crtRB1 3'TE marker, the true F 1 plants were backcrossed to their respective recurrent parent.

The percentage of RPGR of positive plants ranged from The best BC 1 F 1 plant showed a maximum RPGR of In the BC 2 F 1 population, a total of 53 and 87 plants were screened with a crtRB1 3'TE marker.

The best three BC 2 F 1 plants from each cross having maximum RPGR were further selected and selfed to produce the BC 2 F 2 population. A total of and BC 2 F 2 plants were screened with a crtRB1 3'TE marker to identify homozygous plants allele 1 Figure 2.

It revealed 34 homozygous plants in UMI × HP and 31 homozygous plants in UMI × HP The population details and their segregation pattern are presented in Table 1. The numbers of selected plants were further reduced to four and two based on the RPGR and phenotype data, and selfed to produce BC 2 F 3 lines.

UMI × HP based four BC 2 F 3 lines showed The postive plant's RPGR details are presented in Supplementary Tables 1 , 2. Figure 1. Scheme of marker assisted backcross breeding MABB for improving β-carotene concentration in parental lines of CO6 maize hybrid.

Figure 2. Segregation of allele1 and allele 3 in BC 2 F 2 generation using the crtRB1 gene specific marker i. Table 1. Segregation pattern of alleles of the crtRB1 gene in backcrossed and selfed progenies.

Figure 3. Background screening of improved lines using SSR markers. Six improved lines from both crosses were evaluated for the agro-morphological characters Figure 4 and Table 2. The overall performance showed that the traits recorded among the improved lines were on par with their recurrent parents and its phenotypic resemblance percentage ranging from Figure 4.

Morphological resemblance of the parents and the improved lines. Table 2. β-carotene concentration and agronomic performance of the improved lines developed through MABB. The β-carotene concentration was estimated in six improved lines; it showed an increased level of β-carotene ranging from 7.

The β-carotene concentration for the recurrent parents was 0. Introgression of allele1 resulted in an increased level of the kernel β-carotene concentration with a maximum of 9.

We also recorded a minimum level of the kernel β-carotene concentration 7. However, it was higher than their respective recurrent parent. The β-carotene concentration of improved lines is presented in Table 2.

In addition, the β-carotene concentration of the improved lines at different environments are summarized in Supplementary Table 3.

Five hybrid combinations were developed using the improved lines. The agronomic performance and the β-carotene concentration of the five hybrids were evaluated along with their corresponding original hybrid CO6 and a commercial hybrid NK under CYT Figure 5.

Among the five hybrids, ACM-M showed a Thus, ACM-M forwarded to MLT along with eight hybrids to study the GE interaction over the different maize growing regions in Tamil Nadu.

The yield performance of MLT showed that the hybrid ACM-M G3 recorded a value that is comparable to that of its original hybrid CO6 in all the locations. An average yield performance across 10 environments recorded 7, Figure 5. Morphological characteristics of original version and reconstituted hybrid with high β-carotene.

A CO6, B ACM-M Table 3. Agronomic performance and β-carotene value of hybrids developed through MABB under comparative yield trail CYT. Per se performance of the yield for the developed five hybrids ranged from The effect of the genotype and environment is Hence, the variation that exists among the genotypes across the environments paves the way to understanding the yield stability of the hybrids over the varying environments and specific adaption of the hybrids.

Based on the Interaction Principal Component Axis 1 IPCA1 of the AMMI model, the genotypes G1, G2, and G5 recorded values nearer to the zero on the biplot 0. Average Environment Coordination AEC recorded that G3 is placed at the second concentric circle and is ideal in terms of high yielding ability and stability, whereas other genotypes, G2, G6, and G7, placed at the third and fourth concentric circle where the stability gradient is low compared to the genotypes closer to the center axis.

Based on the vertices of the Average Environment Axix AEA , the environments E9 and E4 were considered as highly interactive environments from where the wider adaption of the genotypes are selected. Table 4. Analysis of variance of yield data of nine maize hybrids tested across environments during Kharif and Figure 6.

AMMI biplot for IPCA1 score vs. yield in nine hybrids of maize from 10 environments in the state of Tamil Nadu. G1 NK , G2 CO 6 , G3 ACM-M , G4 ACM-M , G5 CMH , G6 VaMH , G7 VaMH , G8 M GOLD, and G9 CMH E1 Coimbatore , E2 Vagarai , E3 Vridhachalam , E4 Bhavanisagar , E5 Coimbatore , E6 Vagarai , E7 Vridhachalam , E8 Bhavanisagar , E9 Vaigai dam, and E10 Athiyandal.

Figure 7. Stability analysis to evaluate the mean grain yield of nine maize hybrids. A Polygon view of GGE biplot for grouping the environments, B GGE biplot for comparing the test hybrids with ideal environment.

G1 NK , G2 CO 6 , G3 ACM-M , G4 ACM-M , G5 CMH , G6 VaMH , G7 VaMH , G8 M GOLD , G9 CMH , E1 Coimbatore , E2 Vagarai , E3 Vridhachalam , E4 Bhavanisagar , E5 Coimbatore , E6 Vagarai , E7 Vridhachalam , E8 Bhavanisagar , E9 Vaigai dam, and E10 Athiyandal.

CO6 is one of the popular maize hybrids of India, especially in Tamil Nadu. CO6 is known for its high yield, multiple disease resistances, and its suitablity for rainfed and irrigated conditions. However, CO6 is low in pVAC β-carotene, thus, the present investigation was taken up to improve the β-carotene in CO6 through MABB.

Previously, Muthusamy et al. We also used the same MABB scheme to enrich the β-carotene in CO6. Marker-assisted foreground selection using gene-linked markers permits the transfer of the gene of interest with high precision in MABB.

The advantages of using a linked marker for the selection of single or multiple genes simultaneously in MABB have been described in many studies for nutritional traits in maize 7 , 8 , The backcross and selfed-generations from both crosses, UMI × HP and UMI × HP, showed segregation distortion.

The segregation pattern of allele 1 and allele 3 deviated from the expected Mendelian ratio and and allele 1 was diminished. These results are similar to the reports of Babu et al.

There are many facts, such as embryo-specific mutation 24 , segregation distortion regions in the maize genome 25 , mutants like the defective kernel 24 , gametophytic factors 24 , 26 , and genetic background of the target allele 8 , that are reported to be the reason for the segregation distortion.

In this case, the evaluation of a large population for attaining enough foreground positives is essential. Marker-assisted background selection using SSR markers helped in selecting the foreground positive plants with a high RPGR.

We performed the background selection starting from the BC 1 F 1 generation; it hastened the RPGR in advanced lines. Following this approach, after only two generations of backcrossing, it was possible to identify the plants carrying RPGR ranging from These results are in accordance with the previous studies 9 , 27 , The study aims to introgress the crtRB1 gene without troubling the recurrent parental genome by MABB, thus the improved line will be suitable for further use.

For confirming the suitability of the new genotypes, it is essential to characterize and choose the progenies that are closer to the parent for both morphological and nutritional traits as this selection adds value to the MABB program.

Thus, we investigated the agro-morphological characters of the improved lines. It revealed that the agro-morphological characters of the improved lines were on par with the recipient parent. The agro morphological characters among the six improved lines ranged from No language restrictions were imposed in the search.

To identify eligible studies, the titles and abstracts of all articles were first screened. Then, the full articles were read to include the eligible studies.

Moreover, the references of the retrieved articles and reviews were also evaluated. Two researchers reviewed the titles, abstracts and full texts of the retrieved studies independently for relevance evaluation, and disagreements if any were resolved by discussions.

The included studies were required to meet the following criteria: 1 observational studies; 2 the associations of dietary vitamin A and beta-carotene intake with depression; 3 odds ratio OR , relative risk RR or standard mean difference SMD reported.

The exclusion criteria were listed as follows: 1 duplicated or irrelevant articles; 2 reviews, letters or case reports; 3 randomized controlled trials; and 4 non-human studies. The quality of each included study was evaluated in accordance with the Newcastle-Ottawa NOS criteria for non-randomized studies.

It contains 8 items categorized into three dimensions: 1 the selection of study groups; 2 the comparability among different groups; 3 the identification of exposure or outcome of study cohorts, respectively.

Disagreements if any were resolved through discussions until a consensus was reached. The extracted data included the first author, year of publication, location, age, sex, sample size, study design, adjustments, exposure assessment, category of exposure, effect estimates, and diagnostic criteria of depression.

lowest dietary vitamin A and carotene intake category were extracted adjusted for the maximum number of confounding variables. Moreover, the dietary vitamin A and beta-carotene intake mean ± SD was also extracted for depression vs.

control subjects to calculate the SMD. The RR for depression and SMD for dietary vitamin A and beta-carotene intake were the outcome measures in our study. The random-effects model was accepted if significant heterogeneity was observed among the studies; otherwise, the fixed effects model was utilized.

Moreover, subgroup analysis was employed for geographical region, exposure assessment, sex, population, sample size, study design, and adjustment of BMI and energy intake.

Figure 1 presents the study screening process. During the initial literature search, a total of 1, potentially relevant articles for PubMed, for Embase and for Web of Science were retrieved. After eliminating duplicated articles, 1, articles were screened according to the titles and abstracts.

Then, reviews, case reports or letters, 99 non-human studies and 97 randomized control trials studies were excluded. Figure 1. The detailed flow diagram of the study identification and selection in this meta-analysis.

The characteristics and NOS score of all the included studies are shown in Table 1. These studies were published between and Male, female and both male and female participants were recruited in 1 18 , 8 14 , 20 , 26 , 28 , 30 , 31 , 33 , 36 , and 16 13 , 15 — 17 , 19 , 21 — 25 , 27 , 29 , 32 , 34 , 35 , 37 studies, respectively.

The sample size ranged from 41 to 17, for a total number of , The exposure was assessed by food-frequency questionnaire FFQ in 16 studies 13 — 16 , 18 , 20 , 21 , 23 — 25 , 28 , 29 , 31 , 33 , 36 , 37 , and recall method in 9 studies 17 , 19 , 22 , 26 , 27 , 30 , 32 , 34 , The diagnostic criteria of depression were Diagnostic and Statistical Manual of Mental Disorders-IV DSM-IV 15 , 17 , 19 , 24 , Patient Health Questionnaire-9 PHQ-9 27 , 29 , 32 , 34 , Center for Epidemiological Studies Depression Scale CES-D 13 , 14 , 16 , 22 , 28 , 30 , Beck Depression Inventory BDI 18 , 20 , 21 , 26 , 33 , 36 , Geriatric Depression Scale GDS 23 , Clinical Interview Schedule Revised CIS-R 37 , and Depression, Anxiety, Stress Scale DASS 31 , respectively.

Table 1. Characteristics of the individual studies included in this meta-analysis. Table 2 presents the results of subgroup analysis. Figure 2. Forest plot of meta-analysis: Overall multi-variable adjusted RR of depression for the highest vs. lowest category of dietary vitamin A intake.

Table 2. Subgroup analysis of depression for the highest vs. Table 3 presents the results of subgroup analysis. Figure 3. Forest plot of meta-analysis: SMD of dietary vitamin A intake for depression vs.

control subjects. Table 3. Subgroup analysis for SMD of dietary vitamin A intake in depression vs. Table 4 presents the results of subgroup analysis.

Figure 4. lowest category of dietary beta-carotene intake. Table 4. Table 5 presents the results of subgroup analysis. Figure 5. Forest plot of meta-analysis: SMD of dietary beta-carotene intake for depression vs.

Table 5. Subgroup analysis for SMD of dietary beta-carotene in depression vs. A total of 25 observational studies were included in the present meta-analysis. The pooled analysis showed that both dietary vitamin A and beta-carotene intake was inversely associated with depression.

The negative associations of dietary vitamin A and beta-carotene intake with depression can be explained as follow. First, oxidative stress plays a significant role in the pathophysiology of depression 40 , Equipped with extended π-electron system, carotenoids stabilize unpaired electrons after radical quenching.

Second, the levels of IL-6 and TNF-α are significantly increased in depression, which impairs the expression of BDNFs and then contributes to depression Beta-carotene may lead to a reduction in levels of IL-6 and TNF-α mRNA in vivo Third, carotenoids may act through indirect pathways and cellular signaling cascades, such as nuclear factor κB NF-κB , mitogen-activated protein kinase MAPK and nuclear factor erythroid 2-related factor 2 Nrf2 45 , 46 , which are closely associated with the pathology of depression 47 — On the other hand, randomized controlled trials have indicated the potential therapeutic effect of vitamin A supplementation on depression 51 , and the dietary pattern rich in vitamin A may also exert beneficial effect on depression 52 — Taken together, current fundamental and clinical evidence is consistence with our results.

Interestingly, some of our findings are only obtained in females [the females may be more precise and reliable in the exposure assessment 55 ], it may be attributed to the potential genetic sexual differences in diet-related pathology of depression 56 , Importantly, the inverse relationship between dietary vitamin A intake and depression only exists in prospective cohort study, but not cross-sectional study.

Although the number of prospective cohort studies is rather limited only 1 , the factors that matter the dietary vitamin A and beta-carotene intake may change after depression. For instance, depressive subjects may consume less dietary vitamin A and beta-carotene due to the reduced appetite reversed causality.

Moreover, the result of subgroup analysis suggests that BMI and energy intake may also influence the overall result. Taken together, more well-designed prospective cohort studies with sexual specification are still needed.

Since vitamin A and beta-carotene are affordable and accessible nutritional factors, our findings may build an awareness with the potential collaboration between physicians and nutritionists especially in LMICs.

Nevertheless, the safety issue should also be emphasized. The main symptoms include irritability, nausea, blurry vision, vomiting, reduced appetite, hair loss, headaches, papilledema, hemorrhage, muscle pain, weakness, altered mental status, and drowsiness 60 , Therefore, a careful validation for its clinical application is still needed.

Several strengthens in our study should be emphasized. First, this is the first meta-analysis study on the associations of dietary vitamin A and beta-carotene intake with depression based on observational studies.

Moreover, our findings may encourage to build the potential collaboration between physicians and nutritionists for depression management especially in LMICs. Our study is also restricted to the following issues. First, due to the limited evidence, only 1 prospective cohort studies were identified precludes causal relationships.

Second, our results may be influenced by the substantial level of heterogeneity. Third, the classification of exposure and diagnostic criteria of depression varies greatly among individuals. Forth, the adjusted factors were not uniform.

Fifth, the circulating level of vitamin A and beta-carotene is not considered due to the limited evidence. The significance of our study may be weakened by these limitations.

Our results suggest that both dietary vitamin A and beta-carotene intake is inversely associated with depression. However, due to the limited evidence, further well-designed prospective cohort studies with sexual specification are still needed.

YZ and JL conceived the idea and drafted this manuscript and guarantor of the overall content. JD and JL selected and retrieved relevant manuscript, and assessed each study.

All authors revised and approved the final manuscript. This study was supported by the National Natural Science Foundation of China , the National Postdoctoral Science Foundation of China M , the Provincial Outstanding Postdoctoral Innovative Talents Program of Hunan RC , the Provincial Natural Science Foundation of Hunan JJ , the Young Investigator Grant of Xiangya Hospital, the Central South University Q14 , and the FuQing Postdoc Program of Xiangya Hospital, Central South University The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Kessler RC. Epidemiology of women and depression. J Affect Disord. doi: CrossRef Full Text Google Scholar. Yary T, Aazami S. Dietary intake of zinc was inversely associated with depression.

Biol Trace Elem Res. PubMed Abstract CrossRef Full Text Google Scholar. Jung A, Spira D, Steinhagen-Thiessen E, Ilja Demuth I, Norman K.

Zinc deficiency is associated with depressive symptoms-results from the Berlin aging study II. J Gerontol A Biol Sci Med Sci. They measured the FEV1 of participants and measured their beta carotene blood levels. FEV1 measures how much air you can breathe out in one go.

They found that those with high beta carotene levels had much slower decline in FEV1 measures. If you follow a healthy diet rich in beta carotene you do not need supplements.

As mentioned above, supplements can lead to undesirable excesses in beta carotene levels — this cannot occur if your source is from the food you eat. A French study involving adult females published in the Journal of the National Cancer Institute September issue found that smokers with high beta carotene levels had a higher risk of lung cancer and other smoking-related cancers than other smokers.

They also found that non-smokers with high beta carotene intake had a lower risk of lung cancer. Further research has suggested that the high intake among smokers is nearly always due to supplements, and not food intake. Drug interaction refers to a substance interfering in how a medication works, by either making it less effective, increasing its potency, or changing what it is supposed to do.

Long-term alcohol consumption can interact with beta carotene, raising the chances of developing liver problems. Beta carotene supplements are available for purchase online. Speak to a doctor before taking new supplements. Usually, the body turns beta carotene into vitamin A, but not always.

New studies in mice and humans investigate what this means for health. Arugula is healthful, cruciferous leafy green with a peppery taste. It contains essential nutrients that help keep the heart and bones healthy.

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Marker-assisted backcross breeding MABB is an effective approach to improve the nutritional traits in maize. Recently, several studies showed success with improving the nutritional traits in maize hybrids Pusa Vivek QPM9 Improved, Pusa HM4 Improved, Pusa HM8 Improved, and Pusa HM9 Improved using MABB 9 , With this background information, the present study aimed to: i introgress the crtRB1 allele into the parental lines UMI and UMI of CO6 hybrid using MABB; ii evaluate the improved lines agronomic performance and β-carotene concentration; and iii generate the hybrids using the improved lines and evaluate the hybrids in diverse maize growing regions of Tamil Nadu, a southern state of India.

CO6 is a popular maize hybrid well-adapted to different agro-climatic zones of India, especially Tamil Nadu, a southern state of India. Maize inbreds viz. UMI is a female parent for the CO6 hybrid and is suitable for tropical regions; it is characterized as a dent grain type.

UMI is a pollen parent for the CO6 hybrid and is suitable for both tropical and semi-arid regions. It is characterized as a flint type. Both have diverse genetic backgrounds and are placed in different subspecies in accordance with the starch type in the endosperm.

Due to their excellent combining ability, these two inbreds were used to develop the CO6 hybrid. The donor parent was HP from CIMMYT International Maize and Wheat Improvement Center, Mexico , which carries the crtRB1 allele responsible for the high β-carotene concentration.

The conversion process included crossing, two generations of backcrossing, and two generations of selfing. All the field experiments were carried out on the Central farm, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India from to Foreground selection targeted heterozygous plants with allele 1, bp Favorable allele and allele 3, bp Unfavorable allele , or homozygous plants with allele 1, bp Favorable allele.

Background selection was done to select the foreground positive plants with maximum recurrent parent genome recovery RPGR using polymorphic simple sequence repeat SSR markers in backcross and selfed generations.

Two independent crosses, namely UMI × HP and UMI × HP, were made during Rabi to produce F 1 seeds. In Kharif , the F 1 plants were confirmed for their heterozygosity and backcrossed with the recurrent parent to produce BC 1 F 1 progenies.

In Rabi, , the BC 1 F 1 plants that were found to be heterozygous were backcrossed with the recurrent parent to produce BC 2 F 1 progenies. Further, BC 2 F 1 plants were screened to identify heterozygous and the selected plants were selfed to produce BC 2 F 2 progenies during Kharif Agro-morphological character evaluation and β-carotene estimation were carried out in BC 2 F 3 improved lines.

The genomic DNA was isolated from the young leaves using the cetyl trimethyl ammonium bromide CTAB method The DNA was checked for its quantity and quality on 0. A total of simple sequence repeat SSR markers spanning uniformly across the maize genome were used for background selection.

The primer sequences for SSR markers were obtained from the maize genome database www. org and were synthesized by Eurofins Ltd, Bangalore, India. and Pukalenthy et al. All the obtained genotyping results were tested for goodness of fit using the chi-square analysis. Chi-square analysis was done using PROC MIXED of SAS and LSMEANS and ADJUST options were used to find the least square mean values and for the comparison of the p- value.

The percentage of RPGR was calculated following the procedure of Sundaram et al. The seeds obtained from the recurrent and donor parents and the improved lines were subjected to β-carotene estimation.

Estimation of β-carotene was carried out by adopting the Harvest plus protocol The carotenoid compounds were extracted by grinding the seed samples using ice-cold acetone until a fine powder was obtained.

Since the carotenoid compounds undergo photo-oxidation, further processing was done under yellow light 19 , The samples extracted were concentrated in a rotary evaporator at 45°C and made up to 2 ml using methanol prior to separation.

The β-carotene concentration was quantified by high-performance liquid chromatography HPLC. Samples were eluted by C18G A column × 4. Sigma Aldrich, India reconstituted in methanol to five different concentrations 0. The β-carotene concentration was identified by their characteristic spectra and comparison of their retention times with known standard solutions.

Four improved lines from UMI × HP and two improved lines from UMI × HP were crossed during Kharif to generate the five hybrids. These hybrids were evaluated along with the commercial hybrid NK and CO6 during the Rabi and Kharif at the Experimental Farm, Agricultural Research Station, Vaigai dam, Tamil Nadu Agricultural University, India and Maize Research Station, Tamil Nadu Agricultural University, Vagarai, India under comparative yield trial CYT.

Further, one superior hybrid identified from CYT was tested in multi-location trials MLT during Kharif and in diverse maize growing environments of Tamil Nadu along with eight maize hybrids including commercial hybrids. Kharif included four environments viz. Hybrids [namely NK G1 , CO 6 G2 , ACM-M G3 , ACM-M G4 , CMH G5 , VaMH G6 , VaMH G7 , M GOLD G8 , and CMH G9 ] were used in MLT.

All the field trials were conducted in the irrigated condition. The spacing followed was 60 × 25 cm with a plot size of 5 × 3. Trials were carried out using a randomized complete block design RCBD with two replications. Grain yield data were subjected to a combined analysis of variance ANOVA using GEA-R statistical software Additive Main Effect and Multiplicative Interaction AMMI model 22 and Genotype and Genotype by Environmental Interaction GGE effects biplot 23 were employed to analyse the Genotype by Environment GE interaction and to assess grain yield stability based on the principal component analysis PCA.

The crtRB1 allele was introgressed into UMI and UMI using a recurrent backcrossing procedure, combined with foreground and background selection Figure 1. Parental polymorphism screening was conducted between the recurrent and donor parents using a set of SSR markers.

In the cross combinations UMI × HP and UMI × HP, among the SSR markers, and SSR markers were found to be polymorphic markers. These polymorphic SSR markers were used for background selection and RPGR analysis. The F 1 plants were produced from crosses UMI × HP and UMI × HP After confirming heterozygosity with a crtRB1 3'TE marker, the true F 1 plants were backcrossed to their respective recurrent parent.

The percentage of RPGR of positive plants ranged from The best BC 1 F 1 plant showed a maximum RPGR of In the BC 2 F 1 population, a total of 53 and 87 plants were screened with a crtRB1 3'TE marker.

The best three BC 2 F 1 plants from each cross having maximum RPGR were further selected and selfed to produce the BC 2 F 2 population. A total of and BC 2 F 2 plants were screened with a crtRB1 3'TE marker to identify homozygous plants allele 1 Figure 2.

It revealed 34 homozygous plants in UMI × HP and 31 homozygous plants in UMI × HP The population details and their segregation pattern are presented in Table 1.

The numbers of selected plants were further reduced to four and two based on the RPGR and phenotype data, and selfed to produce BC 2 F 3 lines.

UMI × HP based four BC 2 F 3 lines showed The postive plant's RPGR details are presented in Supplementary Tables 1 , 2. Figure 1. Scheme of marker assisted backcross breeding MABB for improving β-carotene concentration in parental lines of CO6 maize hybrid. Figure 2.

Segregation of allele1 and allele 3 in BC 2 F 2 generation using the crtRB1 gene specific marker i. Table 1. Segregation pattern of alleles of the crtRB1 gene in backcrossed and selfed progenies. Figure 3. Background screening of improved lines using SSR markers.

Six improved lines from both crosses were evaluated for the agro-morphological characters Figure 4 and Table 2. The overall performance showed that the traits recorded among the improved lines were on par with their recurrent parents and its phenotypic resemblance percentage ranging from Figure 4.

Morphological resemblance of the parents and the improved lines. Table 2. β-carotene concentration and agronomic performance of the improved lines developed through MABB. The β-carotene concentration was estimated in six improved lines; it showed an increased level of β-carotene ranging from 7.

The β-carotene concentration for the recurrent parents was 0. Introgression of allele1 resulted in an increased level of the kernel β-carotene concentration with a maximum of 9. We also recorded a minimum level of the kernel β-carotene concentration 7. However, it was higher than their respective recurrent parent.

The β-carotene concentration of improved lines is presented in Table 2. In addition, the β-carotene concentration of the improved lines at different environments are summarized in Supplementary Table 3. Five hybrid combinations were developed using the improved lines.

The agronomic performance and the β-carotene concentration of the five hybrids were evaluated along with their corresponding original hybrid CO6 and a commercial hybrid NK under CYT Figure 5.

Among the five hybrids, ACM-M showed a Thus, ACM-M forwarded to MLT along with eight hybrids to study the GE interaction over the different maize growing regions in Tamil Nadu. The yield performance of MLT showed that the hybrid ACM-M G3 recorded a value that is comparable to that of its original hybrid CO6 in all the locations.

An average yield performance across 10 environments recorded 7, Figure 5. Morphological characteristics of original version and reconstituted hybrid with high β-carotene.

A CO6, B ACM-M Table 3. Agronomic performance and β-carotene value of hybrids developed through MABB under comparative yield trail CYT. Per se performance of the yield for the developed five hybrids ranged from The effect of the genotype and environment is Hence, the variation that exists among the genotypes across the environments paves the way to understanding the yield stability of the hybrids over the varying environments and specific adaption of the hybrids.

Based on the Interaction Principal Component Axis 1 IPCA1 of the AMMI model, the genotypes G1, G2, and G5 recorded values nearer to the zero on the biplot 0. Average Environment Coordination AEC recorded that G3 is placed at the second concentric circle and is ideal in terms of high yielding ability and stability, whereas other genotypes, G2, G6, and G7, placed at the third and fourth concentric circle where the stability gradient is low compared to the genotypes closer to the center axis.

Based on the vertices of the Average Environment Axix AEA , the environments E9 and E4 were considered as highly interactive environments from where the wider adaption of the genotypes are selected. Table 4. Analysis of variance of yield data of nine maize hybrids tested across environments during Kharif and Figure 6.

AMMI biplot for IPCA1 score vs. yield in nine hybrids of maize from 10 environments in the state of Tamil Nadu. G1 NK , G2 CO 6 , G3 ACM-M , G4 ACM-M , G5 CMH , G6 VaMH , G7 VaMH , G8 M GOLD, and G9 CMH E1 Coimbatore , E2 Vagarai , E3 Vridhachalam , E4 Bhavanisagar , E5 Coimbatore , E6 Vagarai , E7 Vridhachalam , E8 Bhavanisagar , E9 Vaigai dam, and E10 Athiyandal.

Figure 7. Stability analysis to evaluate the mean grain yield of nine maize hybrids. A Polygon view of GGE biplot for grouping the environments, B GGE biplot for comparing the test hybrids with ideal environment. G1 NK , G2 CO 6 , G3 ACM-M , G4 ACM-M , G5 CMH , G6 VaMH , G7 VaMH , G8 M GOLD , G9 CMH , E1 Coimbatore , E2 Vagarai , E3 Vridhachalam , E4 Bhavanisagar , E5 Coimbatore , E6 Vagarai , E7 Vridhachalam , E8 Bhavanisagar , E9 Vaigai dam, and E10 Athiyandal.

CO6 is one of the popular maize hybrids of India, especially in Tamil Nadu. CO6 is known for its high yield, multiple disease resistances, and its suitablity for rainfed and irrigated conditions.

However, CO6 is low in pVAC β-carotene, thus, the present investigation was taken up to improve the β-carotene in CO6 through MABB. Previously, Muthusamy et al. We also used the same MABB scheme to enrich the β-carotene in CO6. Marker-assisted foreground selection using gene-linked markers permits the transfer of the gene of interest with high precision in MABB.

The advantages of using a linked marker for the selection of single or multiple genes simultaneously in MABB have been described in many studies for nutritional traits in maize 7 , 8 , The backcross and selfed-generations from both crosses, UMI × HP and UMI × HP, showed segregation distortion.

The segregation pattern of allele 1 and allele 3 deviated from the expected Mendelian ratio and and allele 1 was diminished. These results are similar to the reports of Babu et al. There are many facts, such as embryo-specific mutation 24 , segregation distortion regions in the maize genome 25 , mutants like the defective kernel 24 , gametophytic factors 24 , 26 , and genetic background of the target allele 8 , that are reported to be the reason for the segregation distortion.

In this case, the evaluation of a large population for attaining enough foreground positives is essential. Marker-assisted background selection using SSR markers helped in selecting the foreground positive plants with a high RPGR.

We performed the background selection starting from the BC 1 F 1 generation; it hastened the RPGR in advanced lines. Following this approach, after only two generations of backcrossing, it was possible to identify the plants carrying RPGR ranging from These results are in accordance with the previous studies 9 , 27 , The study aims to introgress the crtRB1 gene without troubling the recurrent parental genome by MABB, thus the improved line will be suitable for further use.

For confirming the suitability of the new genotypes, it is essential to characterize and choose the progenies that are closer to the parent for both morphological and nutritional traits as this selection adds value to the MABB program.

Thus, we investigated the agro-morphological characters of the improved lines. It revealed that the agro-morphological characters of the improved lines were on par with the recipient parent. The agro morphological characters among the six improved lines ranged from Moreover, the β-carotene content of the improved lines ranged from 7.

The β-carotene concentration of the improved lines is on par with the β-carotene parent. Similar results were obtained in other studies 10 , The improved lines can be considered as the candidate parents for developing β-carotene hybrids adapted to irrigated and rainfed conditions.

They were successfully used to develop the five hybrid combinations. Among them, ACM-M is a superior hybrid with increased yield and β-carotene concentration over its original hybrid and a commercial hybrid NK under CYT.

Hence, this hybrid was forwarded for MLT along with eight hybrids to study the GE interaction over the different maize growing regions in Tamil Nadu. The yield performance of MLT showed that ACM-M G3 recorded a value that is comparable to that of its original hybrid CO6 G2 in all the locations.

Based on the stability ANOVA, the GE toward the yield was lesser compared to the environment E and it is These results are similar to the earlier study on maize We observed significant genotype by environment interaction GE effect, which revealed that the yield performance of all the hybrids over the environments under study was not consistent.

This situation necessitates understanding the nature and magnitude of genotype by environment effect which may not be possible through standard ANOVA 21 , IPCA1 from the AMMI model gives an idea about the association of the genotypes with the environments under study.

From the above concept the genotype G1, G2, and G5 recorded values closer to the zero, which could be promising genotypes for the yield trait over all the environments.

GGE biplot analysis reflects the similarity existing between the genotypes, environment, and their corresponding response to each other, as well as the interaction between them, making it the best tool to graphically represent the stability of the genotype From the polygon view of GGE biplot, the genotypes G3, G4, G5, and G8 are recorded as a specific adaptation to the respective environments which are present nearer to the vertex of the polygon.

High per se performance, higher vector length, and high stability across the environments are the properties of ideal genotype and the genotypes places closed to the ideal genotype are called desirable genotypes. These are most probably at the second concentric circle of the GGE biplot 23 , with the greatest ability to discriminate genotypes, favoring the selection of superior genotypes.

The GGE biplot revealed that the genotype G3 ACM-M could be recommended for yield trait to all the environments studied since it is placed nearer to the highly interactive environment E4 and also at the second concentric circle on the GGE biplot.

In summary, we have successfully improved the β-carotene concentration in the CO6 hybrid through MABB. The β-carotene enriched hybrid developed in this study will hold great promise for nutritional and food security.

Also, the improved maize inbreds with β-carotene will serve as a potential genetic material for the development of β-carotene rich cultivars in maize breeding programs.

The datasets generated for this study are available on request to the corresponding author. SN conceived and designed the methods and experiments. SN, TD, GK, NT, and RR constructed backcross progenies and managed field work.

TD, BP, SC, JN, DM, KA, and VS conducted phenotype, genotype, and biochemical analyses. The advantage of dietary beta carotene is that the body only converts as much as it needs. Excess vitamin A is toxic. Toxic vitamin A levels can occur if you consume too many supplements.

There are a number of ways that beta carotene can benefit human health. Below, we give some examples:. Beta carotene, like all carotenoids, is an antioxidant. An antioxidant is a substance that inhibits the oxidation of other molecules; it protects the body from free radicals.

Free radicals damage cells through oxidation. Eventually, the damage caused by free radicals can cause several chronic illnesses. Men who have been taking beta carotene supplements for 15 or more years are considerably less likely to experience cognitive decline than other males, researchers from Harvard Medical School reported in Archives of Internal Medicine November issue.

Oxidative stress is thought to be a key factor in cognitive decline, the researchers explained. Studies have shown that antioxidant supplements may help prevent the deterioration of cognition.

Their study, involving 4, men, compared those on beta carotene supplements for an average of 18 years to others who were given placebo. Over the short-term, they found no difference in cognitive decline risk between the two groups of men, but in the long-term it was clear that beta carotene supplements made a significant difference.

The researchers emphasized that there may have been other factors which contributed to the slower decline in cognitive abilities among the men in the beta carotene group.

The BMJ published a report in March which showed that high blood beta carotene levels compensate for some of the damage to the lungs caused by oxygen free radicals.

They measured the FEV1 of participants and measured their beta carotene blood levels. FEV1 measures how much air you can breathe out in one go. They found that those with high beta carotene levels had much slower decline in FEV1 measures.

If you follow a healthy diet rich in beta carotene you do not need supplements. As mentioned above, supplements can lead to undesirable excesses in beta carotene levels — this cannot occur if your source is from the food you eat. A French study involving adult females published in the Journal of the National Cancer Institute September issue found that smokers with high beta carotene levels had a higher risk of lung cancer and other smoking-related cancers than other smokers.

They also found that non-smokers with high beta carotene intake had a lower risk of lung cancer. Further research has suggested that the high intake among smokers is nearly always due to supplements, and not food intake. Drug interaction refers to a substance interfering in how a medication works, by either making it less effective, increasing its potency, or changing what it is supposed to do.

Long-term alcohol consumption can interact with beta carotene, raising the chances of developing liver problems. Beta carotene supplements are available for purchase online.

Speak to a doctor before taking new supplements. Usually, the body turns beta carotene into vitamin A, but not always. New studies in mice and humans investigate what this means for health.

Arugula is healthful, cruciferous leafy green with a peppery taste. It contains essential nutrients that help keep the heart and bones healthy. What are micronutrients?

Read on to learn more about these essential vitamins and minerals, the role they play in supporting health, as well as…. Adding saffron supplements to standard-of-care treatment for ulcerative colitis may help reduce inflammation and positively benefit patients, a new….

My podcast changed me Can 'biological race' explain disparities in health? Why Parkinson's research is zooming in on the gut Tools General Health Drugs A-Z Health Hubs Health Tools Find a Doctor BMI Calculators and Charts Blood Pressure Chart: Ranges and Guide Breast Cancer: Self-Examination Guide Sleep Calculator Quizzes RA Myths vs Facts Type 2 Diabetes: Managing Blood Sugar Ankylosing Spondylitis Pain: Fact or Fiction Connect About Medical News Today Who We Are Our Editorial Process Content Integrity Conscious Language Newsletters Sign Up Follow Us.

Medical News Today. Health Conditions Health Products Discover Tools Connect. All you need to know about beta carotene. By Tim Newman on December 14, What is beta carotene? Uses and benefits Foods Side effects.

It also notes the percentage of your daily value of vitamin A that the food meets. Eating more fruits and vegetables can help you get more beta-carotene. Red, orange, deep yellow, and dark green produce tends to be high in carotenoids.

Severe vitamin A problems can lead to blindness. This is a leading cause of blindness in some parts of the world. But high doses over a long time can lead to carotenemia.

This causes your skin to become yellowish orange. Too much beta-carotene is a problem for some people. This includes people who can't convert beta-carotene to vitamin A.

This can happen to people who have hypothyroidism. Higher doses of vitamin A may increase the risk for fractures in both women past menopause, and in men. High dose supplements with preformed vitamin A are not advised during pregnancy. Too much may cause birth defects or miscarriage.

Orlistat, a medicine for weight loss, decreases fat absorption in the body. Because of this, it may also reduce absorption of beta-carotene and vitamin A. Vitamin A is a fat-soluble vitamin. Don't use vitamin A or beta-carotene supplements if you take any of these medicines. This is because they contain derivatives of vitamin A:.

Search Encyclopedia. Beta-Carotene Other name s vitamin A, b-carotene, provitamin A General Beta-carotene is a type of substance called a carotenoid. Main functions Beta-carotene and vitamin A play a vital part in the reproductive process. Demonstrated uses Beta-carotene and other carotenoids help reduce free radical damage in your body.

Reasons for increased need Poor nutrition is a leading cause of beta-carotene and vitamin A deficiency. These problems can keep you from getting enough vitamin A: Lactose intolerance Celiac disease Sprue Cystic fibrosis Women who are pregnant or breastfeeding may need to take supplements.

Claims Beta-carotene may reduce the risk of some types of cancer, such as prostate cancer. Further research has suggested that the high intake among smokers is nearly always due to supplements, and not food intake.

Drug interaction refers to a substance interfering in how a medication works, by either making it less effective, increasing its potency, or changing what it is supposed to do.

Long-term alcohol consumption can interact with beta carotene, raising the chances of developing liver problems. Beta carotene supplements are available for purchase online. Speak to a doctor before taking new supplements. Usually, the body turns beta carotene into vitamin A, but not always.

New studies in mice and humans investigate what this means for health. Arugula is healthful, cruciferous leafy green with a peppery taste.

It contains essential nutrients that help keep the heart and bones healthy. What are micronutrients? Read on to learn more about these essential vitamins and minerals, the role they play in supporting health, as well as….

Adding saffron supplements to standard-of-care treatment for ulcerative colitis may help reduce inflammation and positively benefit patients, a new…. My podcast changed me Can 'biological race' explain disparities in health? Why Parkinson's research is zooming in on the gut Tools General Health Drugs A-Z Health Hubs Health Tools Find a Doctor BMI Calculators and Charts Blood Pressure Chart: Ranges and Guide Breast Cancer: Self-Examination Guide Sleep Calculator Quizzes RA Myths vs Facts Type 2 Diabetes: Managing Blood Sugar Ankylosing Spondylitis Pain: Fact or Fiction Connect About Medical News Today Who We Are Our Editorial Process Content Integrity Conscious Language Newsletters Sign Up Follow Us.

Medical News Today. Health Conditions Health Products Discover Tools Connect. All you need to know about beta carotene. By Tim Newman on December 14, What is beta carotene?

Uses and benefits Foods Side effects. How we vet brands and products Medical News Today only shows you brands and products that we stand behind. Our team thoroughly researches and evaluates the recommendations we make on our site.

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Since the carotenoid compounds undergo photo-oxidation, further processing was done under yellow light 19 , The samples extracted were concentrated in a rotary evaporator at 45°C and made up to 2 ml using methanol prior to separation.

The β-carotene concentration was quantified by high-performance liquid chromatography HPLC. Samples were eluted by C18G A column × 4. Sigma Aldrich, India reconstituted in methanol to five different concentrations 0.

The β-carotene concentration was identified by their characteristic spectra and comparison of their retention times with known standard solutions. Four improved lines from UMI × HP and two improved lines from UMI × HP were crossed during Kharif to generate the five hybrids.

These hybrids were evaluated along with the commercial hybrid NK and CO6 during the Rabi and Kharif at the Experimental Farm, Agricultural Research Station, Vaigai dam, Tamil Nadu Agricultural University, India and Maize Research Station, Tamil Nadu Agricultural University, Vagarai, India under comparative yield trial CYT.

Further, one superior hybrid identified from CYT was tested in multi-location trials MLT during Kharif and in diverse maize growing environments of Tamil Nadu along with eight maize hybrids including commercial hybrids.

Kharif included four environments viz. Hybrids [namely NK G1 , CO 6 G2 , ACM-M G3 , ACM-M G4 , CMH G5 , VaMH G6 , VaMH G7 , M GOLD G8 , and CMH G9 ] were used in MLT. All the field trials were conducted in the irrigated condition.

The spacing followed was 60 × 25 cm with a plot size of 5 × 3. Trials were carried out using a randomized complete block design RCBD with two replications.

Grain yield data were subjected to a combined analysis of variance ANOVA using GEA-R statistical software Additive Main Effect and Multiplicative Interaction AMMI model 22 and Genotype and Genotype by Environmental Interaction GGE effects biplot 23 were employed to analyse the Genotype by Environment GE interaction and to assess grain yield stability based on the principal component analysis PCA.

The crtRB1 allele was introgressed into UMI and UMI using a recurrent backcrossing procedure, combined with foreground and background selection Figure 1. Parental polymorphism screening was conducted between the recurrent and donor parents using a set of SSR markers. In the cross combinations UMI × HP and UMI × HP, among the SSR markers, and SSR markers were found to be polymorphic markers.

These polymorphic SSR markers were used for background selection and RPGR analysis. The F 1 plants were produced from crosses UMI × HP and UMI × HP After confirming heterozygosity with a crtRB1 3'TE marker, the true F 1 plants were backcrossed to their respective recurrent parent.

The percentage of RPGR of positive plants ranged from The best BC 1 F 1 plant showed a maximum RPGR of In the BC 2 F 1 population, a total of 53 and 87 plants were screened with a crtRB1 3'TE marker.

The best three BC 2 F 1 plants from each cross having maximum RPGR were further selected and selfed to produce the BC 2 F 2 population. A total of and BC 2 F 2 plants were screened with a crtRB1 3'TE marker to identify homozygous plants allele 1 Figure 2.

It revealed 34 homozygous plants in UMI × HP and 31 homozygous plants in UMI × HP The population details and their segregation pattern are presented in Table 1. The numbers of selected plants were further reduced to four and two based on the RPGR and phenotype data, and selfed to produce BC 2 F 3 lines.

UMI × HP based four BC 2 F 3 lines showed The postive plant's RPGR details are presented in Supplementary Tables 1 , 2. Figure 1. Scheme of marker assisted backcross breeding MABB for improving β-carotene concentration in parental lines of CO6 maize hybrid.

Figure 2. Segregation of allele1 and allele 3 in BC 2 F 2 generation using the crtRB1 gene specific marker i. Table 1. Segregation pattern of alleles of the crtRB1 gene in backcrossed and selfed progenies.

Figure 3. Background screening of improved lines using SSR markers. Six improved lines from both crosses were evaluated for the agro-morphological characters Figure 4 and Table 2. The overall performance showed that the traits recorded among the improved lines were on par with their recurrent parents and its phenotypic resemblance percentage ranging from Figure 4.

Morphological resemblance of the parents and the improved lines. Table 2. β-carotene concentration and agronomic performance of the improved lines developed through MABB. The β-carotene concentration was estimated in six improved lines; it showed an increased level of β-carotene ranging from 7.

The β-carotene concentration for the recurrent parents was 0. Introgression of allele1 resulted in an increased level of the kernel β-carotene concentration with a maximum of 9.

We also recorded a minimum level of the kernel β-carotene concentration 7. However, it was higher than their respective recurrent parent.

The β-carotene concentration of improved lines is presented in Table 2. In addition, the β-carotene concentration of the improved lines at different environments are summarized in Supplementary Table 3.

Five hybrid combinations were developed using the improved lines. The agronomic performance and the β-carotene concentration of the five hybrids were evaluated along with their corresponding original hybrid CO6 and a commercial hybrid NK under CYT Figure 5.

Among the five hybrids, ACM-M showed a Thus, ACM-M forwarded to MLT along with eight hybrids to study the GE interaction over the different maize growing regions in Tamil Nadu. The yield performance of MLT showed that the hybrid ACM-M G3 recorded a value that is comparable to that of its original hybrid CO6 in all the locations.

An average yield performance across 10 environments recorded 7, Figure 5. Morphological characteristics of original version and reconstituted hybrid with high β-carotene. A CO6, B ACM-M Table 3.

Agronomic performance and β-carotene value of hybrids developed through MABB under comparative yield trail CYT. Per se performance of the yield for the developed five hybrids ranged from The effect of the genotype and environment is Hence, the variation that exists among the genotypes across the environments paves the way to understanding the yield stability of the hybrids over the varying environments and specific adaption of the hybrids.

Based on the Interaction Principal Component Axis 1 IPCA1 of the AMMI model, the genotypes G1, G2, and G5 recorded values nearer to the zero on the biplot 0.

Average Environment Coordination AEC recorded that G3 is placed at the second concentric circle and is ideal in terms of high yielding ability and stability, whereas other genotypes, G2, G6, and G7, placed at the third and fourth concentric circle where the stability gradient is low compared to the genotypes closer to the center axis.

Based on the vertices of the Average Environment Axix AEA , the environments E9 and E4 were considered as highly interactive environments from where the wider adaption of the genotypes are selected.

Table 4. Analysis of variance of yield data of nine maize hybrids tested across environments during Kharif and Figure 6. AMMI biplot for IPCA1 score vs. yield in nine hybrids of maize from 10 environments in the state of Tamil Nadu. G1 NK , G2 CO 6 , G3 ACM-M , G4 ACM-M , G5 CMH , G6 VaMH , G7 VaMH , G8 M GOLD, and G9 CMH E1 Coimbatore , E2 Vagarai , E3 Vridhachalam , E4 Bhavanisagar , E5 Coimbatore , E6 Vagarai , E7 Vridhachalam , E8 Bhavanisagar , E9 Vaigai dam, and E10 Athiyandal.

Figure 7. Stability analysis to evaluate the mean grain yield of nine maize hybrids. A Polygon view of GGE biplot for grouping the environments, B GGE biplot for comparing the test hybrids with ideal environment.

G1 NK , G2 CO 6 , G3 ACM-M , G4 ACM-M , G5 CMH , G6 VaMH , G7 VaMH , G8 M GOLD , G9 CMH , E1 Coimbatore , E2 Vagarai , E3 Vridhachalam , E4 Bhavanisagar , E5 Coimbatore , E6 Vagarai , E7 Vridhachalam , E8 Bhavanisagar , E9 Vaigai dam, and E10 Athiyandal.

CO6 is one of the popular maize hybrids of India, especially in Tamil Nadu. CO6 is known for its high yield, multiple disease resistances, and its suitablity for rainfed and irrigated conditions.

However, CO6 is low in pVAC β-carotene, thus, the present investigation was taken up to improve the β-carotene in CO6 through MABB. Previously, Muthusamy et al. We also used the same MABB scheme to enrich the β-carotene in CO6. Marker-assisted foreground selection using gene-linked markers permits the transfer of the gene of interest with high precision in MABB.

The advantages of using a linked marker for the selection of single or multiple genes simultaneously in MABB have been described in many studies for nutritional traits in maize 7 , 8 , The backcross and selfed-generations from both crosses, UMI × HP and UMI × HP, showed segregation distortion.

The segregation pattern of allele 1 and allele 3 deviated from the expected Mendelian ratio and and allele 1 was diminished. These results are similar to the reports of Babu et al.

There are many facts, such as embryo-specific mutation 24 , segregation distortion regions in the maize genome 25 , mutants like the defective kernel 24 , gametophytic factors 24 , 26 , and genetic background of the target allele 8 , that are reported to be the reason for the segregation distortion.

In this case, the evaluation of a large population for attaining enough foreground positives is essential. Marker-assisted background selection using SSR markers helped in selecting the foreground positive plants with a high RPGR.

We performed the background selection starting from the BC 1 F 1 generation; it hastened the RPGR in advanced lines.

Following this approach, after only two generations of backcrossing, it was possible to identify the plants carrying RPGR ranging from These results are in accordance with the previous studies 9 , 27 , The study aims to introgress the crtRB1 gene without troubling the recurrent parental genome by MABB, thus the improved line will be suitable for further use.

For confirming the suitability of the new genotypes, it is essential to characterize and choose the progenies that are closer to the parent for both morphological and nutritional traits as this selection adds value to the MABB program. Thus, we investigated the agro-morphological characters of the improved lines.

It revealed that the agro-morphological characters of the improved lines were on par with the recipient parent. The agro morphological characters among the six improved lines ranged from Moreover, the β-carotene content of the improved lines ranged from 7.

The β-carotene concentration of the improved lines is on par with the β-carotene parent. Similar results were obtained in other studies 10 , The improved lines can be considered as the candidate parents for developing β-carotene hybrids adapted to irrigated and rainfed conditions.

Each task consisted of 2 blocks. All 43 male face pairs were presented twice, once in each block, making a total of 86 trials for each task. In block 1, the post-supplementation face was shown on the right for half the face pairs.

The face pairs were presented again in block 2 in the opposite left-right orientation. Block order was counterbalanced across participants.

All faces were rotated and aligned so that the eyes were lying on a horizontal plane at the same height. All images were cropped to × pixels and a black oval mask was applied to cover most of the hair, ears, and neck. An attractiveness score for each face pair was calculated as the percentage of times the post-supplementation face was chosen as the more attractive face.

A perceived health score for each face pair was calculated as the percentage of times the post-supplementation face was chosen as the more healthy looking face.

However, several previous studies have investigated skin color preferences using online samples Lefevre et al. Both samples showed a preference for high carotenoid skin color, with no difference in the preference between the two. This finding suggests that any additional noise due to uncalibrated monitors is relatively small compared to the color variation among the faces.

Markers of DNA oxidation 8-OHdG and lipid peroxidation isoprostane were analyzed in duplicates using competitive enzyme-linked immunoassay ELISA kits from Northwest Life Science Specialties Vancouver, VA.

A significant proportion of urinary isoprostane is conjugated to glucoronic acid, which is not assayable Yan et al. To obtain a more accurate measure of overall isoprostane level, µL of each sample was incubated with 5 µL of beta-glucoronidase for 2 h at 37°C to cleave and free the isoprostanes from their conjugated forms before running the isoprostane assays.

Creatinine was determined in duplicates using colorimetric assay kits from Northwest Life Science Specialties Vancouver. Salivary bacteria killing capacity against Escherichia coli ATCC no. Salivary supernatant was incubated with E. coli for 30 min to facilitate bacteria killing, and then incubated overnight on trypticase soy agar TSA plates to quantify the amount of bacteria remaining see Supplementary Material for details.

Images of the plates were taken together with a ruler as a size reference. We used the program ImageJ to measure the following: total number of colonies in each plate, average area of each colony, and total area of the colonies combined.

Bacteria killing capacity was calculated as the percentage difference in colony number relative to positive controls. Bacteria growth suppression capacity was calculated as the percentage difference in average colony area relative to positive controls. Overall salivary immunity was calculated as the percentage difference in total colony area relative to positive controls.

A lysoplate assay was used to determine salivary lysozyme activity. Micrococcus lysodeikticus ATCC no. Ten microlitres of whole saliva from each sample were added to 80μL of M. lysodeikticus in duplicates in a well plate.

Positive controls containing 10 μL of PBS and 80 μL of M. lysodeikticus were also added to the plate in duplicates.

The plate was incubated at 33°C for 10 min and the absorbance was measured using an M5 SpectraMax microplate reader Molecular Devices, Sunnyvale, CA. Salivary lysozyme activity was calculated as the difference in absorbance between the sample wells and the positive controls. The semen samples were analyzed in 6 replicates immediately upon delivery using the Hamilton-Thorne CEROS Computer Assisted Semen Analysis CASA system Simmons et al.

The system measures total sperm concentration, percentage motile sperm and 7 motility related variables. Seven samples had to be diluted because they were too concentrated for the CASA to analyze see Supplementary Material for details. A portion of the post-supplementation semen sample was stored in accordance with McEvoy et al.

The semen DNA fragmentation level of each sample is measured by the percentage of fragmented sperm cells see supplementary material for details. Principal components analysis PCA was used to summarize the inter-related semen quality data and immune function data.

PC1 was weighted most strongly by bacterial killing and suppression capacity. PC2 was weighted most strongly by lysozyme activity and overall bacteria immunity. PC1 was weighted most strongly by variables related to rapid progressive motility. PC2 was weighted most strongly by variables related to the linearity of the sperm movement.

PC3 was weighted most strongly by high sperm concentration and percentage motile sperm with low levels of left-right head movement. PC3 was found to be influenced by variation in the collection procedure and abnormalities in the sample WHO see Supplementary Material for details.

Therefore, we ran all analyses for PC3 on the residuals after accounting for these variables. Difference scores were calculated for each of the 3 skin color variables yellowness, redness, and lightness , the 2 oxidative stress measures 8-OHdG and isoprostane , the 2 immune function PCs, and the 3 semen quality PCs, by subtracting the pre-supplementation scores from the post-supplementation scores.

The descriptive statistics for the dependent variables, including all the difference scores, 2-alternative forced-choice attractiveness scores, 2-alternative forced-choice perceived health scores, and post-supplementation sperm DNA fragmentation levels are presented in Table 1.

Positive values indicate an increase in a particular measure post-supplementation. During data analysis, the treatment conditions were binary coded to prevent any experimenter biases and the code was broken only after the statistical analyses were finalized.

All variables are presented as pre—post difference scores indicated by Δ with the exception of the 2-alternative forced-choice attractiveness and perceived health scores, which were scored based on the percentage of times the post-supplementation face of a participant was chosen as more attractive or healthy looking, respectively, and the sperm DNA fragmentation levels, which was measured post-supplementation.

One-way Anovas with Treatment beta-carotene, placebo as the between-participants factor were conducted for all the dependent variables, including each of the skin color difference scores yellowness, redness, and lightness , the 2-alternative forced-choice attractiveness score, the 2-alternative forced-choice perceived health score, each of the oxidative stress difference scores, each of the immune function PC difference scores, each of the semen quality PC difference scores and the post-supplementation sperm DNA fragmentation levels.

All residuals were normally distributed. These results indicate that the Treatment effect was due to an increase in yellowness in the beta-carotene group.

These results show that, as predicted, beta-carotene supplementation significantly increased face yellowness and redness but not lightness. Examples of the color variation between pre- and post-supplementation for the beta-carotene and placebo groups are shown in Figure 2.

Boxplots of pre-post changes in skin yellowness, redness, and lightness by Treatment beta-carotene vs placebo. Examples of color variation between pre- and post-supplementation by Treatment condition.

Thus beta-carotene supplementation significantly enhanced facial attractiveness. Boxplots of 2-alternative-forced-choice scores for attractiveness and perceived health by Treatment beta-carotene vs placebo.

Thus beta-carotene supplementation significantly enhanced perceived health. We provide experimental evidence that the carotenoid beta-carotene enhances skin yellowness and redness and increases facial attractiveness in human males. Contrary to the carotenoid trade-off hypothesis, we did not find any effect of beta-carotene on measures of oxidative stress, immune function, semen quality, or sperm DNA fragmentation.

Thus, despite the effects of beta-carotene on facial appearance, we find no evidence that carotenoid-related skin color is an honest signal of health in human males. Carotenoid-based coloration has been shown to influence mate choice in taxa such as birds, fishes, and lizards Endler ; Kodric-Brown ; Olson and Owens ; Møller et al.

Our results suggest that carotenoid-based coloration also serves mate choice functions in humans. First, using a randomized, double-blind, placebo-controlled experimental design, we provide strong experimental evidence that consuming beta-carotene enhances skin yellowness and redness.

Second, we showed that there was a significant effect of beta-carotene supplementation on male facial attractiveness and perceived health. Recent correlational studies have linked beta-carotene intake with increased skin yellowness Stephen et al. Our study provides the first evidence for a causal link between beta-carotene and these changes.

To the extent that attractiveness affects mating success Rhodes et al. According to the carotenoid trade-off hypothesis, coloration signals health because individuals face a trade-off between the use of available carotenoids in coloration versus supporting health.

The assumption that carotenoids affect health has been tested experimentally in numerous species of birds, fishes, lizards, and even insects, but the results have been equivocal Aguilera and Amat ; Blount et al.

For humans, we found that although beta-carotene made the participants look healthier, there was no evidence that it enhanced actual health. Beta-carotene supplementation did not affect innate immune function, oxidative stress, or semen quality, all measures that have been linked theoretically to the proposed antioxidant capacity of carotenoids.

Moreover, for each aspect of health, we used multiple measures, which should be superior to using single measures Adamo ; Halliwell and Whiteman Our results suggest that, rather than indicating actual health changes, the changes in perceived health due to beta-carotene supplementation may reflect an attractiveness halo effect Eagly et al.

It is possible that beta-carotene supplementation might have an effect on health in a population that is under greater physiological health stress or greater dietary restrictions than our sample.

From a life-history perspective, physiological trade-offs are more apparent when individual or environmental conditions are limiting Stearns Our participants were all relatively healthy individuals recruited from a university community.

It is possible that we found an effect of beta-carotene on facial appearance but not health because the participants simply did not require additional carotenoids to support their health and devoted all the supplemented beta-carotene to appearance.

It would be informative for future studies to examine a population that is under greater physiological or dietary stress. Although we did not find a significant effect of beta-carotene on any of our health measures, carotenoids could still be linked to health via indirect mechanisms.

For example, Hartley and Kennedy postulated that carotenoid coloration might actually signal the presence of other antioxidants that protect carotenoids from oxidative damage, which would otherwise cause carotenoids to lose their color i.

This hypothesis is also known as the carotenoid protection hypothesis. In support of this hypothesis, experimental studies have found that dietary supplementation of nonpigmentary antioxidants increase carotenoid-based coloration in species such as zebra finches, Taeniopygia guttata Bertrand et al.

It would be interesting to examine the carotenoid protection hypothesis in humans by investigating the effect of consuming nonpigmentary antioxidants on skin yellowness and redness. Another possibility is that carotenoids only affect health when they are paired with other nutrients.

Almbro et al. They argued that because vitamin E is converted to radical species when it is used as an antioxidant, beta-carotene might serve to recycle the radicalized vitamin E, thus enhancing overall antioxidant status.

Therefore, a potential future direction would be to examine whether beta-carotene affects health in humans when paired with vitamin E. In most species, carotenoid-based coloration is sexually selected via female mate choice for male carotenoid ornamentation, and most species show sexual dimorphism in carotenoid coloration.

A recent study reported that human skin color is also sexually dimorphic Carrito et al. However, mate selection in humans occurs in both directions and carotenoids influence appearance in both sexes Stephen et al. Therefore, it would be interesting to see the extent to which our findings could be replicated in women.

It would also be interesting for future studies to examine whether beta-carotene influences health over a longer supplementation period. We chose the week duration partly because spermatogenesis plus epididymal transit in humans takes a total of 82 days ~ Therefore, we should have been able to observe any effects of beta-carotene on semen quality after 12 weeks of supplementation.

Previous studies examining the effects of antioxidant supplementation on semen quality and oxidative stress have used similar supplementation durations Møller and Loft ; Showell et al. However, it remains possible that we might observe significant effects of beta-carotene on health in a relatively healthy population like the one in the present study with a longer supplementation duration.

Zareba et al. Given this finding, it would be interesting to examine the long-term effect of beta-carotene supplementation on health by repeating the present study with a supplementation period of 1 year or more.

In summary, we report the first double-blind, placebo-controlled experimental study on the effect of carotenoids on human facial appearance and health. We found that beta-carotene alters skin color by enhancing yellowness and redness to enhance facial attractiveness in human males.

However, we found no evidence that carotenoids improve actual health. Together, our results suggest that carotenoid-based coloration may have been sexually selected in humans, but we have no evidence to suggest that it is an honest signal of health. The study is supported by the ARC Centre of Excellence in Cognition and its Disorders CE , ARC Professorial Fellowships to L.

DP and G. DP , an ARC Discovery Outstanding Researcher Award to G. DP and student research grants awarded to Y. by The Australasian Society for the Study of Animal Behaviour ASSAB and European Human Behaviour and Evolution Association EHBEA.

designed the study, conducted the experiment, analyzed the data, interpreted the results and drafted the manuscript. and L. were involved in designing the study, interpreting the results, and revising the manuscript.

Data accessibility: Analyses reported in this article can be reproduced using the data provided by Foo et al. Adamo SA. How should behavioural ecologists interpret measurements of immunity?

Anim Behav. Google Scholar. Aguilera E Amat JA. Carotenoids, immune response and the expression of sexual ornaments in male greenfinches Carduelis chloris. Alaluf S Heinrich U Stahl W Tronnier H Wiseman S. Dietary carotenoids contribute to normal human skin color and UV photosensitivity.

J Nutr. Almbro M Dowling DK Simmons LW. Effects of vitamin E and beta-carotene on sperm competitiveness. Ecol Lett. Alonso-Álvarez C Galván I. Free radical exposure creates paler carotenoid-based ornaments: A possible interaction in the expression of black and red traits.

PLoS ONE. Amann RP. The cycle of the seminiferous epithelium in humans: A need to revisit? J Androl. Andersson M. Sexual selection. Princeton NJ : Princeton University Press.

Google Preview. Bertrand S Fivre B Sorci G. Do carotenoid-based sexual traits signal the availability of non-pigmentary antioxidants?

J Exp Biol. Blount JD. Carotenoids and life-history evolution in animals. Arch Bichem Biophys. Blount JD Metcalfe NB Birkhead TR Surai PF. Carotenoid modulation of immune function and sexual attractiveness in zebra finches. Carrito M de L Santos IMB dos Lefevre CE Whitehead RD Silva CF da Perrett DI.

The role of sexually dimorphic skin colour and shape in attractiveness of male faces. Evol Hum Behav. Changizi MA Zhang Q Shimojo S. Bare skin, blood and the evolution of primate colour vision. Biol Lett. Chen J Song Y Zhang L. Effect of lycopene supplementation on oxidative stress: an exploratory systematic review and meta-analysis of randomized controlled trials.

J Med Food 16 : — Coetzee V Perrett DI. Effect of beta-carotene supplementation on African skin. J Biomed Opt. Costantini D Møller AP. Carotenoids are minor antioxidants for birds. Funct Ecol. Darwin C. The descent of man and selection in relation to sex.

London : John Murray. Dowling DK Simmons LW. Reactive oxygen species as universal constraints in life-history evolution. Proc R Soc B : — Eagly AH Ashmore RD Makhijani MG Longo LC. What is beautiful is good, but…: A meta-analytic review of research on the physical attractiveness stereotype.

Psychol Bull. Endler JA. Natural and sexual selection on color patterns in poeciliid fishes. Env Biol Fish. Fisher RA. The genetical theory of natural selection. Algal cells and E.

coli cells were harvested by centrifugation at rpm for 8 min at 4 °C and then frozen by liquid nitrogen for further processing. To construct the plasmid expressing lycopene β-cyclase in E. coli , the vector pET-Duet-1 was used as the backbone.

After codon-optimization based on E. PCR was performed using SuperFi DNA polymerase Invitrogen Life technologies, Carlsbad, CA, USA as recommended. The restriction enzyme digestion Thermo Scientific FastDigest and vector construction using T4 DNA ligase Invitrogen were performed as the manual.

To construct the plasmid expressing lycopene β-cyclase in microalga C. reinhardtii , the vector pDb was used, which contains the psaD promoter and psaD terminator to drive the expression of target gene Additional file 1 : Fig.

pDb also contains the bleomycin reporter cluster that can be used to screen transformants. CrtY was codon-optimized for better expression according to the nucleotide preferences of C. reinhardtii nuclear genome. Additionally, a chloroplast signal peptide CTP was fused to the N-terminal of CrtY.

The codon-optimized CTP - CrtY was synthesized and cloned into the pDb at the NheI site to form pDb-CrtY. The synthesis and subclone of target nucleotides were performed by GenScript Biotech Corp.

Nanjing, China. The accuracy of nucleotides in each plasmid was confirmed by sequencing. To produce target carotenoids in E. coli , plasmids pET-DsLcyb1 and pET-CrtY were transferred into the chemical competent cell of lycopene-producing E.

coli using heat-shock method [ 25 ]. To produce target carotenoids in C. reinhardtii , the plasmid pDb-CrtY was transferred into algal cells using glass-bead method with few modifications [ 26 ].

The algal transformation process was performed as described, with additional information that NotI was used to linearize plasmids. The proliferated plasmid DNA was extracted from E. coli strain DH5α using Omega Plasmid Mini Kit II Omega Bio-Tek, USA.

Genomic DNA was extracted from algal cells using M5 HiPer Plant Genomic DNA Kit Mei5 Biotechnology, Beijing, China and total RNA was extracted using SteadyPure Plant RNA Extraction Kit Accurate Biotech.

The first strand of cDNA was synthesized by PrimeScriptTM RT reagent Kit with gDNA Eraser Takara, Dalian, China. All qPCRs were performed at least in triplicates. To extract carotenoids from E. coli and algal cells, harvested cells were dried using a freeze-dryer for 24 h.

The carotenoids extraction, identification, and quantification in E. coli were processed as described previously [ 27 ]. The carotenoids determination in algal cells was described as following. The dried pellet of algal cells was crushed into powder, and then 10 mg were weight out and transferred into a 10 ml centrifuge tube.

After adding 3 mL of cold methanol, cells were sonicated to release carotenoids. Since there are non-target pigments presented in the extracts form algal cells, such as chlorophylls and lutein, a different HPLC detection procedure was applied, using the YMC C30 carotenoid column 4.

For the quantification, a calibration curve with coefficiency at higher than 0. Shanghai, China. To determine the growth of transgenic microalgae, the OD was measured in a period of 6 days.

The seed culture at the OD of 0. The algal seed cells were diluted by TAP liquid medium into a final OD of 0. The measurement was performed on Epoch2 microplate reader Bio-Tek Instruments, Winooski, VT, USA. All experiments were performed with at least three biological replicates.

Data were presented as the mean with standard deviation. To compare the lycopene β-cyclase driven from bacterium and Dunaliella salina on β-carotene production, their performance was investigated in E.

After cultivation and induction, the E. coli cells BL21 DE3 without any additional foreigner genes had no additional color Fig. coli cells producing lycopene displayed a red color Fig. It is observed that the cell pellet of lycopene-produced E. coli containing either DsLcyb1 or CrtY showed the yellow color Fig.

Further pigment analysis using HPLC revealed that lycopene-produced E. coli transformed with alga-driven DsLcyb1 or bacterium-driven CrtY generated β-carotene, at the level of 0.

Statistically, CrtY performed better and produced appreciate 1. The detection and quantification of β-carotene productivity in E. coli by HPLC analysis. A The cell pellet of E. coli BL21 DE3 ; B the cell pellet of lycopene-produced E. coli ; C the cell pellet of lycopene-produced E.

coli with plasmid pET-DsLcyb1; D the cell pellet of lycopene-produced E. coli with plasmid pET-CrtY; E the amount of β-carotene in lycopene-produced E. coli with plasmid pET-DsLcyb1 or pET-CrtY. To figure out if the motivated CrtY found in E. coli could promote the β-carotene biosynthesis in microalgae, the codon-optimized CrtY was transferred into C.

reinhardtii genome. The PCR was performed to evaluate the successful integration of CrtY into C. reinhardtii nuclear genome, using genomic DNA isolated from colonies grown under antibiotic selection stress.

reinhardtii genome, the positive transformants should generate two PCR fragments, including the bp of native psaD gene and the bp of CrtY gene. Results suggested that the negative control NC, the wild type of C.

As a result, algal colonies Y35, Y38, Y43, Y51, and Y58 generated two fragments that were in the same size as NC and P, suggesting the successful integration of CrtY in their genome.

M refers to the DNA ladder; P refers to the plasmid DNA of pDbCrtY, as the positive control; NC refers to the wild type of C. reinhardtii , as the negative control; ddH 2 O refers to the double distilled water, as the no template control; Y30, Y35, Y36, Y37, Y38, Y43,Y49, Y51, and Y58 refer to the algal colonies.

Subsequently, to confirm the successful expression of CrtY in positive transformants of C. reinhardtii , the qPCR was conducted. Hence, to normalize the relative expression level of CrtY in transformants, the transformant Y43 was employed as the control.

Results suggested that there were significant differences on the relative expression level of CrtY among transformants Fig.

The high expression level of CrtY was found in Y35 and Y58, which is about Transformants Y38 and Y51 had similar transcripts of CrtY , that were about 2. The relative expression levels of C. reinhardtii transformants based on qPCR analysis.

The expression level of actin was set as the internal control and the expression level of CrtY in Y43 was used for data normalization. To determine the effect of expressing CrtY in the transgenic C.

reinhardtii on carotenoids biosynthesis, pigments were extracted from algal cells suffering from the high light treatment which has been reported as a stimulation to promote carotenoids biosynthesis.

The amounts of β-carotene and lutein are expected to increase and reduce in transgenic algal cells, respectively. Based on the HPLC analysis, the β-carotene content in transgenic C.

reinhardtii was varied from reinhardtii was Therefore, all tested transgenic C. reinhardtii produced significant higher amount of β-carotene than the wild type Fig. The highest amount of β-carotene was found in Y51, which had 2.

Compared with β-carotene, the lutein content was much lower, ranging from 4. Statistical analysis revealed that only Y35, Y38, and Y51 had significant higher amount of lutein than the wild type, at the confidence of 0. By comparing the data, the highest lutein production was found in Y38, which was only 1.

The amount of β-carotene A and lutein B in the wild type and transgenic C. reinhardtii based on the HPLC analysis. NC indicates the wild type of C. reinhardtii , as the negative control.

Y35, Y38, Y43, Y51, and Y58 indicate different strains of transgenic C. ns indicates no statistical significance at the level of 0. Moreover, to check if the overexpression of CrtY affects the growth of microalgae, the growth curve of transgenic C. reinhardtii was measured.

Results suggested that except Y38, which showed obvious growth defect at Day3, all other transgenic C. reinhardtii showed no difference on the growth with the wild type at all determined points Fig.

Moreover, the growth of Y38 was recovered at Day 4 and maintained similar growth status as the wild type. Hence, it was concluded that the overexpression of CrtY had no defective effect on the microalga growth. The growth curve of the wild type and transgenic C.

NC stands for the wild type of C. Y35, Y38, Y43, Y51, and Y58 refer to different strains of transgenic C. β-Carotene is a terpenoid with high economic value [ 7 ]. With the advantage of well-elucidated pathway of β-carotene biosynthesis, lots of efforts were placed to bio-engineer its production with high yield and low cost, mainly using E.

coli and yeast as cell factory [ 8 , 9 , 10 ]. However, only few researches on the production of β-carotene in microalgae were reported, even though microalgae were considered as the green cell factory for drug-related products, due to the advantages of low cultivation cost, fast growth, and fixation of CO 2 as the carbon source [ 28 ].

Aiming to construct a microalgae strain with high β-carotene productively, a set of experiments was designed in this study, using microalgae Chlamydomonas reinhardtii as the platform.

As documented, the biosynthesis of β-carotene starts from IPP isopentenyl pyrophosphate , followed by various enzymes including isopentenyl pyrophosphate isomerase, geranylgeranyl diphosphate synthase, phytoene desaturase, phytoene synthase, and lycopene cyclase, the C5 blocks were built into C40 carotene [ 1 ].

The lycopene cyclase [EC: 5. Four families of lycopene cyclase have been reported and their similarities with each other are low [ 19 , 20 , 21 ]. Among them, lycopene β-cyclase introduces one β-ring at one end of lycopene to form γ-carotene, and then introduces another β-ring to the other end to form β-carotene [ 21 ].

The lycopene β-cyclases from carotenogenic bacteria CrtY and from plants, algae, and cyanobacteria CrtL-b and LCY-b were proved to have good activity for carotenoids production in plants and engineered bacteria [ 21 ].

In microalgae, it has been reported that the microalga D. Besides, the bacterial CrtY has been frequently used to produce β-carotene in E. coli [ 23 ]. Therefore, this study selected CrtY from bacteria Pantoea agglomerans and LCY-b from microalga Dunaliella salina DsLcyb1 as candidates, aiming to improve the β-carotene production in C.

reinhardtii by overexpressing an effective lycopene cyclase. According to the results obtained in this study, it was found that CrtY has better activity than DsLcyb1 on β-carotene production in E. coli Fig. Furthermore, the bacterial CrtY was attempt to integrate into the nuclear genome of C.

reinhardtii , with the expect to competitively and dominantly convert lycopene into β-carotene rather than α-carotene. Thereby, the transgenic C. reinhardtii was expected to have increased β-carotene and reduced lutein content. The thickness of the inner layers of the carotid arteries can be measured noninvasively using ultrasound technology.

This measurement of carotid intima-media thickness is considered a reliable marker of atherosclerosis A number of case-control and cross-sectional studies have found higher blood concentrations of carotenoids to be associated with significantly lower measures of carotid artery intima-media thickness Additionally, higher plasma carotenoids at baseline have been associated with significant reductions in risk of cardiovascular disease in some prospective cohort studies but not in others In a US national survey, National Health And Nutrition Examination Survey NHANES , serum total carotenoid concentration was inversely associated with blood concentrations of two cardiovascular risk factors, C-reactive protein CRP and total homocysteine NHANES also found an inverse association between total dietary carotenoid intake sum of α-carotene, β-carotene, β-cryptoxanthin, lycopene, lutein, and zeaxanthin and risk of hypertension While the results of several prospective studies indicate that people with higher intakes of carotenoid-rich fruit and vegetables are at lower risk of cardiovascular disease , , it is not yet clear whether this effect is a result of carotenoids or other factors associated with diets high in carotenoid-rich fruit and vegetables.

Based on the results of these randomized controlled trials, the US Preventive Health Services Task Force found good evidence to suggest that β-carotene supplements provided no benefit in the prevention of cardiovascular disease in healthy adults Moreover, a recent meta-analysis of 12 randomized, placebo-controlled trials found that β-carotene supplementation increased mortality related to cardiovascular disease RR , 1.

Thus, although diets rich in β-carotene have generally been associated with reduced cardiovascular disease risk in observational studies, there is no evidence that β-carotene supplementation reduces cardiovascular disease risk Supplemental lycopene : Several randomized controlled trials have examined whether supplementation with lycopene, tomato products, or tomato extracts might benefit cardiovascular health by improving blood pressure, lipid profiles, or function of the vascular endothelium.

A recent meta-analysis of eight trials found no effect of supplemental lycopene on systolic or diastolic blood pressure ; no benefits were found in healthy subjects or in hypertensive subjects The meta-analysis also showed no effect of lycopene supplementation on total cholesterol 10 trials , LDL cholesterol 11 trials , or HDL 11 trials cholesterol Moreover, some, but not all , short-term trials have indicated supplemental lycopene might improve function of the vascular endothelium in healthy subjects , ; however, large, long-term placebo -controlled studies are needed.

An experimental study found that the administration of β-cryptoxanthin to ovariectomized mice limited bone resorption by inhibiting osteoclast differentiation but had no effect on osteoblast -driven bone formation Yet, evidence of a protective role of β-cryptoxanthin — and other individual carotenoids — against bone loss in humans is scarce.

In the prospective Framingham Osteoporosis Study, data analysis of participants found no association between β-cryptoxanthin intake and changes in bone mineral density BMD over a four-year period The year follow-up of participants in the Framingham Osteoporosis Study participants showed those in the highest tertile of total carotenoid intake median intake: No such association was reported with serum concentrations of other individual carotenoids.

More recently, in a cross-sectional analysis of the European Prospective Investigation into Cancer and Nutrition-Norfolk cohort, higher dietary intakes of β-carotene and combined lutein and zeaxanthin were linked to higher bone density at the heel in women No associations of dietary carotenoid intake and heel bone density were found in men, and serum concentration of carotenoids was not linked to heel bone density in either men or women While a few studies have found a protective association between higher carotenoid intake and osteoporosis or bone fracture, the available studies are observational.

Whether carotenoid supplementation may help prevent bone loss and reduce the risk of osteoporosis in older individuals is currently unknown; randomized controlled trials would be needed to address this question. It is important to note that high-dose supplementation with preformed vitamin A retinol has been associated with adverse effects on bone health see the article on Vitamin A.

Observational studies have suggested that dietary lutein may be of benefit in maintaining cognitive health , and a cross-sectional study of 4, older adults associated higher blood lutein concentrations with improved cognition, including memory and executive function As stated above, among the carotenoids, lutein and its isomer zeaxanthin are the only two that cross the blood- retina barrier to form macular pigment in the eye.

Lutein also preferentially accumulates in the brain , A few studies have suggested that lutein and zeaxanthin concentrations in the macula correlate with brain lutein and zeaxanthin status and therefore might be used as a biomarker of cognitive health Additionally, in the Georgia Centenarian Study, the analysis of cross-sectional data from 47 centenarian decedents showed a positive association between post-mortem measures of brain lutein concentrations and pre-mortem measures of cognitive function Brain lutein concentrations were found to be significantly lower in individuals with mild cognitive impairment compared to those with normal cognitive function The most prevalent carotenoids in the human diet are α-carotene, β-carotene, β-cryptoxanthin, lycopene, lutein, and zeaxanthin Most carotenoids in foods are found in the all-trans form see Figure 1 and Figure 2 above , although cooking may result in the formation of other isomers.

The relatively low bioavailability of carotenoids from most foods compared to supplements is partly due to the fact that they are associated with proteins in the plant matrix Chopping, homogenizing, and cooking disrupt the plant matrix, increasing the bioavailability of carotenoids 3.

For example, the bioavailability of lycopene from tomatoes is substantially improved by heating tomatoes in oil , For information on other factors that affect carotenoid bioavailability, see above and the Moran et al.

review 5. α-Carotene and β-carotene are provitamin A carotenoids, meaning they can be converted in the body to vitamin A.

Thus, it would take 12 µg of β-carotene from food to provide the equivalent of 1 µg 0. Orange and yellow vegetables like carrots and winter squash are rich sources of α- and β-carotene.

Spinach is also a rich source of β-carotene, although the chlorophyll in spinach leaves hides the yellow-orange pigment. Some foods that are good sources of α-carotene and β-carotene are listed in Table 2 and Table 3 Like α- and β-carotene, β-cryptoxanthin is a provitamin A carotenoid.

Orange and red fruit and vegetables like sweet red peppers and oranges are particularly rich sources of β-cryptoxanthin. Some foods that are good sources of β-cryptoxanthin are listed in Table 4 Lycopene gives tomatoes, pink grapefruit, watermelon, and guava their red color.

Lycopene is not a provitamin A carotenoid because it cannot be converted to retinol. Some foods that are good sources of lycopene are listed in Table 5 Although lutein and zeaxanthin are different compounds, they are both classified as xanthophylls and nonprovitamin A carotenoids see Figure 2 above.

Both pigments are present in a variety of fruit and vegetables. Dark green leafy vegetables like spinach and kale are particularly rich sources of lutein but poor sources of zeaxanthin Although relatively low in lutein, egg yolks and avocados are highly bioavailable sources of lutein.

Good sources of dietary zeaxanthin include yellow corn, corn-based products, bell peppers, and egg yolk Some foods containing lutein and zeaxanthin are listed in Table 6 For more information on the carotenoid content of certain foods, search USDA's FoodData Central database.

Dietary supplements providing purified carotenoids and combinations of carotenoids are commercially available in the US without a prescription. Carotenoids are best absorbed when taken with a meal containing fat.

Supplements containing a mixture of carotenoids may include α-carotene. As a provitamin A carotenoid, supplemental α-carotene can contribute to fulfill vitamin A requirements. It is not known whether the relative bioavailability of supplemental α-carotene is greater than that of dietary α-carotene.

Supplements containing a mixture of carotenoids may include α-cryptoxanthin. As a provitamin A carotenoid, supplemental α-cryptoxanthin can contribute to fulfill vitamin A requirements. It is not known whether the relative bioavailability of supplemental α-cryptoxanthin is greater than that of dietary α-cryptoxanthin.

β-Carotene is sold as individual supplements and also found in supplements marketed to promote visual health Commercially available β-carotene supplements usually contain between 1. As a provitamin A carotenoid, β-carotene may be used to provide all or part of the vitamin A in multivitamin supplements.

The provitamin A activity of β-carotene from supplements is much higher than that of β-carotene from food: it takes only 2 micrograms [µg] 0. Of note, the β-carotene content of supplements is often listed in international units IU rather than µg: 3, µg 3 mg of supplemental β-carotene provides 5, IU of vitamin A.

Lycopene has no provitamin A activity. Synthetic lycopene and lycopene from natural sources, mainly tomatoes, are available as nutritional supplements containing up to 15 mg of lycopene per softgel capsule Lutein and zeaxanthin are not provitamin A carotenoids.

Lutein and zeaxanthin supplements are available as free carotenoids non-esterified or as esters esterified to fatty acids. Both forms appear to have comparable bioavailability Many commercially available lutein and zeaxanthin supplements have much higher amounts of lutein than zeaxanthin Supplements containing only lutein or zeaxanthin are also available.

Although β-carotene can be converted to vitamin A, the conversion of β-carotene to vitamin A decreases when body stores of vitamin A are high see Absorption, Metabolism, and Bioavailability. This may explain why high doses of β-carotene have never been found to cause vitamin A toxicity.

Although the reasons for these findings are not yet clear, the potential risk of lung cancer in smokers and other high-risk groups supplemented with high-dose β-carotene outweigh any possible benefits for chronic disease prevention It should be noted that there is no evidence that β-carotene supplementation may harm nonsmokers.

Carotenodermia is not associated with any underlying health problems and resolves when supplementation with β-carotene is discontinued or dietary carotene intake is reduced. Lycopenodermia : High intakes of lycopene-rich food or supplements may result in a deep orange discoloration of the skin known as lycopenodermia.

Because lycopene is more intensely colored than the carotenes, lycopenodermia may occur at lower doses than carotenodermia Unlike vitamin A, high doses of β-carotene taken by pregnant women have not been associated with increased risk of birth defects However, the safety of high-dose β-carotene supplements in pregnancy and lactation has not been well studied.

The safety of carotenoid supplements other than β-carotene in pregnancy and lactation has not been established, so pregnant and breast-feeding women should obtain carotenoids from food rather than supplements. There is no reason to limit the consumption of carotenoid-rich fruit and vegetables during pregnancy The cholesterol -lowering agents, cholestyramine Questran and colestipol Colestid , can reduce absorption of fat- soluble vitamins and carotenoids, as can mineral oil and Orlistat Xenical , a drug used to treat obesity Colchicine, a drug used to treat gout , can cause intestinal malabsorption.

Increasing gastric pH through the use of proton-pump inhibitors Omeprazole, Lansoprazole may decrease the absorption of a single dose of a β-carotene supplement , but the effect is unlikely to be clinically significant A three-year randomized controlled trial in patients with documented coronary heart disease CHD and low serum high density lipoprotein HDL concentrations found that a combination of simvastatin Zocor and niacin increased HDL2 levels, inhibited the progression of coronary artery stenosis , and decreased the frequency of cardiovascular events, including myocardial infarction and stroke Surprisingly, when an antioxidant combination of 1, mg of vitamin C, mg of RRR -α-tocopherol vitamin E , µg of selenium, and 25 mg of β-carotene daily was taken with the simvastatin-niacin combination, the protective effects were diminished.

Since the antioxidants were taken together in this trial, the individual contribution of β-carotene cannot be determined. In contrast, a much larger randomized controlled trial of simvastatin and an antioxidant combination of mg of RRR -α-tocopherol, mg of vitamin C, and 20 mg of β-carotene daily in more than 20, men and women with CHD or diabetes mellitus found that the antioxidant combination did not diminish the cardioprotective effects of simvastatin therapy over a five-year period These contradictory findings indicate that further research is needed on potential interactions between antioxidant supplements and cholesterol-lowering agents, such as niacin and statins.

One study in adults found that those who consumed more than 4. However, advising people who use plant sterol- or stanol-containing margarines to consume an extra serving of carotenoid-rich fruit or vegetables daily prevented decreases in plasma carotenoid concentrations The relationships between alcohol consumption and carotenoid metabolism are not well understood.

There is some evidence that regular alcohol consumption inhibits the conversion of β-carotene to retinol Increases in lung cancer risk associated with high-dose β-carotene supplementation in two randomized controlled trials were enhanced in those with higher alcohol intakes 40 , The results of metabolic studies suggested that high doses of β-carotene compete with lutein and lycopene for absorption when consumed at the same time However, the consumption of high-dose β-carotene supplements did not adversely affect serum carotenoid concentrations in long-term clinical trials Originally written in by: Jane Higdon, Ph.

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